Annual Report (2018)

No.30
The NOVARTIS Foundation (Japan)
for the Promotion of Science
平成30年度

財団年報 第30号

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Contents
Ⅰ．Introduction はじめに ……………………………………………………………………… 5
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ．Reports from the Recipients of Novartis Research Grants

研究報告 ………………………………………………………………………………………… 7
1. Regulatory function of stress responsive splicing controlled by long non-coding
RNA ………………………………………………………………………………………… 8
Tetsuro Hirose
Institute for Genetic Medicine, Hokkaido University

2. How is recombination suppression achieved in the first place between sex
chromosomes? ………………………………………………………………………………… 11
Kiyoshi Kikuchi
University of Tokyo, Graduate School of Agricultural and Life Sciences

3. Identification of novel neural processing mechanism by using transcriptome
analysis ………………………………………………………………………………………… 14
Akane Ohta, Natsune Takagaki, Atsushi Kuhara
Institute of integrative neurobiology & Department of Biology, Konan University

4. Analysis of starch grain formation using live imaging technique ………………………… 18
Ryo Matsushima
Institute of Plant Science and Resources, Okayama University

5. The role of RNA binding protein PSF in prostate cancer progression …………………… 21
Ken-ichi Takayama
Department of Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology

6. Selective information routing in the hippocampal network ……………………………… 25
Kenji Mizuseki
Osaka City University Graduate School of Medicine

7. Chemical approach to developing anticancer agents of marine alkaloid origin ………… 28
Takehiko Yoshimitsu
Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, andPharmaceutical Sciences, Okayama University

8. Catecholamine-induced senescence of endothelial cells and bone marrow cells promotes
cardiac dysfunction in mice…………………………………………………………… 31
Tohru Minamino
Department of Cardiovascular Biology and Medicine
Niigata University Graduate School of Medical and Dental Sciences

9. Modulation of three-dimensional chromatin structure by Piwi-piRNAs ………………… 34
Yuka Iwasaki
Keio University School of Medicine

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10. Catalytic Synthesis of Spiroketals Using Water as the Oxygen Source Triggered byRearrangement of Internal Alkynes ………………………………………………………… 37
Mutoh, Yuichiro
Tokyo University of Science

11. Molecular analysis for the pathophysiological significance of brain hypoxic response
and application for the CNS diseases ……………………………………………………… 41
Hisashi Shirakawa
Kyoto Univ., Grad. Sch. Pharm. Sci.

12. Elucidation of the onset mechanism of myeloproliferative neoplasms through theerythropoietin receptor ……………………………………………………………………… 44
Megumi Funakoshi-Tago
Faculty of Pharmacy, Keio University

13. Induction and visualization of adult cardiomyocytes proliferation through growth
factor receptor activation …………………………………………………………………… 47
Maretoshi Hirai
Kansai Medical University, Department of Pharmacology

14. Analysis of the function and genomic abnormality of tumor suppressor gene PHLDA3
in neuroendocrine tumors- Towards development of a novel genetic diagnosis method for neuroendocrine
tumors - ……………………………………………………………………………………… 50
Rieko Ohki
National Cancer Center Research Institute

15. Disease specific regulation of the histone methylation in acute myeloid leukemia …… 53
Takeshi Ueda
Kindai University Faculty of Medicine

16. Treatment of metastatic tumors by systemic injection of tumor-retargeted oncolytic
herpesviruses ………………………………………………………………………………… 55
Hiroaki Uchida
Division of Bioengineering, Advanced Clinical Research Center,
The Institute of Medical Science, The University of Tokyo

17. Revealing cell membrane functions through in-vitro analysis by tip-enhanced Raman
spectroscopy in liquid ……………………………………………………………………… 58
Prabhat Verma
Department of Applied Physics, Osaka University

18. Elucidation of the immune-nerve-metabolic systems network in cardiac homeostasis
and failure …………………………………………………………………………………… 61
Ichiro Manabe
Chiba University

19. Roles of synaptic plasticity and replay during sleep in memory consolidation ……… 63
Yasunori Hayashi
Department of Pharmacology
Kyoto University Graduate School of Medicine

20. Study on the Mechanism of Sex Determination in Germline Stem Cells ……………… 66
Minoru Tanaka
Graduate School of Science, Nagoya University

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21. ROR1-mediated caveolae formation and caveolar endocytosis in lung cancer ……… 69
Tomoya Yamaguchi
Kumamoto University

22. Regulation of energy metabolism by environmental cue and epigenome ……………… 73
Yoshihiro Matsumura
Research Center for Advanced Science and Technology, The University of Tokyo

23. Mitochondrial dynamics and antiviral innate immunity ………………………………… 76
Takumi Koshiba
Department of Biology, Faculty of Science, Kyushu University

24. Analysis of epigenetic regulation toward establishment of a stable culture method ofneural progenitor cells ……………………………………………………………………… 78
Noriaki Sasai
Nara Institute of Science and Technology

25. Elucidating the regulatory mechanism of histone modification status in colorectal ancer
stem cells to maintain stemness and its application for drug development …………… 80
Ryuji Hamamoto
Division of Molecular Modification and Cancer Biology,
National Cancer Center Research Institute

26. Lymphoma progression promoted by the breakdown of the system for convergence of
inflammation ………………………………………………………………………………… 83
Yoshio Katayama
Hematology, Kobe University Hospital

27. Role of CRIMI as a novel regulator for osteoblast functions revealed using steoporotic
mutant mice …………………………………………………………………………………… 86
Tatsuya Furuichi
Co-Department of Veterinary Medicine, Faculty of Agriculture, Iwate University

28. Peroxisome biogenesis disorders: Molecular mechanism of pathogenesis in theerebellum ……………………………………………………………………………………… 88
Yukio Fujiki
Medical Institute of Bioregulation, Kyushu University

29. Optically transparent bamboo-cellulose/collagen nanocomposite having antibacterial
action ………………………………………………………………………………………… 91
Yoko Okahisa-Kobayashi
Kyoto Institute of Technology

30. Establishment of therapeutic genome editing techniques applicable for post-mitotic
neurons ………………………………………………………………………………………… 93
Izumi Oinuma
Graduate School of Life Science, University of Hyogo

31. Vascular intraluminal pressure-dependent switching machinery of ngiogenic mode … 96
Koichi Nishiyama
International Research Center for Medical Sciences, Kumamoto University

32. Novel therapeutic strategy for heart failure targeting PKG/TORC signaling and its
intracellular localization. …………………………………………………………………… 99
Taishi Nakamura
Kumamoto University Hospital

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33. Virus-mediated suppression of host innate immune response ………………………… 102
Hiroyuki Oshiumi
Department of Immunology, Faculty of Life Sciences, Kumamoto University

34. Treg Related Exosomal microRNA Involving to the Spatial and Chronologic Evolutionof CRC ……………………………………………………………………………………… 105
Koshi Mimori
Kyushu University Beppu Hospital

35. Elucidation of the sialyl-Tn antigen-mediated survival mechanism of hypoxic tumor
cells ………………………………………………………………………………………… 107
Kazuaki Ohtsubo
Department of Analytical Biochemistry, Faculty of Life Sciences, Kumamoto University

Ⅲ．Reports from the Recipients of Grants for International Meetings
研究集会報告………………………………………………………………………………… 110
1. The 6th Symposium on ATP1A3 in Disease ……………………………………………… 111
Masayuki Sasaki
National Center of Neurology and Psychiatry, National Center Hospital

2. 29th International Symposium on Chirality [Chirality 2017; ISCD-29] ……………… 113
Toshikazu Takata
Tokyo Institute of Technology, Japan

3. The 15th International Conference on Na, K-ATPase and Related Transport ATPases……… 115
Haruo Ogawa
Institute of Molecular and Cellular Biosciences, The University of Tokyo

4. 31st Annual Meeting of the Japanese Society for Biological Sciences in Space(JSBSS 31st) ……………………………………………………………………………… 117
Akihisa Takahashi
Gunma University Heavy Ion Medical Center

5. International Conference on“The Origin of Life –Synergy among the RNA, Protein and Lipid Worlds –“…… 119
Masayuki Imai
Tohoku University

Ⅳ．The 31st (fiscal year 2017) Promotion Report

第31期（2017年度）助成事業報告 ……………………………………………………… 121

Ⅴ．The 31st (fiscal year 2017) Financial Report

第31期（2017年度）財務報告 …………………………………………………………… 128

Ⅵ．List of the Board Members

役員名簿 ……………………………………………………………………………………… 130

Ⅶ．Information from the Secretariat

事務局便り …………………………………………………………………………………… 134

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Introduction

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
We have compiled here the research accomplishments of the grantees of the NOVARTISFoundation (Japan) for the Promotion of Science in FY2016. The Foundation was originallyestablished on September 4, 1987 with the generous donation of JPY 1 billion from CibaGeigy AG, Switzerland, for the purpose of “contributing to academic development and thus
improving public health and welfare of the people by subsidizing to promote creative researchand international exchange in the fields of biological and life sciences and related chemistry”.
Since then, the Foundation has granted nearly JPY 2 billion to approximately 1,700 researchcases in 30 years. Research funds provided by the Foundation may be only a part of the cost of
the entire research, however, we have continued to subsidize in belief that we could contributeto the development of excellent research. Joy of grantees could be felt in the acknowledgmentof the papers received by the Secretariat, and we are pleased with our humble contribution.
The Foundation supports creative research in the fields of biological and life sciences, related
chemistry and information science. Numerous entries submitted are examined by specialized
researchers in a strict, fair and impartial manner. While we value originality as selection criteria,
considerations are given to those start-up laboratories in urgent needs of research funds, aswell as age and women researchers. In each case, we believe that excellent research is selected.
In fact, we are informed that the quality of the research funded by the Foundation is highly
esteemed by researchers in Japan, proving our purposes have not been wrong. We hope thatthese researches will lead the development of life sciences, and eventually to the developmentof new drugs and therapies.
Speaking of 2016, the Kumamoto earthquake occurred on April 14th and 16th was observed
the maximum seismic intensity 7. Kumamoto and Oita prefectures had 267 deaths (including
direct death and related deaths). A complete destruction of houses or a semi-destruction
exceeded 43,000 buildings. It suffered extensive damage. I prayed for the reconstruction andawarded five Novartis research grants to Kumamoto and Oita researchers as earthquake disasterframe. I hope that it helped the research in a harsh.
This Annual Report summarizes the excellent research outcomes. It is an outstandingachievement accomplished within a limited period of one year. We are deeply grateful toeveryone who supported the activities of the Foundation, including the selection committee
members who have selected these excellent researches.

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はじめに

代表理事

金子

章道

ここに2016年度にノバルティス科学振興財団研究助成金を受けられた方々の研究報告を収録いたしました。当財団は1987年９月４日、スイス、チバガイギー社からの10億円のご寄附をもとに、「生物・生命科学および関連する化学の領域において、創造的な研究ならびに国際交流への助成を行うことにより、学術の振興を図り国民の健康と福祉の向上に寄与する」ことを目的に設立されました。爾来30年間に1,700件
近く、金額にしておよそ20億円の助成を行ってまいりました。当財団が提供する研究
費は、研究全体に要する費用の一部かも知れませんが、優れた研究の発展に少しでも寄与できればと考え、助成して参りました。事務局に寄せられた論文等の謝辞からも受賞者の喜びが伝わり、多少なりとも貢献できているものと嬉しく思っております。
当財団は、生物・生命科学、関連する化学および情報科学の領域における創造的な研究を助成しております。数多く寄せられた応募を専門の研究者により厳正に公正
かつ公平に審査しております。選考基準と致しまして独創性を重視しておりますが、研究室を新たに立ち上げ研究資金に逼迫しているケースや年齢、女性研究者にも配慮しており、いずれも優れた研究が選択されたものと存じます。事実、当財団で助成を受けた研究の質が高いと国内の研究者の間で評価されていることを聞くにつけ、我々の目指すところは間違っていないことを確信する次第であります。このような研究が生命科学の発展、ひいては新しい治療薬の開発や治療法の開発に発展することを期待しております。
2016年と言えば、４月14日及び16日に発生した最大震度７を観測する熊本地震
にて、熊本県と大分県は死者267人（直接死、関連死などを含め）、住宅の全壊・半壊は43,000棟を超えるなど、甚大な被害を受けました。その復興を祈念しまして、震災枠として熊本及び大分の研究者にノバルティス研究奨励金、5件を授与しました。厳しい環境の中、研究の一助になられたものと思います。
この年報にはこのような優れた研究の成果をまとめております。１年間と限られた時間の中で達成した立派な業績であります。なお、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をはじめ、財団の活動を支えて下さっている関係者の皆様に深く感謝いたします。

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

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Regulatory function of stress responsive splicing controlled
by long non-coding RNA
Tetsuro Hirose
hirose@igm.hokudai.ac.jp
Institute for Genetic Medicine, Hokkaido University

Summary Abstract
Satellite III long noncoding RNA (Sat III lncRNA) is the architectural RNA to construct nuclear
stress bodies (nSBs) that are formed under heat stress condition. We identified multiple protein
components of nSBs that include SR splicing factors and their modifier protein. Sat III knockdown
study revealed that nSBs are required for efficient phosphorylation of SR proteins that control
splicing of specific mRNAs in the stress recovery phase.
Key Words：noncoding RNA, stress response, splicing regulation

Introduction
Sat III lncRNA is a primate-specific lncRNA which is transcribed from the Sat III repeats inpericentromeric regions. Sat III repeats are transcriptionally silent in normal unstressed condition,
however, it is transcribed under heat stress. Sat III lncRNA recruits specific RNA-binding proteins(RBPs) including specific SR proteins and SWI/SNF chromatin remodeling complexes to assemble
a massive ribonucleoprotein complex called nuclear stress body (nSB) (Ref1). nSBs are detectable
for several hours even after the stress removal. nSBs presumably regulate pre-mRNA splicing
under certain stress conditions. However, the precise structure and function of nSBs remains
uninvestigated.

Results
1. Identification of nSB protein components by ChIRP-MS
We first isolated nSBs by ChIRP (Chromatin Isolation by RNA Purification) from the heat shocked
HeLa cells. ChIRP was carried out using antisense oligonucleotide (ASO) complementary to therepeat sequence of Sat III lncRNA. Silver staining of the proteins in the ChIRP fraction with Sat
III ASO revealed that multiple proteins were specifically precipitated under heat stressed cells but
not from normal unstressed cells. Mass spectrometric analysis (ChIRP-MS) identified multiple
RNA binding proteins (RBPs) including Serine/Arginie rich splicing factors (SR proteins), HNRNP
proteins and Scaffold attachment factors as the Sat III-interacting proteins or the nSB components.
Most of the identified nSB proteins are localized in nuclear speckles under normal unstressed
conditions, suggesting that nuclear speckle are partially remodeled and its components relocate tonSBs by interacting with Sat III lncRNA upon heat stress. Interestingly, in addition to the RBPs, theirmodifier proteins were identified as the nSB components. Immunofluorescence combined with RNAFISH confirmed that one modifier protein was co-localized with Sat III lncRNA in nSBs.

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2. nSBs control SR protein re-phosphorylation in post-stress recovery phase
SR proteins are known to be rapidly dephosphorylated upon heat stress and gradually
rephosphorylated after the stress removal. Here, we hypothesized that nSBs regulate thephosphorylation state of the nSB-localized SR proteins by recruiting the modifier protein. We
investigated the effect of Sat III knockdown on phosphorylation state of SR proteins during andafter heat stress. The phosphorylation state was monitored by loading the proteins on Phos-tag SDSPAGE. Sat III knockdown caused marked delay in the modifier-dependent rephosphorylation ofnSB-localized SR proteins after stress removal. However, it hardly affected their stress-induced
dephosphorylation, suggesting that Sat III lncRNA facilitates rephosphorylation of nSB-localized
SR proteins in post-stress recovery phase by recruiting the modifier protein. This result raised
an intriguing possibility that Sat III lncRNA controls pre-mRNA splicing after stress removal bycontrolling the phosphorylation state of specific SR proteins.
3. nSBs control pre-mRNA splicing in stress recovery phase
Recent studies have revealed that multiple partially-unspliced pre-mRNAs that retain specificintron(s) are localized in the nucleus. Some of these nuclear retained pre-mRNAs undergo splicing
upon heat stress that leads to production of mature mRNAs. We investigated the role of Sat III
lncRNA in the heat shock-responsive intron removal from the nuclear retained partially-unspliced
pre-mRNAs. In the control HeLa cells, the nuclear pool of these partially-unspliced pre-mRNAs
were mostly depleted by the heat shock-induced splicing and conversely restored within a few hours
after the stress removal. In contrast, this restoration was markedly delayed in the Sat III knockdown
cells. Moreover, we confirmed that nSB-localized SR proteins and the enzymatic activity of themodifier protein were also required for the rapid restoration of the nuclear pool of the partiallyunspliced pre-mRNAs. These data suggest that nSB is required for efficient recovery of accumulationof partially unspliced pre-mRNA through modification of SR proteins upon stress removal. RNAseq analysis identified multiple pre-mRNAs that are analogously controlled by nSBs during stressrecovery phase through modification of the nSB-localized SR proteins.

Discussion & Conclusion
Our findings suggest that Sat III lncRNA-dependent nSB accelerates restoration of the pool of
specific nuclear-retained pre-mRNAs, which was depleted upon heat stress beforehand, throughfacilitating rephosphorylation of nSB-localized SR proteins by specific modifier protein in the poststress recovery phase. Sat III lncRNA as the architectural RNA of nSBs is primate specific lncRNA
which lacks in other mammalian species including rodents such as mouse. It is intriguing to pursue
the pattern of splicing control in heat stress condition as well as stress recovery phase in mouse cells.
Sat III lncRNA is transcribed from Satellite III repeats in pericentromiric region which is silenced
in unstressed cells. The repeated sequence may correspond to the recognition motifs of specific SR
proteins raising the interesting possibility that evolution of satellite repeats gives some advantage to
produce lncRNAs to capture specific splicing regulatory proteins under heat stress.

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References
1. Kawaguchi T, Tanigawa A, Naganuma T, Ohkawa Y, Souquere S, Pierron G, Hirose T. SWI/SNF
chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear
bodies. Proc Natl Acad Sci USA. 2015 Apr 7;112(14):4304-9. doi: 10.1073/pnas.1423819112..

一般の皆様へ
21世紀のポストゲノム時代に入り、ヒトゲノムの75% もの領域から正体不明なノンコーディング

RNA（lncRNA）が産生されていることが明らかになり、その機能に注目が集まっている。Sat IIIと呼ばれる lncRNA は、熱ストレスが細胞に加わった際に合成され、核内ストレス体という顆粒状の核内構造体を組み立てる機能を果たしている。実施者は、今回この核内ストレス体の果たす機能を調べた結果、Sat III lncRNA が核内ストレス体内に係留している SR タンパク質という制御因子のリン酸化状態をコントロールして、遺伝子発現過程の一段階である RNA スプライシングを制御していることを発見した。このメカニズムは、lncRNA が細胞のストレス状態からの回復を効率よく行うために働いていることを示しており、ヒトの身体のストレス遭遇時に重要な役割を果たしている可能性が浮上した。

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How is recombination suppression achieved in the first place
between sex chromosomes?
Kiyoshi Kikuchi
akikuchi@mail.ecc.u-tokyo.ac.jp
University of Tokyo, Graduate School of Agricultural and Life Sciences

Summary Abstract
A fundamental question concerning sex chromosome evolution is what has led to the lack ofrecombination between the pair of chromosome. Large-scale chromosomal inversions have been
assumed to be a major cause of the suppressed recombination. In this study, we characterized the sexdetermining region of a pair of closely-related species of fish, and identified a novel sex-determining
gene. Moreover, we found that their sex-determining regions exhibit differential recombination
patterns, despite that their sequences are very similar each other. Our results are not consistent
with the inversion hypothesis and, thus, suggest epigenetic mechanisms as a potential trigger of therecombination suppression.
Key Words：Evolution, recombination suppression, sex chromosome

Introduction
A major question concerning sex chromosome evolution is what has led to the lack ofrecombination between X and Y (or Z and W) chromosomes. Large-scale chromosomal inversions
have been assumed to be a major cause of the suppressed recombination1. However, recent reports
including ours suggest that recombination suppression is variable near the sex-determining locus,
implying that the suppression is initiated by mechanisms other than large-scale inversions, and thatthe inversions may be the effect rather than cause of recombination suppression. Since sex-limited
chromosomes of classic model species (such as mammalian Y and Drosophila Y) are enriched forrearrangements and repeats elements, research on newly formed sex chromosome systems is needed
to understand how the initial recombination suppression was established.

Results
1) In order to understand how recombination suppression was initially established, we searched for
newly formed sex chromosome systems in many fish species, and found a pair of closely-related
species (Carangidae) that potentially show differential recombination patterns around the sexdetermining locus.
2) To know whether the recombination suppression around the sex-determining locus has developedin the two species, we examined extents of linkage disequilibrium across the sex-determining
locus by performing genome-wide (five individuals for each sex and species) sequencing and sexdetermining-region-wide sequencing (approximately 50 individuals for each sex and species). We
found that species A exhibits no sign of suppression of recombination around the sex-determining
region, while species B shows a marked decrease in recombination ratio in the region spanningapproximately 300 Kb. This result indicates that the sex chromosome systems of these two closelyrelated species is indeed suitable for studying an initial phase of recombination suppression between
a pair of sex chromosomes in vertebrates.

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The Novartis Foundation(Japan)
for the Promotion of Science3) As a first step to establish the sex chromosomes of the two species as a model system in the fieldof sex chromosome evolution as well as to conduct rigorous analyses of the sex-determining region
in the near future, we performed genome-wide and region-wide association assay for identifying thesex-determining gene of the two species. We found that a single nucleotide polymorphism (SNP) in asteroidogenic gene Hsd17b1 is the sole polymorphisms almost perfectly associated with phenotypicsex in both species2. This sex-determining SNP causes a non-synonymous change from glycineon the W chromosome to glutamic acid on the Z near the catalytic center of Hsd17b1. Subsequentbiochemical and molecular dynamics analyses suggested that the amino acid substitution disrupts
the hydrogen-bond network among steroids and the catalytic residues of the Hsd17b1. Consequently,inter-conversion between 17-keto and 17β-hydroxy steroids is attenuated in the Z-type HSD17B1
protein. In situ hybridization of developing gonads indicated that Hsd17b1 mRNA is expressed inboth undifferentiated and differentiating gonads of both genotypic sexes. In the differentiating ovary,
both Z- and W-type Hsd17b1 is expressed in pre-granulosa cells, whereas Cyp19a1a, another key
component of estrogens synthesis, is predominantly expressed in pre-theca cells. In the differentiating
testis, only Z-type is expressed in pre-Sertoli cells, and no induction of Cyp19a1a was observed.
Based on these results, we conclude that homozygosity for the Hsd17b1 allele on the Z chromosome
results in testis formation due to the reduced enzymatic activity of its gene product and the resultant
shortage of estrogens, whereas Z/W heterozygosity promotes the development of ovaries due to theactive-type Hsd17b1 on the W chromosome.
4) To know whether the suppression of recombination between the Z and W chromosomes in species
B is due to large-scale inversions, we performed targeted genome sequencing of the sex-determining
regions from two species by using long-reads technologies, and constructed contigs/scaffolds of thetargeted region from Z and W chromosomes. We then compared the contigs/scaffolds within species(between Z and W) as well as between species (e.g. between Z of species A and W of species B).
We found no evidence of the large-scale inversions in those sequences of both species, althoughaccumulation of repeats elements was observed in species B. This result clearly indicates that thedecreased recombination ratio observed in species B is not initiated by the large-scale inversions.

Discussion & Conclusion
In this study, we first showed that a pair of closely-related species (Carangidae) exhibit differential
recombination patterns around the sex-determining locus. We then identified a novel master-sexdetermining gene and its precise genomic position in these species. We finally obtained a strong
evidence suggesting that large-scale inversions and sequence differences between sex chromosomes
are not the initial mechanisms that reduce recombination. One possible mechanism is an epigenetic
modification around the sex-determining locus as the sequence of Z and W are very similar. Since
the region required for the epigenetic analyses is sufficiently small, and the location of the sexdetermining gene has been identified in the pair of closely-related species, these fishes will be
an excellent material to study the effect of epigenetic modifications on the evolution of reduced
recombination in sex chromosomes.

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References
1) Wright AE, Dean R, Zimmer F, Mank JE (2016) Nature Commun. 7, 12087.
2) Koyama T, Kikuchi K, et al. Revisiting the steroid theory: Steroidogenic Hsd17b1 is a genetic sex
determiner in fish (submitted).

一般の皆様へ
ヒトの Y 染色体は著しく退化しており、大多数の遺伝子を失っています。このような退化したY 染色体は多くの動植物にみられます。この性染色体の退化は「組み換え抑制」てによってもたらされますが、「その原因は染色体間の逆位である」と説明されてきました。しかし、この説は、ヒトやハエの退化しきった性染色体をもとにした説であり、真の原因はわかっていません。我々は、退化直前・直後の性染色体をもつ魚類群を発見しました。これらを調べたところ、逆位が組み換え抑制のトリガー
ではなさそうだということがわかりました。抑制がある種とない種で、性決定領域の DNA 配列は類似していることから、エピゲノム機構が、組み換え抑制に関わる可能性が高いと考えられました。

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Identification of novel neural processing mechanism
by using transcriptome analysis
Akane Ohta, Natsune Takagaki, Atsushi Kuharao_akaneiro@me.com
Institute of integrative neurobiology & Department of Biology, Konan University

Summary Abstract
Temperature acclimation is essential system for living and proliferation of animals, yet their neuralmechanisms have not been well known. We are using temperature experience-dependent cold
acclimation of nematode C. elegans, as a model for studying temperature acclimation. ASJ sensory
neuron that has been known as a light sensing neuron senses and memorizes temperature in cold
acclimation. In this study, we conducted RNA-sequencing analysis of ASJ by using single cell RNAsequencing analysis.
Key Words：Temperature memory, acclimation, C. elegans

Introduction
Cold tolerance in C. elegans is a phenomenon that 20°C-cultivated animals cannot survive at 2°C,
whereas 15°C-cultivated animals can survive at 2°C. ASJ sensory neuron that has been known asa light and pheromone sensing neuron senses temperature and regulates cold tolerance throughinsulin signaling (Ohta, Ujisawa et al., Nature commun, 2014). Our previous analyses suggest thatcold tolerance is regulated by tissues network containing neurons, intestine and sperm, in whicha feedback regulation from sperm to ASJ thermosensory neuron is essential (Sonoda, et al., Cell
Reports, 2016). However, we have not known thermo-sensing molecules on ASJ and how ASJ iscontrolled by sperm signaling remain poorly understood.

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Results1
Cold tolerance of C. elegans is regulated by a pair of head sensoryneruon, ASJ (Figure1)(Ohta,
Ujisawa et al., Nature commun, 2014; Ujisawa et. al., PLOS ONE, 2016; Sonoda, et al., Cell Reports,2016). In the ASJ sensoryneuron, temperature is received by the unidentified-temperature receptor
that activates multiple trimeric G protein alpha subunits (Gα)(Figure1). Gα activate two types ofguanylyl cyclases, which increase concentration of cGMP in ASJ neuron, then opens cGMP-gatedcation channel composed by TAX-4 and TAX-2. Increment of intracellular calcium concentration
regulates secretion of insulin like molecules from synaptic region of ASJ neuron. These insulins
encoded by daf-28 and ins-6 genes are received by the intestine and neuron, in which insulinsignaling pathway negatively regulates cold tolerance thorough FOXO-type transcriptional factor
encoded by daf-16 gene (Figure1). The insulin signaling in intestine affects sperm that regulates ASJ
neuronal activity. On these sequential molecular signaling, a secretory signaling from sperm to head
sensoryneuron has not been known.

To identify new genes involved in the temperature sensation and feedback from sperm to ASJ
in temperature tolerance, we conducted RNA-sequencing analysis comparing mRNA expression
levels between ASJ and a gustatory neuron by using single cell RNA-sequencing analysis (Sugi &Ohtani, BBRC2014). PAB-1 is a polyA binding protein that binds with polyA site of mRNA. We
expressed PAB-1 gene in ASJ sensoryneuron specifically, by using the ASJ specific promoter trx1. This PAB-1 protein was tagged with 3*FLAG-tag for immunoprecipitation assay. The transgenic
animal containing extrachromosomal array, trx-1p:: 3*FLAG-pab-1, were cultivated at 20 degree.
Then mRNA in ASJ was in vivo cross-linked with FLAG-tagged PAB-1 protein. We homogenizedthese animals and isolated mRNA binding with FLAG-tagged PAB-1 protein by anti-FLAG-tag withmagnetic beads. By using this mRNA from ASJ, we constructed cDNA library for RNA sequencing
analysis by using next generation DNA sequencer. Also, cDNA library from a gustatory neuron
was constructed as a control analysis against ASJ. Because, the concentrations of mRNA
from ASJ or ASE were very low levels, we amplified these mRNA to cDNA by using wholetranscriptome amplification kit.

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Results2
By comparing gene expression levels between ASJ and a gustatory neuron in 20°C cultivatedanimals, we found the expression levels of ~600 genes such as neuropeptides, neuropeptide
receptors, insulin were altered. We measured cold tolerance phenotypes of many mutants defective inthose genes, and found that mutants defective in collagen, metalloprotease, cation channels showedabnormal cold tolerance. In these mutants, mutant animals defective in metalloprotease encoded by
nas-9 gene showed strong abnormality in cold tolerance (Figure2). After cultivation at 20 degree,
wild-type animals can not survive at 2 degree (Figure2A, B). By contrast, about 40% of nas-9 mutant
animals can survive at a degree, after cultivation at 20 degree (Figure2B). Previously reported, nas9 encodes an astacin-like metalloprotease and a nas-9::GFP promoter fusion reporter construct
is expressed in embryos and in the hypodermis though larval to adult stage. However, expressionof nas-9 gene in ASJ was not reported. Our RNA sequencing analysis revealed that astacin-likemetalloprotease NAS-9 is expressed in ASJ. We therefore hypothesized that NAS-9 functions in
ASJ thermosensory neuron and is involved in neural function or development of ASJ neuron, whichmaybe required for cold tolerance.

Discussion & Conclusion
Temperature tolerance system is important for animal, but its mechanisms are intricate. In
C. elegans, sensoryneuron senses temperature and temperature information is transmitted todownstream tissues through insulin signaling. In the nervous system, ASJ sensoryneuron acts as athermosensory neuron in cold tolerance.
The results in this study showed that important molecules for cold tolerance in thermosensory
neuron. Especially, astacin-like metalloprotease NAS-9 could function in ASJ and could be involvedin neural function or development of ASJ neuron, which may be required for cold tolerance. We
believe that the molecular and cellular analysis of cold tolerance in C. elegans should shed lightinto understanding fundamental mechanisms of temperature tolerance and acclimation in animals
including human.

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References
1. Light and pheromone-sensing neurons regulate cold habituation through insulin signaling in C.
elegans. Ohta, A., Ujisawa T., Sonoda S., Kuhara, A., Nature commun., 5: 4412, doi: 10.1038/
ncomms5412, 2014
2. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis
elegans. Ujisawa, T., Ohta, A., Uda-Yagi, M., Kuhara A.,
PLOS ONE, 11(10): e0165518., 1-20, 2016
3. Sperm affects head sensory neuron in temperature tolerance of Caenorhabditis elegans. Sonoda S.,
Ohta, A., Maruo, A., Ujisawa T., Kuhara, A., Cell Reports, 16, 1, 56–65, 2016

一般の皆様へ温度は生物の生死に直結する環境情報である。本申請者はこれまでに、C. elegans の低温耐性を指標に、温度応答のシンプルな実験系を確立した (Ohta et al., Nature commun, 2014)。具体的には、頭部の感覚ニューロンが温度を感知し、インスリンを分泌することで、腸などに働きかけ、温度耐性を獲得させる。さらに、腸が精子に働きかけ、精子が頭部の温度受容ニューロンをフィードバック制御する (Sonoda, Ohta et al., Cell reports, 2016)。本研究を通じて我々は、低温耐性に関して、温度受容ニューロンで機能する新規の分子を同定した。

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Analysis of starch grain formation using live imaging technique
Ryo Matsushimarmatsu@okayama-u.ac.jp
Institute of Plant Science and Resources, Okayama University

Summary Abstract
Conventionally, the iodine staining has been used to observe of starch grains. This iodide staining
method can observe the shape of starch grains, but cannot visualize amyloplast, which is an plantorganelle that synthesizes starch grains. In this study, transgenic plants expressing green fluorescentproteins accumulating in amyloplast were produced in barley and rice (AmyloTarget-GFP plants).
The AmyloTarget-GFP plants produced in this study can directly visualize amyloplasts and at thesame time visualize starch grains indirectly. It will be an important research tool for analyzing
mutants related to starch grains in the future.
Key Words：Starch grains, Rice, Barley, Green fluorescent protein, Imaging

Introduction
Starch grains are particles formed by starch synthesized in plant cells. The starch grains aresynthesized inside the plant organelle called amyloplast which is one type of plastid. Since
amyloplast is almost occupied by starch grains, it is believed that shape of starch grains should be
the shape of amyloplast, but there are rare cases in which amyloplast is directly visualized. In orderto clarify the shape of amyloplasts synthesizing starch grains, this study used fluorescent proteins tovisualize amyloplast in barley and rice and observe them to obtain new findings.

Results
The morphological diversity of starch grains has been reported over 100 years ago. The diversity
is remarkable especially in the endosperm of Poaceae families1). Morphologically, starch grains areclassified as either compound or simple starch grains2). Rice starch grains in endosperm are formed
as assemblies of small starch particles3). This is compound starch grains. On the other hand, barleydevelops simple starch grains that is composed of a single starch particle. Both rice and barley areimportant crops, but the morphology of starch grains is in contrast. Therefore, comparative imaging
of starch grains development of the two important crops will lead to clarify the mechanism thatdefines the shape of the starch grains. So, I used rice and barley to construct transgenic plants thatvisualize starch grains by green fluorescent proteins (GFP).
First, transit peptide (AmyloTarget) that effectively transport proteins to amyloplasts wereidentified. Then, the nucleotide sequence encoding AmyloTarget was fused to GFP gene. The
AmyloTarget-GFP gene was inserted downstream of the promoter and transformed into rice andbarley. The transformed plants will be referred to as AmyloTarget-GFP plants. The rice AmyloTargetGFP plants were grown at 28-30°C and the barley AmyloTarget-GFP plants were grown for 1315°C. Significant GFP fluorescence signals were observed in rice and barley with AmyloTargetGFP gene introduced. In the case of rice, GFP fluorescence was observed in the state of brown rice.
The presence or absence of AmyloTarget-GFP gene was evaluated based on the presence or absence

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for the Promotion of Scienceof a fluorescent signal, and a homozygote was isolated from the segregation ratio. The developing
seeds were focused because it is the synthesis stage of starch grains. Thin sections were preparedfrom developing seeds and observed by confocal laser scanning microscope. In the case of barley,
it was found that a plurality of simple starch grains was present in a single amyloplast at the early
developing stages. In the later stage, it was observed that large starch grains and small starch grains
were present in the same amyloplast. This result implies that amyloplast containing several starchgrains exists in the endosperm of barley. In the case of rice, it was found that amyloplast having
several compound starch grains exists. This result also means that amyloplast containing several
starch grains is developed in rice. Transmission electron microscopic observation of developing
seeds of non-transgenic rice and barley also gave images showing similar characteristics. That means
the results is not the effect of transformation.
In the AmyloTarget-GFP plants, GFP is transported inside the amyloplast, but it does not penetrateinside the starch grains. Therefore, the part of the starch grains inside the amyloplast are blackspots. This result implies that starch grain can be visualized indirectly in AmyloTarget-GFP plants.
In the AmyloTarget-GFP plants, GFP fluorescence was also observed in chloroplasts of leaves inboth rice and barley, and the chloroplasts could be visualized. This means that the AmyloTargetGFP plants produced in this study can be useful research tools not only for amyloplasts but also formorphological analysis of chloroplasts.

Discussion & Conclusion
In this study, AmyloTarget-GFP plants were successfully constructed in rice and barley. In
AmyloTarget-GFP plants, amyloplast can be directly visualized, and starch grains can be indirectly
visualized in living endosperm cells. I believe that AmyloTarget-GFP plant is important research tool
to clarify the mechanism to differentiate between compound and simple starch grains. Sequential
observation will be needed to clarify when and how the morphological differentiation occur during
seed development. In addition, I have already isolated several rice and barley mutants defective instarch grain morphologies3). Several mutants had genetic lesions in starch biosynthetic enzymes.
Introduction of AmyloTarget-GFP gene into these mutants, amyloplasts and starch grains can bevisualized in mutant backgrounds. Therefore, the observation system constructed in this study should
also be important when analyzing starch-related mutants.

References
1) Tateoka T (1962)
Starch grains of endosperm in grass systematics.
Bot Mag Tokyo 75: 377-383
2) Matsushima R, Yamashita J, Kariyama S, Enomoto T, Sakamoto W (2013)
A phylogenetic re-evaluation of morphological variations of starch grains among Poaceae
species.
J Appl Glycosci 60: 37-44
3) Matsushima R, Maekawa M, Fujita N, Sakamoto W (2010)
A rapid, direct observation method to isolate mutants with defects in starch grain morphology inrice.
Plant Cell Physiol 51: 728-741

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一般の皆様へ澱粉粒とは、植物細胞内で合成された澱粉が形成する粒子のことである。澱粉粒は、アミロ

プラストという植物オルガネラの内部で合成される。澱粉粒の形状は植物種によって多様性を示し、その形状は澱粉の利用用途と精製効率を規定する重要形質と考えられている。しかし、
澱粉粒の形状が決定される仕組みは未解明である。本研究では、蛍光タンパク質を用いて、生きた細胞内でアミロプラスト並びに澱粉粒を可視化する観察系を構築した。今後、澱粉粒やアミロプラストを形態学的に解析する上で、極めて重要な研究ツールになり得るだろう。

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The role of RNA binding protein PSF in prostate cancer progression
Ken-ichi Takayama
takayama@tmig.or.jp
Department of Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology

Summary Abstract
New therapeutic approaches are necessary for treating hormone-refractory prostate cancer.
Activation of androgen receptor (AR) and its variants expression along with the downstream signals
are mostly important for disease progression. Our high-speed sequence analyses identified global
PSF-binding transcripts. Interestingly, mRNAs of spliceosome-related genes are putative primarytargets of PSF. Their gene expressions are upregulated by PSF in hormone-refractory prostatecancer. Moreover, PSF coordinated these spliceosome proteins to form a complex to promote AR
splicing and expression. Thus, targeting PSF and its related pathways implicates the new therapeutic
possibility for hormone-refractory prostate cancer.
Key Words：Androgen receptor, prostate cancer, RNA-binding protein, PSF

Introduction
Androgen receptor (AR) regulates many genes central to the identity and behavior of prostatecancer cells (1). AR functions in a ligand-dependent manner in many prostate cancers, and androgendeprivation therapy is effective in inhibiting tumor growth. However, most of the patients acquire
resistance to this therapy, and eventually suffer from castration-resistant prostate cancer (CRPC) due
to abberant expression and enhanced activity of AR and its variant, AR-V7 (2-5). PSF has a uniquestructure possessing both DNA- and RNA-binding domains, implicating in transcription and nuclear
RNA processing (6, 7). However, the transcriptional and post-transcriptional specific targets and theclinical significance of these proteins in prostate cancer progression still remain to be elucidated.

Results
To explore other functions of PSF in prostate cancer, we next analyzed RNAs bound with PSF
using deep-sequence analysis. First, we performed RNA immunoprecipitation coupled with deep
sequencing (RIP-seq) in LNCaP cells treated with vehicle or DHT. By mapping sequence tags to
RefSeq, GENCODE and NONCODE, we identified putative PSF target genes (Fold > 2, P-value <10-5). We further performed RIP-seq in CRPC model cells (LTAD and 22Rv1). In LTAD and 22Rv1
cells, we found more binding genes, which significantly overlapped with those of LNCaP cells. In
addition, we observed that DHT treatment changed the binding transcripts of PSF. We found that
androgen-regulated genes such as CAMKK2 are enriched among DHT-specific target genes of PSF.
In contrast to the PSF targets identified by ChIP-seq, pathway analysis showed that spliceosome
genes are assumed to be the primary targets of PSF at RNA level in LNCaP cells, suggesting thisgene cluster is specifically regulated by PSF as an RNA-binding protein. Prostate cancer-associatedsignals were also significantly included in these pathways.
To understand how PSF effects on the binding transcripts, we performed directional RNA-seq
to investigate the gene regulation by PSF. These results revealed that PSF mainly increases the

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expression of its binding transcripts. In addition, knockdown of PSF resulted in a marked decreasein DHT-induced expression of AR target genes. Taken together, non-coding signals associated withprostate cancer, spliceosome genes, and AR-regulated genes by binding to these gene transcripts toincrease stability.
We next investigated the PSF function in CRPC model cells. Importantly, the pathway analysis
of PSF-binding genes in 22Rv1 cells indicated the importance of PSF in induction of spliceosome
genes, as observed in LNCaP cells. In addition, the interaction of PSF with prostate cancer-associatedsignals was also enhanced. For example, the binding of PSF to the introns of SchLAP1 and AR)
was enhanced in LTAD and 22Rv1 cells compared with LNCaP cells, suggesting the role of PSF in
expressing these important signals in CRPC.
We then investigated the role of PSF in AR splicing and post-transcriptional regulation. First,
we confirmed the binding of PSF with AR and AR-V7, an AR-splicing variant associated withCRPC development, by RIP-assays. In addition, western blot analyses showed that AR-V7
protein expression was detected in VCaP and 22Rv1 cells. Interestingly, PSF expression was also
upregulated in these cell lines compared with LNCaP and LTAD, in which AR-V7 protein could not
be detected. To determine whether PSF induces AR-V7 protein production or not, we performed
qRT-PCR and western blot analysis by using siRNAs targeting PSF and its associated factor, NONO.
Surprisingly, PSF knockdown in 22Rv1 cells inhibited full-length AR and AR-V7 mRNA and protein
dramatically. Analysis of mRNA stability of AR and AR-V7 by inhibiting transcription indicated that
PSF and NONO are involved in the post-transcriptional regulation of these mRNAs.
We further investigated whether upregulation of spliceosome genes could enhance splicing activity
cooperating with PSF and NONO in CRPC. Our immunnoprecipitation and western blotting analysis
indicated that PSF interacts with these spliceosome components, and was responsible for the complex
formation of spliceosome. In addition, immunofuorescnece analysis showed that this interaction
occurred in the nucleus. RIP-assay also indicated that these factors binding to AR transcripts and act
as an integrator for PSF-mediated splicing of AR transcripts. Taken together, these results revealed
that PSF also orchestrated its target splicing factors at protein levels to form a complex for splicing
and protein expression in CRPC.

Discussion & Conclusion
We indicated the importance of PSF in the regulation of splicing machinery in the progressionof aggressive prostate cancer. Our discovery revealed a novel mechanism in which a broad rangeof splicing components were aberrantly regulated for cancer progression. The wide-ranging
upregulation of the splicing pathway in metastatic prostate cancer could affect the splicing
complexes in cancer. RNA splicing is mediated by an assembly, rearrangement and disengagementof a set of small nuclear ribonucleoprotein (snRNP) complexes (U1, U2 and either U4/5/6 or U11/
U12) or other proteins onto the pre-mRNAs (8, 9). The broad coverage of the wide spectrum of thesecomponents in our study suggests the involvement of PSF in enhancing this machinery. Interestingly,
frequent mutations of splicing pathway have been reported in several malignancies by whole-exome
sequencing (10-12). These clinical analyses provide an intriguing insight into the mechanism ofcancer progression by splicing machinery since RNA splicing system has essential cellular roles
for the diversity of protein species using a limited number of genes. Loss of splicing activity by
mutations would induce severe developmental abnormality and tumorigenesis (13). Conversely, suchmutation has been rarely identified in prostate cancer (14). Enhanced splicing machinery could be

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indispensable for the cancer progression in some types of malignancies (15). Moreover, the present
study would illustrate the specificity of PSF function and splicing machinery in prostate cancer.
In summary, our global analysis of PSF functions revealed its target signals based on the RNAbinding ability in addition to cell cycle regulator control by epigenetic silencing of the transcription
with histone modification. Importantly, we proposed a new mechanistic link between RNA-binding
proteins and AR as well as prostate cancer-associated signals, indicating the clinical and biologicalimpact of PSF in the development of abnormal splicing machinery in hormone-refractory prostatecancer. Considering the potential function of AR and AR-variants in the development of metastatic
CRPC, PSF and its associated factors could have the potential for therapy targets.

References
1. Yuan X, Cai C, Chen S, Chen S, Yu Z, et al (2014). Androgen receptor functions in castrationresistant prostate cancer and mechanisms of resistance to new agents targeting the androgen axis.
Oncogene. 33(22):2815-25.
2. Chen CD, Welsbie DS, Tran C, Baek SH, Chen R, et al (2004). Molecular determinants ofresistance to antiandrogen therapy. Nat Med. 10(1):33-9.
3. Wang Q, Li W, Zhang Y, Yuan X, Xu K, et al (2009). Androgen receptor regulates a distinct
transcription program in androgen-independent prostate cancer. Cell. 138(2):245-56.
4. Takayama K, Horie-Inoue K, Katayama S, Suzuki T, Tsutsumi S, et al (2013). Androgenresponsive long noncoding RNA CTBP1-AS promotes prostate cancer. EMBO J. 32:1665–1680.
5.
6. Cosker KE, Fenstermacher SJ, Pazyra-Murphy MF, Elliot HL, Segal RA (2016). The RNA binding
protein SFPQ orchestrates an RNA regulon to promote axon viability. Nat Neurosci. 19:690-696.
7. Lee M, Sadowska A, Bekere I, Ho D, Gully BS, et al (2015). The structure of human SFPQ
reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation.
Nucleic Acids Res. 43(7):3826-40.
8. Wahl MC, Will CL, Luhrmann R (2009). The spliceosome: design principles of a dynamic RNP
machine. Cell 136, 701–718.
9. Lee Y, Rio DC (2015). Mechanisms and regulation of alternative pre-mRNA splicing. Annu Rev
Biochem 84:291–323.
10. Quesada V, Conde L, Villamor N, Ordonez GR, Jares P, et al (2012). Exome sequencing identifies
recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia. Nat
Genet 44:47–52.
11. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, et al (2011). Frequent pathway mutationsof splicing machinery in myelodysplasia. Nature 478:64–9.
12. Supek F, Minana B, Valcarcel J, Gabaldon T, Lehner B (2014). Synonymous mutations frequently
act as driver mutations in human cancers. Cell 156:1324–35.
13. Daguenet E, Dujardin G, Valcarcel J (2015). The pathogenicity of splicing defects: mechanistic
insights into pre-mRNA processing inform novel therapeutic approaches. EMBO Rep 16:1640–
55.
14. Robinson D, Van Allen EM, Wu YM, Schultz N, Lonigro RJ, et al (2015). Integrative clinicalgenomics of advanced prostate cancer. Cell. 2015;161(5):1215-28.
15. Dvinge H, Kim E, Abdel-Wahab O, Bradley RK (2016). RNA splicing factors as oncoproteins
and tumour suppressors. Nat Rev Cancer 16:413–30.

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一般の皆様へ
前立腺がんは世界的にみて男性に発生するがんとして最も多いがんです。男性ホルモンである

アンドロゲンによりがんの成長、病気の進行を促進することが知られておりアンドロゲンを抑制する薬剤が治療に用いられるものの治療抵抗性に陥ることが問題となっています。治療抵抗性の出現にはアンドロゲンの受容体の発現上昇が知られていますが、その機序がよくわかっていません。遺伝子の発現には DNA からの転写により生成される RNA から蛋白合成を通じて行われます。本研究は RNA 結合タンパク質である PSF がアンドロゲン受容体および RNA の成熟に関与する遺伝子群の RNA に作用することでその安定性、発現を促進していることを明らかとしま

した。RNA 成熟に関与する遺伝子群の発現上昇が引き起こされアンドロゲン受容体の活性化、がんの悪性化につながる新たな作用機序を見出しています。

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Selective information routing in the hippocampal network
Kenji Mizuseki
mizuseki.kenji@med.osaka-cu.ac.jp
Osaka City University Graduate School of Medicine

Summary Abstract
The subiculum is a major output hub of the hippocampal formation. However, information outflow
from the subiculum to various downstream regions remains poorly characterized. This research
involved the use of a combination of large-scale recordings and optogenetics to establish a method
for the antidromic identification of projection neurons in freely behaving rats. This method allowed
us to investigate information outflow from the subiculum to each downstream region.
Key Words：hippocampus, subiculum, optogenetics, routing, memory

Introduction
The hippocampal formation, which comprises the hippocampus proper, dentate gyrus, andsubiculum, is crucial for learning and memory. Historically, although most studies have focusedon the hippocampus proper, recent evidence has highlighted the substantial contribution of thesubiculum to interregional connectivity and behavioral performance. In fact, from an anatomical
point of view, the subiculum is a major output hub of the hippocampal formation. However,information outflow from the subiculum to various downstream regions remains poorly
characterized. Thus we investigated the target-specific information outflow from the subiculum usinglarge-scale recordings and antidromic identification of projection neurons in freely behaving rats.

Results
Although the subiculum is a major output structure in the hippocampal formation (Naber andWitter, 1998), the type of information transferred from the subiculum to each downstream region
remains elusive. The main reason for this gap is that by using currently available electrophysiologicalrecording methods we cannot classify the recorded neurons based on the brain region(s) towhich each neuron sends axon(s) (Ciocchi et al., 2015; Saiki et al., 2018). We overcame thislimitation by combining large-scale recordings and optogenetics, thus establishing a method tosimultaneously record from over 100 neurons and identify the target brain areas of approximately
10% of the recorded neurons (Fig 1). For this purpose, we used adeno-associated viruses to express

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channelrhodopsin (ChR2) in the subicular neurons in freely behaving rats. Simultaneously, we
implanted optic fibers in the target brain areas of the subiculum (the nucleus accumbens, retrosplenialcortex, intralaminar and anteroventral thalamic nuclei, and medial mammillary body) and stimulated
the axons of ChR2-expressing subicular neurons using blue light. The spikes induced by photostimulation at the axons retrogradely propagated into the cell bodies in the subiculum. By recordingfrom the subiculum using high-density silicon probes (up to 256 channels), we detected retrogradely
propagated spikes and thereby identified the neurons directly projecting into that target area. The
collision test was performed to ensure that the spikes recorded at the cell bodies during photostimulation were indeed back-propagated spikes generated at the axons and not indirectly generated
via synaptic interactions. The collision test is based on the following rationale: if a spike generated
at the axon of a given neuron by blue-light stimulation occurs within several milliseconds before orafter a spike spontaneously generated at the cell body of that neuron, the spike generated at the axon
propagates to the cell body and that generated at the cell body propagates to the axon, thus resulting
in a collision; therefore back-propagated spikes generated at the axon are not recorded at the cellbody. When blue light-induced spikes are due to synaptic interactions, collisions will not occur.
Therefore, blue light-induced spikes can be detected at the cell body even if spikes are generated at
the soma at very similar time points as light-induced spikes. To ensure the efficacy of the collision
test, we first putatively identified the neurons projecting into the target area. We then selected thechannel at which largest spike waveform of those neurons were recorded and used ON-LINE spike
detection algorithm to identify the spikes recorded from these channels. Using these spikes, we
triggered blue-light stimulation at the target area, thus speeding up the collision test. So far, we havebeen able to identify the target areas of approximately 10% of the recorded neurons. To increase
this proportion, we are currently working to increase the efficacy of this method by optimizing
the following: ChR2 expressions in the subiculum, blue light power, optical stimulation sites, andspacing between silicon probe recording sites, thus increasing the number of recorded neurons.
Moreover, we established recording systems that enable recording from free-moving rats during
various behavioral tasks, thus revealing the type of information represented by individual subicular
neurons. We established an alternation task with more than several seconds of delay, which is ahippocampus-dependent memory task. We also established behavioral tasks using zig-zag and radial
mazes, the open field test, and a linear track to reveal the information represented by individual
subicular neurons.
Fig 1. Channelrhodopsin (ChR2) was expressed in the subiculum neurons using adeno-associatedvirus, (B) then we recorded from the subiculum using a 256 channels silicon probe, (C) and stimulate
a projection target brain region using blue light. By detecting spikes generated at the axon by lightstimulation and back-propagated to the soma with short latency (<15 ms) and jitter (<1 ms), weclassified neurons by their projection target regions.

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Discussion & Conclusion
We have established a method for antidromic identification of projection neurons in freely behaving
rats by combining large-scale recordings and optogenetics. The use of high-density silicon probes(up to 256 channels) for large-scale recordings enabled us to simultaneously record from over 100
subicular neurons. In addition, we expressed ChR2 in subicular neurons and stimulated target brain
areas using blue light. The detection of blue light-induced back-propagated spikes at the cell bodies
in the subiculum allowed us to identify the target areas of approximately 10% of the recorded
subicular neurons. We also established behavioral tasks that allow us to characterize the information
represented by subicular neurons. Using this developed method to identify the target areas of therecorded neurons and behavioral tasks, we are currently investigating target-specific information
outflow from the subiculum.

References
Naber, P.A., and Witter, M.P. (1998). Subicular efferents are organized mostly as parallel
projections: a double-labeling, retrograde-tracing study in the rat. The Journal of comparative
neurology 393, 284-297.
Ciocchi, S., Passecker, J., Malagon-Vina, H., Mikus, N., and Klausberger, T. (2015). Braincomputation. Selective information routing by ventral hippocampal CA1 projection neurons. Science
348, 560-563.
Saiki, A., Sakai, Y., Fukabori, R., Soma, S., Yoshida, J., Kawabata, M., Yawo, H., Kobayashi, K.,
Kimura, M., and Isomura, Y. (2018). In Vivo Spiking Dynamics of Intra- and Extratelencephalic
Projection Neurons in Rat Motor Cortex. Cerebral cortex (New York, NY : 1991) 28, 1024-1038.

一般の皆様へ
海馬と呼ばれる脳の領域は、個人的な経験を思い出す能力であるエピソード記憶に重要な役割を果たします。本研究では、海馬からどの脳領域へどのような情報が伝達されるのかを明らかにするための基盤的な技術を開発しました。今後は開発した技術を用いて、海馬と他の脳領域がどのように情報をやり取りして記憶をサポートしているのかを明らかにしたいと考えています。アルツハイマー病などの認知症では、ごく初期から海馬に変性がみられます。海馬における情報処理のメカニズムを少しでも明らかにし て 、将 来 的 に 認 知 症 の 新 規 治 療 法 を 確 立 す る た め の 基 盤 を 作 り た い と 考 え て い ま す 。

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Chemical approach to developing anticancer agents ofmarine alkaloid origin
Takehiko Yoshimitsu
yoshimit@okayama-u.ac.jp
Division of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry, andPharmaceutical Sciences, Okayama University

Summary Abstract
Agelastatin A (AA) is a natural antitumor alkaloid isolated from the marine sponge Agelas
dendromorpha. We have established a new method for the chemical synthesis of AA and its analogs.
Biological assessment of AA analogs has led to the discovery of novel compounds that exert
antiproliferative activity against cancer cell lines. Structure-activity relationship (SAR) studies on
AA analogs have provided important insights into the molecular design for the further developmentof AA-based medicinal leads.
Key Words：natural product, chemical synthesis, anticancer, agelastatin A

Introduction
Agelastatin A (AA), a tetracyclic alkaloid isolated from the marine sponge Agelas dendromorpha,
has shown potent antiproliferative activity against various cancer cell lines by inhibiting the Wnt/
β-catenin pathway responsible for cellular processes. Moreover, AA exhibits anti-osteopontin (OPN)
activity and has been touted as an anti-OPN agent.1 As various cancers, particularly aggressive andmetastatic cancers, significantly express OPN, there has been avid interest in the development of AA
derivatives as a new therapeutic agent for such cancers.

Results
1) Development of new divergent synthetic routes to AA and its analogs:
Our previous structure-activity relationship (SAR) studies on AA have revealed that themodification of the N1-substituent on the D-ring system yields new analogs with potent biologicalactivity.2,3 Therefore, we endeavored to establish a new synthetic protocol that streamlines accessto D-ring analogs bearing various N1-substituents. Our study on the chemical synthesis commenced
with known amino alcohol derivative 2 (>99% ee) that serves as the C-ring motif of the AA molecule(Scheme 1). Starting material 2 was subjected to five-step synthetic manipulations to produce
tricyclic ketone 3 that corresponds to the ABC ring system of AA. Then, a sequential transformation
involving enolization and bromination was applied to ketone 3 to deliver TBS enol ether 4 that isready for the installation of the pivotal nitrogen functionality of the D-ring system. Whereas the key
nitrogenation reaction was extremely challenging, we eventually devised a new means that features
radical azidation to deliver azide 5, which bears the necessary functionalities to fully access the
D-ring system. Hydrogenation of compound 5 afforded amine (structure not indicated), which was
further subjected to carbamoylation and deprotection to afford 7. The final bromination of 7 smoothly
furnished AA (1). Furthermore, the synthetic protocol wherein various carbamoylation reagents were
employed eventually culminated in the facile syntheses of novel AA analogs (8, 9, and 10) with suchN1-substituents as n-propyl, n-hexyl, and i-propyl groups.

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The Novartis Foundation(Japan)
for the Promotion of Science2) Identification of novel potent AA analogs by SAR studies:
New AA analogs 8, 9, and 10 synthesized by the above-mentioned protocol were tested for their invitro cytotoxicity toward various cancer cell lines, including DU145 (prostate cancer cells). The SAR
study suggested that the structural modification of the N1-substituent with linear alkyl chains was
somewhat tolerable and AA analogs 8 and 9 obtained by the structural modification retained their
biological activity. In contrast, the introduction of a bulky alkyl substituent at the N1-position led tosignificant loss of activity. These SAR studies provide important insights into the molecular designfor the further development of AA-based lead compounds.
3) Synthesis of chemical probes to identify molecular target underlying biological activity:
Having established a new protocol to access AA analogs with varying N1-substituents, we madefurther efforts to synthesize chemical probes that would enable identification of the molecular target
responsible for the biological activity of AA. Common intermediate 5 was subjected to reduction,
carbamoylation with 11, deprotection, and bromination to afford biotinylated compound 12. In
addition, compound 5 was coupled with alkyne fragment 13 to provide propargyl derivative 14,
which was reacted with an alkyne fragment (structure not shown) to furnish compound 15. Third
chemical probe 16 was synthesized by connecting propargyl derivative 14 to an azide fragment(structure not shown). However, the biological assessment of chemical probes 12, 15, and 16
revealed that none of these derivatives exhibited any promising antiproliferative activity. This result
provides an additional insight into the SAR of AA analogs, namely, the introduction of linear polar
substituents at the N1-position may be unfavorable to the biological function of AA.

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Discussion & Conclusion
The present study was undertaken to develop new anticancer leads derived from the marine
natural alkaloid agelastatin A (AA). We established divergent synthetic routes to AA and its analogs,
wherein a radical azidation reaction enabled facile construction of the nitrogen-containing moleculararchitecture of AA. Furthermore, AA analogs that exhibit antiproliferative activity against cancer
cell lines were identified by the SAR study. Three chemical probes that possess a biotin tag on N1nitrogen were also synthesized. However, biological assessment of the probes revealed their lackof antiproliferative activities, suggesting that such structural modifications are deleterious to the
biological activity of AA.

References
1) C. K. Mason, S. McFarlane, P. G. Johnston, P. Crowe, P. J. Erwin, M. M. Domostoj, F. C.
Campbell, S. Manaviazar, K. J. Hale, M. El-Tanani, Mol. Cancer Ther., 2008, 7, 548-558.
2) Z. Li, D. Shigeoka, T. R. Caulfield, T. Kawachi, Y. Qiu, T. Kamon, M. Arai, H. W. Tun, and T.
Yoshimitsu, Med. Chem. Commun., 2013, 4, 1093-1098.
3) (a) E. P. Stout, M. Y. Choi, J. E. Castro, T. F. Molinski, J. Med. Chem. 2014, 57, 5085-5093.;(b) M. Jouanneau, B. McClary, J. C. P. Reyes, R. Chen, Y. Chen, W. Plunkett, X. Cheng, A. Z.
Milinichik, E. F. Albone, J. O. Liu, D. Romo, Bioorg. Med. Chem. Lett., 2016, 26, 2092-2097.; (c)A. H. Antropow, K. Xu, R. J. Buchsbaum, M. Movassaghi, J. Org. Chem., 2017. 82. 7720-7731.

一般の皆様へ
海綿から見出されたアゲラスタチンＡは，抗がん剤の開発において大変魅力的な物質として興味を集めています。これまで我々は，アゲラスタチンＡの化学構造を人工的に改変し、抗がん剤のリード化合物を創り出す研究を進めてきました。本研究では、こうした創薬研究の基盤を成す化学

合成法の開発、化学合成によって得た化合物の中から有望な物質を見出すこと、そして、アゲラス

タチンＡの作用機序の解明に役立つ分子プローブの合成に取り組みました。その結果，各種誘導体の新規合成法の開発並びにリード化合物の設計指針となる知見を得ることに成功しました。

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Catecholamine-induced senescence of endothelial cells andbone marrow cells promotes cardiac dysfunction in mice
Tohru Minamino
tminamino@med.niigata-u.ac.jp
Department of Cardiovascular Biology and Medicine
Niigata University Graduate School of Medical and Dental Sciences

Summary Abstract
In the present study, we found that catecholamine infusion induced senescence of endothelial
cells and bone marrow cells, and promoted cardiac dysfunction in mice. Continuous infusion of
isoproterenol induced cardiac inflammation and cardiac dysfunction. Expression of p53, a master
regulator of cellular senescence, was increased in the cardiac tissue and bone marrow cells of thesemice. Suppression of cellular senescence by genetic deletion of p53 in endothelial cells or bonemarrow cells led to improvement of isoproterenol-induced cardiac dysfunction. Our results indicatethat catecholamine-induced senescence of endothelial cells and bone marrow cells plays a pivotal
role in the progression of heart failure. Suppression of catecholamine-p53 signaling is crucial for
inhibition of remodeling in the failing heart.
Key Words：Chronic inflammation, p53, cellular senescence

Introduction
Previous studies have suggested a central role of cellular senescence in the progression ofpathologic changes in the failing heart. It is well known that the sympathetic nervous system is
activated in patients with heart failure, and this change is associated with poor clinical outcomes.
Sympathetic activation increases the levels of various catecholamines, such as epinephrine andnorepinephrine, but the contribution of these catecholamines to cellular senescence associated withheart failure remains to be determined.

Results
To investigate whether catecholamine infusion induced cellular senescence, we administeredisoproterenol to mice via an infusion pump, leading to the development of cardiac hypertrophy
associated with cardiac systolic dysfunction. Under these conditions, there was an increase in p53
and the transcript of its target molecule, p21 (Cdkn1a), in cardiac tissues. We previously detectedp53-induced senescence of endothelial cells and bone marrow cells in a murine model of LV
pressure overload1. These findings led us to analyze p53 expression in bone marrow cells under
hyperadrenergic conditions in the present study. After continuous infusion of isoproterenol, there
was a marked increase of p53 and its transcript p21 (Cdkn1a) in bone marrow cells. These results
suggested that the hyper-adrenergic state associated with heart failure promotes p53-induced cellularsenescence in cardiac tissue and in the bone marrow. Chronic sterile inflammation develops inpatients with heart failure and promotes pathologic changes2-4. In agreement with this report, wefound that isoproterenol infusion increased the infiltration of mononuclear cells and Mac3 positive
cells into the left ventricle, and was associated with increased LV expression of transcripts for

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inflammatory markers. Isoproterenol infusion also increased transcripts for integrin alpha-L (Itgal) inbone marrow cells. These results suggested that activation of adrenergic signaling per se contributes
to elevation of integrin expression by bone marrow cells and to increased cardiac tissue expressionof intercellular adhesion molecule-1 (ICAM-1), which may promote sterile inflammation andremodeling in the failing heart.
Our data indicated that activation of adrenergic signaling resulted in up-regulation of p53 inboth cardiac tissue and bone marrow. In addition, transcripts of Icam1 and Itgal were respectively
increased in cardiac tissue and bone marrow by a hyperadrenergic state. We previously reported
that p53 positively regulates the expression of these adhesion molecules at the transcriptional level,
inducing cardiac inflammation and remodeling in a murine model of LV pressure overload1. To
obtain further direct evidence that p53 expression in endothelial cells and/or bone-marrow cells
contributes to progression of catecholamine-induced heart failure, we performed isoproterenol
infusion in endothelial cell (EC) - or bone marrow cell (BM)-p53 knockout (KO) mice. EC-p53 KO
mice were generated by crossing Tie-2 Cre mice with Trp53flox/flox mice, and Tie2-Cre+ Trp53flox/flox (EC-p53
KO) mice and their littermate controls (Trp53flox/flox mice) were analyzed. Infusion of isoproterenol
led to cardiac dysfunction and dilatation in control mice, while these changes were ameliorated in
EC-p53 KO mice. We generated a BM-p53 KO model by transplanting bone marrow cells fromsystemic p53 KO mice into wild-type mice after irradiation, while control mice were generated bytransplanting bone marrow cells from wild-type mice into irradiated wild-type mice. Isoproterenol
infusion caused systolic dysfunction along with cardiac dilatation in the control groups, while suchchanges were ameliorated in BM-p53 KO mice. These results suggested that up-regulation of p53
signaling in endothelial cells and bone marrow cells by a hyper-adrenergic state induces cardiac
dysfunction.

Discussion & Conclusion
In this study, we showed that catecholamine-induced senescence of endothelial cells and bonemarrow cells had a pivotal role in the progression of pathological changes associated with heart
failure. In a murine model of isoproterenol-induced heart failure, infusion of isoproterenol led to anincrease of p53 expression in both cardiac tissue and bone marrow, while genetic suppression of p53
in these organs ameliorated cardiac dysfunction.
While β-blockers were initially contraindicated for patients with heart failure, these drugs havesince become first-line therapy for this critical condition. Carvedilol is a non-selective β-blocker
with lower affinity for the β1-adrenergic receptor and higher affinity for the β2-adrenergic receptor.
Among various β-blockers (including selective and non-selective drugs), carvedilol is recognized as
first-line therapy for heart failure5, 6. Our findings suggest that β-blockers like carvedilol may havea beneficial effect via suppression of β2-adrenergic receptor signaling and prevention of cellularsenescence in the hyper-adrenergic state associated with heart failure.
In coclusion, inhibition of catecholamine-induced cellular senescence is important for suppressionof pathologic changes in the failing heart.

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References
1. Yoshida Y, Shimizu I, Katsuumi G, Jiao S, Suda M, Hayashi Y and Minamino T. p53-Induced
inflammation exacerbates cardiac dysfunction during pressure overload. Journal of molecular andcellular cardiology. 2015;85:183-98.
2. Mann DL. Inflammatory mediators and the failing heart: past, present, and the foreseeable future.
Circ Res. 2002;91:988-98.
3. Heymans S, Hirsch E, Anker SD, Aukrust P, Balligand JL, Cohen-Tervaert JW, Drexler H,
Filippatos G, Felix SB, Gullestad L, Hilfiker-Kleiner D, Janssens S, Latini R, Neubauer G, Paulus
WJ, Pieske B, Ponikowski P, Schroen B, Schultheiss HP, Tschope C, Van Bilsen M, Zannad F,
McMurray J and Shah AM. Inflammation as a therapeutic target in heart failure? A scientificstatement from the Translational Research Committee of the Heart Failure Association of theEuropean Society of Cardiology. Eur J Heart Fail. 2009;11:119-129.
4. Majmudar MD, Keliher EJ, Heidt T, Leuschner F, Truelove J, Sena BF, Gorbatov R, Iwamoto Y,
Dutta P, Wojtkiewicz G, Courties G, Sebas M, Borodovsky A, Fitzgerald K, Nolte MW, Dickneite
G, Chen JW, Anderson DG, Swirski FK, Weissleder R and Nahrendorf M. Monocyte-Directed
RNAi Targeting CCR2 Improves Infarct Healing in Atherosclerosis-Prone Mice. Circulation.
2013;127:2038-+.
5. Packer M, Coats AJS, Fowler MB, Katus HA, Krum H, Mohacsi P, Rouleau JL, Tendera M,
Castaigne A, Roecker EB, Schultz MK, DeMets DL and Randomized CP. Effect of carvedilol onsurvival in severe chronic heart failure. New Engl J Med. 2001;344:1651-1658.
6. Poole-Wilson PA, Swedberg K, Cleland JGF, Di Lenarda A, Hanrath P, Komajda M, Lubsen J,
Lutiger E, Metra M, Remme WJ, Torp-Pedersen C, Scherhag A, Skene A and Investigators C.
Comparison of carvedilol and metoprolol on clinical outcomes in patients with chronic heart
failure in the Carvedilol Or Metoprolol, European Trial (COMET): randomised controlled trial.
Lancet. 2003;362:7-13.

一般の皆様へ
本研究で私たちは、心不全に伴って心臓で炎症が惹起される機序について明らかにしました。

このような炎症の亢進が、心不全悪化につながっていることから、今後新たな治療の標的となりうると考えられます。

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Modulation of three-dimensional chromatin structure by Piwi-piRNAs
Yuka Iwasaki
Iwasaki@keio.jp
Keio University School of Medicine

Summary Abstract
The piRNA (PIWI-interacting RNA) pathway in Drosophila ovarian somatic cells represses
transposable elements (TEs) transcriptionally to maintain the genome integrity. Here, we described
the link between piRNA mediated silencing and regulation of chromatin structure. We performed
Hi-C experiment, capable of detecting 3D structure of the genome, together with series of ChIP-seq
and RNA-seq analyses. This revealed that piRNAs silence their target TEs by dynamic regulation ofchromatin 3D structure coupled with modulation of histone marks. Additionally, we obtained a list of
candidate proteins, such as insulators, which may play a key role in this regulation.
Key Words：Chromatin structure, Epigenetic regulation, Transcriptional regulation, RNA silencing,
non-coding RNA

Introduction
The piRNA pathway in Drosophila ovarian somatic cells represses TEs transcriptionally to maintainthe genome integrity. The Piwi-piRNA complex establishes heterochromatic H3K9me3 marks on
TEs and their neighboring regions to silence them. Additionally, we have recently shown that thelinker histone H1 is necessary for Piwi-piRNA to repress TEs. We proposed a model in which PiwipiRNA complexes exert TE silencing through H1 association with specific chromatin regions,
resulting in modulation of chromatin accessibility.

Results
The piRNA pathway in Drosophila ovarian somatic cells represses TEs transcriptionally tomaintain the genome integrity [Iwasaki et al., Annu Rev Biochem (2015)]. The Piwi-piRNA complex
establishes heterochromatic H3K9me3 marks on TEs and their neighboring regions to silence them.
Additionally, we have recently shown that the linker histone H1 is necessary for Piwi-piRNA torepress TEs [Iwasaki et al., Mol Cell (2016)]. We proposed a model in which Piwi-piRNA complexes
exert TE silencing through H1 association with specific chromatin regions, resulting in modulationof chromatin accessibility.
In line with this and the fact that some non-coding RNAs are known to effect chromatin boundaries[Cohen et al., WIREs RNA, (2014)], we have analyzed the involvement of Piwi-piRNA on the 3D
chromatin organization of Drosophila OSC (Ovarian Somatic Cells) cell line, using Hi-C (Chromatin
conformation capture sequencing) method [Lieberman-Aiden et al., Science (2009)]. This revealed
that depletion of Piwi induces long-range interactions within a chromosome arm. Also, we analyzed
TADs (Topologically associating domains) of OSCs using Hi-C data, and revealed the decreaseof intra-TAD interaction and increase of inter-TAD interaction upon loss of Piwi. Importantly, thedecrease of intra-TAD interaction and increase of inter-TAD interaction correlated with the densityof Piwi-piRNA target TEs in each TAD. This suggests that Piwi is essential for maintaining the

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The Novartis Foundation(Japan)
for the Promotion of Sciencestructure of chromatin, which possibly is mediated by the regulation of target TEs.
Additionally, we performed ChIP-seq experiment of H3K9me3, H3K27Ac, and H3K4me3 histonemarks upon depletion of Piwi. This revealed that H3K9me3 repressive marks were correlated withdecrease of intra-TAD interaction, where H3K27Ac and H3K4me3 active histone marks were inverse
correlated with decrease of intra-TAD interaction induced by Piwi knock down. Importantly, we
analyzed the transcription level of the genes harbored in each TADs by using RNA-seq analysis. This
also suggested that decrease of intra-TAD interaction upon Piwi knock down was inverse correlatedwith average expression level of the genes located in TADs. These results suggest that Piwi modulate
chromatin level in a manner, which couples histone marks, and also transcription levels.
We further performed the Hi-C experiment using Drosophila ovary, in order to see whether
chromatin modulation examined by using OSC cell line could still be seen in vivo. We could observe
the decrease of intra-TAD interaction and increase of inter-TAD interaction in Piwi mutant flies, but
in much stronger level than in OSCs. This may be due to the fact that in case of using mutant files,
we can observe the effect of Piwi on TADs prior to the primary formation of TADs. We will further
analyze the data to discuss in vivo effect of Piwi on chromatin structure.
Also, in order to screen for the protein,which is involved in this modulationof chromatin structure by Piwi, we
performed ChEP (Chromatin Enrichment
for Proteomics) analysis [Kustatscher etal., Nat Prot (2014)], to obtain a list ofproteins whose chromatin associationlevel is affected by depletion of Piwi.
This analysis revealed that Piwi might
affect on the chromatin binding of
insulator proteins, which are known as
the genetic boundary elements. We havealready raised several antibodies for these candidate insulator proteins. We will further analyze theimpact of Piwi on these factors and discuss how Piwi can modulate 3D chromatin structure.

Discussion & Conclusion
Our combined Hi-C, ChIP-seq, and RNA-seq data analysis using OSC cell line suggests that Piwi
is essential for maintaining the structure of chromatin, which possibly is mediated by the regulationof target TEs. The results also suggest that Piwi modulate chromatin level in a manner, which
couples histone marks, and also transcription levels. Further analysis performed using Drosophila
ovary, suggested the similar decrease of intra-TAD interaction and increase of inter-TAD interactionin Piwi mutant flies, but in much stronger level. This may be due to the fact that in case of using
mutant files, we can observe the effect of Piwi on TADs prior to the primary formation of TADs.
We will further analyze the data to discuss in vivo effect of Piwi on the chromatin structure. Also,
we obtained insulator proteins as candidates from ChEP analysis. This analysis suggested that Piwi
might affect on the chromatin binding of insulator proteins, which are known as the genetic boundary
elements. We have already raised several antibodies for these candidate insulator proteins. We willfurther analyze the impact of Piwi on these factors and discuss how Piwi can modulate 3D chromatin
structure.

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References
1. Iwasaki YW et al., PIWI-Interacting RNA: Its Biogenesis and Functions. Annu Rev Biochem.
2015. 84: 405-433
2. Iwasaki YW et al., Piwi Modulates Chromatin Accessibility by Regulating Multiple Factors
Including Histone H1 to Repress Transposons, Mol Cell. 2016. 63:408–419
3. Cohen AL et al., Noncoding RNAs and the borders of heterochromatin, WIREs RNA 2014. doi:
10.1002/wrna.1249
4. Lieberman-Aiden E et al., Comprehensive mapping of long-range interactions reveals folding
principles of the human genome. Science. 2009. 326:289-93
5. Kustatscher G et al., Chromatin enrichment for proteomics. Nature Protocols 2014. 9: 2090–2099

一般の皆様へ
生物の設計図であるゲノムは、自身のコピー数をどんどん増やしてしまう「利己的な遺伝子」

として知られるトランスポゾンの転移により、傷ついてしまいます。piRNA と呼ばれる小さなRNA が、このトランスポゾンを抑え込む働きをしており、この制御が正常に機能しないと不妊等が引き起こされることが知られています。そこで本研究では、piRNA がどのようにトランスポゾンを抑制するかを解析しました。研究成果として、piRNA によるトランスポゾンの抑制においては、核内でゲノム（クロマチン）がどのように折りたたまれているかが制御されることが重要だということが分かりました。

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Catalytic Synthesis of Spiroketals Using Water as the Oxygen Source
Triggered by Rearrangement of Internal Alkynes
Mutoh, Yuichiro
ymutoh@rs.tus.ac.jp
Tokyo University of Science

Summary Abstract
A ruthenium-catalyzed synthesis of [5,6]-spiroketals that involves a very rare 1,2-carbon migrationof internal alkynes is reported. Mechanistic studies revealed that this reaction proceeds via adisubstituted vinylidene complex as the keyintermediate by 1,2-carbon migration of the eneynones.
Key Words：Spiroketal, Internal alkyne, Vinylidene, Ruthenium, Rearrangement

Introduction
Catalytic processes that involve the vinylidene rearrangement of internal alkynes with a carbon
substituent on their alkyne terminus by 1,2-carbon migration has not yet been reported thoughexperimental1 and theoretical2 investigations into stoichiometric internal alkyne-to-vinylidene
rearrangements have only recently been reported.
Meanwhile, we have developed the first catalytic reaction that involves the vinylidene
rearrangement of internal alkynes by 1,2-carbon migration.3 These results encouraged us to develop
new and useful reactions that involve 1,2-carbon migration. Herein, we report a ruthenium-catalyzed
synthesis of [5,6]-spiroketals, which are core structures of biologically active natural compounds
such as γ-rubromycin and berkelic acid.4

Results
Scheme 1 shows working hypothesis for the catalytic synthesis of [5,6]-spiroketals 2 that involves
vinylidene rearrangement of eneynone 1. A cationic ruthenium complex affects the internal alkyneto-vinylidene rearrangement of 1, and the vinylidene complex 3 is formed. Hydration of dihydropyran
moiety in 3 under an acidic condition generates hemiacetal 4. Subsequent oxycyclization of 4, in
which the hydroxy group nucleophilically attack to the electrophilic α-carbon atom of a vinylidene
ligand, and [5,6]-spiroketals are formed. Finally, the protonation (decomplexation) of 5 furnishes thedesired [5,6]-spiroketals 2 with concomitant regeneration of the ruthenium catalyst.

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Scheme 1. Working hypothesis

Initially, I investigated the reaction of 1 using [CpRuCl(dppe)]/NaBAr F 4 ‧2H 2 O (Ar F =3,5-(CF3)2C6H3) as the catalytic system, which was found to be highly active in the previous studies.3
When a mixture of 1, [CpRuCl(dppe)] (10 mol%), and NaBArF4‧2H2O (12 mol%) was stirred under
heating conditions, the desired
1,2-aryl migration/hydration/cyclization product, [5,6]-spiroketal 2, was formed, albeit in ca. 10 %yield (Scheme 2). The [5,6]-spiroketal structure was unambiguously determined by a single-crystal
X-ray diffraction analysis of 2-NO2. Unfortunately, screening of catalysts, acids, and solvents did not
improve the yield of the product.
Scheme 2. Ru-catalyzed synthesis of [5,6]-spiroketal 2 involving 1,2-carbon migration

To gain insight into the underlying mechanistic aspects of the present reaction, I examined thestoichiometric reaction between 1, [CpRuCl(dppe)], and NaBArF4‧2H2O at 70 °C in PhCl (Scheme
3). After stirring for 2 h, vinylidene complex 3 was isolated in 45% yield. The molecular structureof 3 was unambiguously determined by a single-crystal X-ray diffraction analysis that confirmed theanticipated 1,2-carbon migration to form the corresponding disubstituted vinylidene intermediate.
The isolation of the vinylidene complex implies that the hydration/cyclization of 1 proceeds slower
than the vinylidene rearrangement of 1 under the applied reaction conditions.

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Scheme 3. Stoichiometric internal alkyne-to-disubstituted vinylidene rearrangement of 1 on
[CpRu(dppe)]+ that leads to vinylidene complex 3

Next, hydration of vinylidene complex 3 was examined. When a solution of 3 was stirred at
90°C in the presence of H3PO4 (8.5% aqueous solution, 2 equiv), [5,6]-spiroketal complex 4 was
isolated in 67% yield (Scheme 4). The molecular structure of 4 was unambiguously determinedby a single-crystal X-ray diffraction analysis. The formation of 4 from 3 indicates that thevinylidene rearrangement of internal alkynes is the crucial step in the present catalytic synthesis of[5,6]-spiroketals.
Scheme 4. Hydration/cyclization of 3 that leads to [5,6]-spiroketal complex 4.

The final step of the catalytic cycle was also confirmed (Scheme 5). Thus, reaction of ruthenium
complex 4 with HCl afforded the desired [5,6]-spiroketal 2 in 84% yield as well as [CpRuCl(dppe)]in 95% yield.
Scheme 5. Decomplexation of 4 using HCl that leads to 2 and [CpRuCl(dppe)]

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Discussion & Conclusion
I have demonstrated a ruthenium-catalyzed formation of [5,6]-spiroketals, which proceed via
a very rare 1,2-carbon migration of the internal alkynes. Stoichiometric reactions revealed thatthe disubstituted vinylidene complex that is directly derived from the internal alkyne is the key
intermediate in this spiroketal synthesis. This study shows that the vinylidene rearrangement ofinternal alkynes by 1,2-carbon migration can be integrated into useful catalytic reactions that involve
carbon-carbon bond formation reactions. Further studies directed toward the catalytic formation ofvarious spiroketals that involve 1,2-carbon migration is ongoing.

References(1) (a) Shaw, M. J.; Bryant, S. W.; Rath, N. Eur. J. Inorg. Chem. 2007, 2007, 3943−3946. (b) Mutoh,Y.; Ikeda, Y.; Kimura, Y.; Ishii, Y. Chem. Lett. 2009, 38, 534–535.
(2) Otsuka, M.; Tsuchida, N.; Ikeda, Y.; Kimura, Y.; Mutoh, Y.; Ishii, Y.; Takano, K. J. Am. Chem.
Soc. 2012, 134 (42), 17746–17756.
(3) Watanabe, T.; Mutoh, Y.; Saito, S. J. Am. Chem. Soc. 2017, 139, 7749–7752.
(4) Wilsdorf, M.; Reissig, H.-U. Angew. Chem., Int. Ed. 2012, 51, 9486–9488.

一般の皆様へ
本研究では、これまでの常識では考えられなかったルテニウム触媒をもちいる内部アルキンの1,2−転位を起点とする［5,6］– スピロケタールの触媒的新規合成法の開発に挑戦しました。
［5,6］– スピロケタールは、ベルケリン酸やγ - ルブロマイシンなど抗腫瘍生物活性物質の活性を担う

重要な骨格です。今後の薬理学研究に向けてスピロケタールライブラリーを構築するためには、それらを自在に合成できる新たな反応の開発が必要不可欠です。低収率ではありますが、［5,6］– スピロケタール新しい合成法の開発に成功し、段階的に反応を行うことによって反応機構についても明らかとしました。

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Molecular analysis for the pathophysiological significance of brain
hypoxic response and application for the CNS diseases
Hisashi Shirakawa
shirakaw@pharm.kyoto-u.ac.jp
Kyoto Univ., Grad. Sch. Pharm. Sci.

Summary Abstract
Chronic cerebral hypoperfusion is manifested in a wide variety of CNS diseases, including
neurodegenerative and mental disorders that are accompanied by chronic mild-hypoxic condition
and resultant cognitive impairment. Here, we used a chronic cerebral hypoperfusion mouse model
to investigate whether transient receptor potential ankyrin 1 (TRPA1), a Ca2+-permeable cation
channel, which can respond to mild hypoxic conditions. When male mice were subjected to chronic
cerebral hypoperfusion, cognitive dysfunction and white matter injury at day 14 were observed
only in TRPA1-knockout mice compared but not wild-type mice, suggestting that hypoxia-sensitive
TRPA1 may play a protective in chronic cerebral hypoperfusion-related disorders.
Key Words：Hypoperfusion, Hypoxia, CNS diseases, TRPA1

Introduction
Chronic cerebral hypoperfusion, resulting in an inadequate supply of blood to the brain andinducing hypoxic condition, is elicited by ageing and a variety of risk factors such as atherosclerosis,
hypertension, and obesity. It has been previously suggested that chronic cerebral hypoperfusion ishighly related to dementia diseases such as Alzheimer’s disease and vascular dementia, and other
CNS diseases. Therefore, the precise molecular mechanism of chronic cerebral hypoperfusioninduced cognitive impairment needs to be identified.

Results
In this study, we investigated the pathophysiological role of TRPA1, a Ca2+-permeable channel
that is expressed in neuron, glial cells and endothelial cells and is involved in responding to hypoxic
conditions, using a mouse chronic cerebral hypoperfusion model with bilateral common carotid
artery stenosis (BCAS) by the 0.18 mm internal diameter microcoils, as previously described (Temmaet al., 2017). Cognitive assessment was performed by novel object recognition test. Experiments
were conducted in a dimly illuminated situation (30 Lx), with mice being habituated to the black box
for 3 days (10 min a day) before the training. In the training session, two different objects (a yellow
triangular prism and a blue quadrangular pyramid) were placed in the box and mice were allowed
to freely interact with the objects for 10 min. Six hours after the training session, the test session
was performed, and the blue quadrangular object was replaced by a wooden ball to serve as a novel
object. Exploratory time was defined as the time spent exploring the blue quadrangular object in the
training session and the wooden ball in the test session, and was considered an indicator of locomotor
activity. Exploratory preference was defined as the ratio of the exploratory time versus the total timespent exploring both of the objects, and was considered to be an indicator of recognition memory as
previously described (Miyanohara et al., 2018).

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When mice were subjected to BCAS, the rCBF 60 min after the operation was successfully
decreased to approximately 60% compared with the sham operation, with the change not differing
between the wild-type (WT) and TRPA1-knockout (TRPA1-KO) mice. Exploratory preferences
to the two different objects were around 50%, with no significant difference being observed in the
training sessions in both genotypes. In the test session, exploratory preferences to the novel object
after the training session and interval did not differ between the groups at day 14 after BCAS,
whereas at day 14 there were significant decreases in BCAS-operated TRPA1-KO mice, but not
in BCAS-operated WT mice. At day 28, there were significant decreases in both genotypes. These
results suggest that blockade of TRPA1 can accelerate the development of cognitive impairment
induced by chronic cerebral hypoperfusion.
Next, we have checked tissue injury in BCAS-operated mice. White matter damage is acharacteristic of a variety of CNS diseases, and is associated with cognitive impairment in dementia
cases and other psychological disorders. To evaluate the involvement of TRPA1 in white matterinjury due to chronic cerebral hypoperfusion, myelin staining was performed in the corpus callosum
at day 14 after BCAS. In BCAS-operated TRPA1-KO mice, there was a significant decrease in
myelin density. By contrast, such a decrease was not observed in BCAS-operated WT mice.
To investigate whether stimulation of TRPA1 affects chronic cerebral hypoperfusion-induced
cognitive impairment, we systemically administered cinnamaldehyde (CA), a pungent compoundin cinnamon and also a TRPA1 agonist, into BCAS-operated mice. In the test session of the novel
object recognition test, daily CA-treated mice showed significant reductions in cognitive disturbancewhen compared with vehicle-treated mice.

Discussion & Conclusion
Within CNS, TRPA1 is expressed in expressed in neuron, glial cells (presumably astrocyte andoligodendrocyte) and cerebral endothelial cells and sensitive to hypoxic conditions. Here wedemonstrate that cognitive dysfunction and white matter injury at day 14 were observed onlyin TRPA1-KO mice but not in WT mice. Moreover, daily treatment with a TRPA1 agonist CA
significantly improved cognitive perturbation. Our data indicate that TRPA1 plays a protective rolein the development of chronic cerebral hypoperfusion-induced cognitive impairment, and propose
a new hypothesis in which TRPA1 stimulation specifically improves the pathology with chronic
cerebral hypoperfusion. These findings shed light on the understanding of the mechanisms of chronic
cerebral hypoperfusion-related CNS diseases, and are expected to provide a novel therapeutic
molecule for cognitive impairment in CNS diseases, including dementia.

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References
Temma T, Yamazaki M, Miyanohara J, Shirakawa H, Kondo N, Koshino K, Kaneko S, Iida H.
J Cereb Blood Flow Metab. 37:3334-3343, 2017.
Sequential PET estimation of cerebral oxygen metabolism with spontaneous respiration of 15O-gas
in mice with bilateral common carotid artery stenosis.
Miyanohara J, Kakae M, Nagayasu K, Nakagawa T, Mori Y, Arai K, Shirakawa H, Kaneko S. J
Neurosci. 38:3520-3533, 2018.
TRPM2 Channel Aggravates CNS Inflammation and Cognitive Impairment via Activation of
Microglia in Chronic Cerebral Hypoperfusion.

一般の皆様へ
脳血流の軽度な低下は、脳梗塞や心不全にかかったり、継続的なストレスを受けたりする

ことで起き、さらに高血圧・糖尿病・脂質異常症などの生活習慣病による動脈硬化により慢性化されます。これまでに我々は、軽度な脳血流の低下であっても長く続くことで白質傷害を主に介して、軽度な認知機能障害になることを示してきましたが、本研究により低酸素感受性を有する TRPA1チャネルがこの軽度な認知機能障害に対して保護的な役割を果たしていること、
並びにこの TRPA1を刺激することにより病態を改善できる可能性を示すことができました。
今後はこの TRPA1チャネルを介した保護機構をより詳細に解析したいと思います。

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Elucidation of the onset mechanism of myeloproliferative neoplasms
through the erythropoietin receptor
Megumi Funakoshi-Tagotago-mg@pha.keio.ac.jp
Faculty of Pharmacy, Keio University

Summary Abstract
In myeloproliferative neoplasms (MPN), erythropoietin (Epo)-independent signaling through Epo
receptor (EpoR) can be caused by JAK2 V617F mutant. Previously, we found that phosphorylationof three tyrosine residues, Y343, Y460, and Y464 in EpoR, are required for the JAK2 mutant to
exhibit its oncogenic activity. In the present study, to understand how JAK2 mutant induced cellulartransformation through the phosphorylation of EpoR, we purified EpoR complex in cells expressing
JAK2 V617F mutant and identified the some components of EpoR complex such as STAT5, Grb2
PI3K and CIS.
Key Words：myeloproliferative neoplasms (MPNs), JAK2 V617F mutant, EpoR, STAT5Introduction
A somatic mutation in JAK2, V617F, has been identified in the majority of patients with MPNs.
Although we found that phosphorylation of EpoR was critical for JAK2 mutant-provoked cellulartransformation, the mechanisms by which EpoR contributes to JAK2 mutant-induced oncogenic
signaling. It is expected that the phosphorylation of tyrosine residues in EpoR provides a platform forthe recruitment and activation of signaling mediators which are critical for JAK2 mutant-provokedtransformation. Therefore, to define the signaling mechanisms by which the combination of the
JAK2 mutant and EpoR causes the pathogenesis of MPN, we tried to identify the interacting proteins
with EpoR.

Results
In Ba/F3 cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellulardomain of EpoR were phosphorylated. We performed a mutational analysis of the eight EpoR
tyrosine residues and found that the Y343F/Y460F/Y464F mutant in which Y343, Y460 and Y464
were substituted with phenylalanine completely lost the ability to induce JAK2 mutant-induced cellproliferation and tumor formation. These results indicated that phosphorylation at Y343, Y460, andY464 in EpoR was required for the JAK2 mutant to exhibit its oncogenic activity (Ueda et al. J Biol
Chem 2017).
In order to investigate the roles of phosphorylated tyrosine residues in EpoR, we constructed EpoR
mutants such as 8YF in which all eight tyrosine residues was substituted with phenylalanine, 7YF(7YF-Y343, 7YF-Y460, 7YF-Y464) harboring one intact tyrosine residue and 5YF-Y343/460/464
harboring three intact tyrosine residues and examined the effects of these mutants on JAK2 mutantinduced transformation.
We showed that EpoR recruited a number of signaling molecules such as a transcription factor,
STAT5, an adaptor protein, Grb2, phosphoinositide 3-kinase (PI3K) and a suppressor of cytokine

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for the Promotion of Sciencesignaling, CIS in Ba/F3 cells expressing JAK2 mutant. We next investigated whether EpoR mutants
could recruit these molecules.
Whereas EpoR associated with STAT5,
Grb2, and PI3K, 8YF failed to associatewith these molecules. Interestingly, notonly EpoR but also 7YF-Y343, 7YF-Y460,
and 5YF-Y343/460/464 interacted with
STAT5, suggesting that the phosphorylated
Y343 and Y460 are critical for the
recruitment of STAT5 in cells expressing
the JAK2 mutant. Both 7YF-Y460 and
5YF-Y343/460/464 interacted with Grb2,
suggesting that the phosphorylation of
Y460 seemed to be sufficient for the
recruitment of Grb2 in cells expressing
JAK2 mutant. Furthermore, these EpoR
mutants failed to associate with PI3K,
suggesting that PI3K-Akt pathway seemed
to be dispensable for JAK2 mutantinduced oncogenic signals (Fig. 1).
Previous study reported that activation of STAT5 and ERK1/2 through Grb2 was involved in cellproliferation and tumor formation. We next examined the influence of mutations in tyrosine residues
in EpoR on JAK2 mutant-induced activation of STAT5 and ERK1/2. As a result, it was revealed
that three tyrosine phosphorylation at Y343, Y460 and Y464 are necessary for the full activationof STAT5. In addition, whereas the co-expression of EpoR and 5YF-Y343/460/464 with the JAK2
mutant significantly induced the phosphorylation of ERK1/2, the co-expression of 8YF and 7YF
mutants failed to induce the phosphorylation of ERK1/2. Interestingly, co-expression of 6YFY343/464 and 6YF-Y460/464 induced the phosphorylation of ERK1/2 to the same extent as EpoR.
These results suggest that the phosphorylation of Y464 is insufficient for the activation of ERK1/2,
but the phosphorylation of either Y343 or Y460 in addition to Y464 could induce the activationof ERK1/2 in the cells expressing the JAK2 mutant (Fig. 1). We also found that CIS interactedwith phosphorylated Y401 in EpoR, resulting in the inhibition of JAK2 mutant-provoked cellulartransformation.
Collectively, we demonstrated that three tyrosine residues in EpoR, Y343, Y460 and Y464, areessential for cellular transformation through their mediation of activation of STAT5 and ERK1/2.
We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.
Furthermore, we showed that CIS functions as a novel type of tumor suppressor in JAK2 mutantinduced tumorigenesis by interacting phosphorylated EpoR.

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Discussion & Conclusion
Several studies have reported that the physical interaction with EpoR is indispensable for the JAK2
mutant to induce the pathogenesis of MPN, however; the JAK2 mutant-induced signaling pathway
through EpoR has not been elucidated. In this study, we first found that three tyrosine residues in
EpoR, Y343, Y460 and Y464, are essential for cellular transformation through their mediationof activation of STAT5 and ERK. We also found that CIS prevented the JAK2 mutant-induced
tumorigenesis by interacting phosphorylated EpoR at Y401.
Collectively, the results of this study and previous findings indicate the importance of EpoR asa suitable target for therapeutic drugs against MPN and also that the functional role of EpoR in
oncogenic signaling induced by the JAK2 mutant needs to be elucidated in more detail.

References
1: Ueda F, Tago K, Tamura H, Funakoshi-Tago M. Three Tyrosine Residues in the
Erythropoietin Receptor Are Essential for Janus Kinase 2 V617FMutant-induced Tumorigenesis.
J Biol Chem. 2017 Feb 3;292(5):1826-1846.
2: Funakoshi-Tago M, Moriwaki T, Ueda F, Tamura H, Kasahara T, Tago K.
Phosphorylated CIS suppresses the Epo or JAK2 V617F mutant-triggered cell proliferation
through binding to EpoR. Cell Signal. 2017 Feb;31:41-57.
3: Uchihara Y, Kidokoro T, Tago K, Mashino T, Tamura H, Funakoshi-Tago M.
A major component of vitamin E, α-tocopherol inhibits the anti-tumor activity of crizotinib
against cells transformed by EML4-ALK. Eur J Pharmacol. 2018 Apr 15;825:1-9.

一般の皆様へ
慢性骨髄増殖性腫瘍 (MPN) の効果的な治療薬を開発する上で、未だ不明な点の多い MPN の発症メカニズムを解明し、理解することが重要です。今回の研究では、MPN の原因となることが知られているチロシンキナーゼ JAK2変異体が、サイトカイン受容体 EpoR を介して STAT5や

ERK などのシグナル伝達分子を活性化し、細胞増殖や腫瘍形成を誘導するメカニズムを明らかにしました。本研究を通して、EpoR が MPN 治療薬の標的分子となりうる可能性が高いことが示されました。

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Induction and visualization of adult cardiomyocytes proliferation
through growth factor receptor activation
Maretoshi Hirai
hiraimar@hirakata.kmu.ac.jp
Kansai Medical University, Department of Pharmacology

Summary Abstract
Cardiac myocytes lose their cell cycle activity after birth. After myocardial infarction, cardiac
function is impaired due to loss of cardiac myocytes. In this project, we tried to develop novel
strategy to induce cell cycle activity in mature cardiac myocytes, thus increase their number,
improve contractile function. For this purpose, we succeeded in generating mouse line conditionallyoverexpressing growth factor receptor. It is currently ongoing work to examine if adult cardiac
myocytes can activate cell cycle activity by this gene modulation.
Key Words：Cardiac myocytes, Proliferation, Growth factor receptor, Gene-targeted mouse

Introduction
Heart is an integral organ to maintain our life. Among various types of cells in heart, cardiac
myocytes play major role in heart beating. However, soon after birth, cardiac myocytes lose
proliferative potential. Thereby, once heart loses cardiac myocytes through myocardial infarction,
cardiac myocytes are not capable of proliferating, leading to contractile dysfunction. In this research,
we attempted to induce proliferative potential in adult cardiomyocytes by overexpressing growth
factor receptor in cardiac myocytes, trying to develop novel regenerative therapy for post infarctheart failure.

Results
Soon after birth, cardiac myocytes lose cell cycle activity, cease proliferating whithin the firstweek postnatally. Loss of cardiac myocytes due to myocardial infarction leads to heart contractile
dysfunction. To prevent heart failure after myocardial infarction, we attempted to postnatlly activate
cell cycle activity, thereby increase cell number of cardiac myocytes. For this purpose, we try to
generate mouse line conditionally overexpressing one of the growth factor receptor.
First, we designed a novel targeting vector to target into Rosa locus, where we deleted transcription
initiation sites so that endogenous transcription for Rosa lncRNA loci does not disturb transcriptionof our transgene which is driven by CAG promoter. To overexpress the transgene selectively andspecifically in cardiac myocytes in heart, we put floxed-Neo-polyA casssette between CAG promoter
and our transgene, then planned to cross with cardiac myocyte specific troponin T-Cre mouse line.
We also put 2A sequence at the carboxyl-terminus, followed by histone fused mCherry coding
sequence (H2B-mCherry), which enable us to visualize nuclei of cardiac myocytes overexpressing
the growth factor receptor.
We optimized targeting strategy to ES cells using Crispr/Cas9. By utilizing pEGxxFP psystem,
we confirmed double strand break efficiently happens in our target Rosa locus with our gRNAs.
As the size of gene we would like to knock-in was 9 ~ 10 kbp, we estimated that the efficiency of

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proper targeting might be very low. At this time point, it was not clear how long homologous arm
is necessary for knock-in strategy with Crispr/Cas9. Generally, a couple of hundreds of nucleotide
sequence were considered to be sufficient to knock-in EGFP coding sequence with Crispr/Cas9,
however, thinking of the large size of our knock-in gene, we prepared 2 kbp of homologous sequenceeach side, total 4 kbp of homologous arm. Next, we performed gene-transduction to ES cells by
lipofection. To select transfected ES cells, we used Puromycin for 48 hours, as Puro resistant gene
expresses by the plasmid co-transfected with our targeting vector and Crispr/Cas9-gRNA expressing
vector. Although our targeting vector contained Neo resistant gene, we did not use Neomycin (G419)
for selection, thinking of future application of our targeting approach. For future design of targeting
vector, we would not like to put Neo resistant gene with a promoter intervened with FRT sequence,
so that we do not have to take out the resistant gene with Flpase, and avoid extra consumption oftime.
After all, we succeded in obtaining two positive ES clones among from 32 clones we picked,
followed by injection of ES cells into 8-cell stage embryos. We obtained chimeric mice, thensucceeded in germline transmission, obtained conditionally overexpressing mouse line. This mouseline was crossed with cardiac myocyte specific Troponin – T Cre mouse.
We sectioned heart tissue, then immunostained with anti-troponin-T antibody to label cardiac
myocytes, anti-PDGFR-alpha antibody to label fibroblasts, and CD31 antibody to label endothelial
cells. By immuno-staining, we confirmed that H2B-mCherry was expressed selectively andspecificlly in cardiac myocytes, as we expected. In future experiments, we will dissociate cardiac
myocytes from postnatal heart to define the change of the number of cardiac myocytes byoverexpression of the growth factor receptor. We will also inject EdU intrapertoneally to assess thecell cycle activity, confirm how our transgene promote cell cycle activity.

Discussion & Conclusion
It has been very inefficient to knock-in large size of gene to targeted locus before Crispr/Cas9 wasintroduced. In this project, by optimizing suitable culture conditions, designing proper targeting
vector, and utilizing Crispr/Cas9, we succeeded in efficiently obtaining gene-targeted ES cell lines.
By injectiing of ES cells into 8-cell stage embryos, we generated novel conditionally overexpressing
knock-in line, which might activate cell cycle, even in adult cardiac myocytes. Our knocked-in genecan also be overexpressed in any cell lineage of any tissue by using suitable Cre mouse line, which
would be commercially available. By future experiments, we will reveal whether growth factor
receptor activation might be able to modulate cell cycle activity, and induce proliferation of mature
cardiac myocytes. This result might open the door for the possiblity of promissing novel therapeuticstrategy for heart failure after myocardial infarction.

References“Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte
Proliferation.”
Hirai M, Chen J, Evans SM.
Circulation Research, Vol. 118, No. 1, 20-28: Jan 8, 2016

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一般の皆様へ
心筋細胞は生後細胞周期の休止期に入りその後増殖することはない。そのため心筋梗塞で心筋細胞が失われると心機能障害をきたし心不全を発症してしまう。本研究ではマウスで成長因子受容体シグナルを活性化することにより、成熟心筋細胞の増殖を誘導しうるかどうかを明らかにすることを目的とした。本研究の結果、心筋細胞で成長因子受容体を過剰発現させ、その細胞を1細胞レベルで直接可視化できるマウスの作製に成功した。今後、このマウスを用いて、成熟心筋細胞の増殖を誘導することが可能であれば、梗塞後心不全に対する新規治療の開発への展望が開けるだろう。

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Analysis of the function and genomic abnormality oftumor suppressor gene PHLDA3 in neuroendocrine tumors- Towards development of a novel genetic diagnosis method
for neuroendocrine tumors Reiko Ohki
rohki@ncc.go.jp
National Cancer Center Research Institute

Summary Abstract
We have found that: the PHLDA3 gene encodes a novel repressor of oncogene Akt and functions as
a tumor suppressor gene in NET of lung and pancreas (1, 2); in these NET, the function of PHLDA3
is frequently lost due to genomic abnormality; and cases in which PHLDA3 function is lost are
shown to be highly malignant and have poor prognosis (2, 3). Based on our findings, we perform:
Genomic analysis using human NET samples with clinical information; and Analysis of NET model
animals.
Key Words：NET, PHLDA3, Akt

Introduction
Neuroendocrine tumors (NET), which is a rare cancer, occurs in various organs of the whole body.
Due to its rareness, the mechanism of tumorigenesis is not sufficiently elucidated, and there are noeffective diagnostic methods or treatments.
In this project, we will analyze the world's largest NET cohort / new NET mouse model, and clarify
the tumor-suppressing pathway common to NET across organs, and also clarify the relationshipbetween patient prognosis, drug efficacy and genomic abnormality. From the results obtained, we
will develop a novel diagnosis and treatment method, and respond to unmet medical needs of NET
patients.

Results
We have proceeded with the genomic analysis using human NET samples with detailed clinicalinformation, and the analysis of NET model animals. Specifically, we used specimens of: 150 casesof Pancreatic NET; 203 cases of Pituitary NET; 69 cases of Colorectal NET; and 30 cases of Thyroid
NET. And, we have performed the genomic analysis focusing on whether or not any PHLDA3
genetic abnormality exists. Although the results are still preliminary, we have obtained results thatis reasonable for considering that all of the analyzed NETs have PHLDA3 genetic abnormality, andthus PHLDA3 is a tumor suppressor gene common to all NETs (3, 4).
1. Genomic analysis of the NETs
We hold the world's largest pancreatic, pituitary, colon, thyroid and parathyroid NET specimencohort with over 450 cases. Using the specimens, analysis of PHLDA3 gene abnormality (LOH,
methylation, point mutation), comprehensive whole exon sequence analysis, and RNA-seq have been
performed.

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Through these analyses, in addition to PHLDA3 genetic abnormality, we have identified genes
mutated together with PHLDA3. We are now analyzing the intracellular pathway involved in NET
onset.
2. Analysis of NET model animals
We have established an unique pancreatic NET model mice (PHLDA3 and MEN1 double-deficientmice) that mimic the genomic abnormality of human NET, and model mice of pituitary and thyroid
NET are currently being prepared. The pancreatic NET model mice and model cell lines derived fromthe model mice have been treated with drugs used for therapy of pancreatic NET, and the clinicalpredictability of these models are now being verified. At the same time, we are now exploring theusefulness of combination therapy using the inhibitors of the pathways identified in project 1.
3. Development of NET diagnostic method using the PHLDA3 geneWe have analyzed the relationship between genetic abnormality of PHLDA3 and patient prognosis /therapeutic drug response, and are now developing a new NET diagnostic method. We havealso conducted a research to develop a diagnostic method based on the combination of genetic
abnormality of genes found in project 1 and PHLDA3.

Discussion & Conclusion
We will continue to proceed the genomic analysis using human NET samples with detailed
clinical information and the analysis NET model animals, for elucidating the essential condition of
Neuroendocrine tumors (NET), which is a rare cancer. And we aim at further development of NET
diagnostic method using PHLDA3 gene for a novel prognosis and treatment option.
As for ethical considerations, we will compliance to the ethical guidelines related to study of humangenome and gene analysis, and the ethical guidelines related to epidemiological study. With reference
to the genetic recombination experiments, we obey “Act on securing biodiversity by regulation on
the use of genetically modified organisms, etc.” and related laws and regulations. With reference tothe animal experimentation, we compliance with basic guidelines and perform the experimentationaccording to the animal examination code with due consideration for the experiment animals.

References
1. Tatsuya Kawase*, Rieko Ohki#, *, Tatsuhiro Shibata, Shuichi Tsutsumi, Naoko Kamimura, Johji
Inazawa, Tsutomu Ohta, Hitoshi Ichikawa, Hiroyuki Aburatani, Fumio Tashiro and Yoichi Taya.
(*These authors equally contributed to the work, #corresponding author) PH domain-only protein
PHLDA3 is a p53-regulated repressor of Akt.Cell, Vol. 136, pp. 535-550, 2009.
2. Rieko Ohki#, Kozue Saito, Yu Chen, Tatsuya Kawase, Yukie Aita, Nobuyoshi Hiraoka, Raira
Saigawa, Maiko Minegishi, Goichi Yanai, Hiroko Shimizu, Shinichi Yachida, Naoaki Sakata,
Akihiko Yokoyama, Ryuichiro Doi, Tomoo Kosuge, Kazuaki Shimada, Benjamin Tycko,
Toshihiko Tsukada, Yae Kanai, Shoichiro Sumi, Hideo Namiki, Yoichi Taya, Tatsuhiro Shibata
and Hitoshi Nakagama. (#corresponding author) PHLDA3 is a novel tumor suppressor of
pancreatic neuroendocrine tumors. Proc Natl Acad Sci USA, 111 (23), E2404-E2413, 2014.
3. Masahiro Takikawa and Rieko Ohki (corresponding author).
A vicious partnership between AKT and PHLDA3 to facilitate neuroendocrine tumors. Cancer
Science, 108(6):1101-1108, 2017.

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4. Mitsuhiro Tomosugi, Tatsuya Kawase, Kohei Tominaga and Rieko Ohki, Tumor Suppressor GenePHLDA3, One of the Most Important Molecules to Conquer Pancreatic Neuroendocrine Tumor.
Tan to Sui, Vol. 38, pp1345-1355, 2017

一般の皆様へ
神経内分泌腫瘍は全身の様々な臓器に生じますが、希少がんであるため研究開発は遅れており、有効な診断法・治療法がありません。

私たちは、肺と膵臓の神経内分泌腫瘍において、PHLDA3 遺伝子は高頻度にゲノム異常によって機能が失われていることを発見し、PHLDA3 遺伝子が肺と膵臓の神経内分泌腫瘍において、がん抑制遺伝子として機能している事を明らかにしました。さらに、PHLDA3 機能が失われている症例は悪性度が高く予後不良である事が示されました。これらの知見を元に、神経内分泌腫瘍のヒトや動物のサンプルを解析し、その本態を解明し、治療と診断に貢献
したいと考えております。

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Disease specific regulation of the histone methylationin acute myeloid leukemia
Takeshi Ueda
takeshiueda@med.kindai.ac.jp
Kindai University Faculty of Medicine

Summary Abstract
In the present study, we demonstrate the role of Lysine demethylase 4B (KDM4B)-mediatedepigenetic regulation in the disease progression of acute myeloid leukemia (AML).
Key Words：Epigenetics, Hematologic malignancy

Introduction
KDM4B is an epigenetic regulator that functions as a histone demethylase for di- and tri-methylated
H3K9 as well as di- and tri-methylated H3K36 and H1.4K26. Although KDM4B has so far beenreported to promote tumor development in several types of cancers, the tissue specificity and context
dependency still remain largely uncertain.

Results
To investigate the role of KDM4B in hematopoiesis, Kdm4b flox/flox (fl/fl) mice were crossed totransgenic mice expressing Cre recombinase from the interferon-responsive Mx1 promoter (Mx1-Cremice). To induce Cre expression, Mx1-Cre/ Kdm4b fl/fl mice and littermate controls (Kdm4b fl/fl)
were injected with polyinosine-polycytosine, which mimics a viral infection. With this protocol, Cremediated recombination through the loxP sites effectively excised exon 5 of the Kdm4b gene andgenerated a Kdm4b-null allele (Kdm4bΔ). Western blot analysis showed that Cre induction broughta clear reduction in the amount of Kdm4b protein in bone marrow (BM) cells. We characterized
hematopoietic stem cell (HSC) compartment in the BM of Kdm4bΔ/Δ and control mice using surface
marker analysis, and found similar absolute cell numbers between these two genotypes. There wasno significant difference in hematopoietic colony forming capacity in semisolid media by use ofc-Kit+ HSC/progenitor cells from BM of Kdm4bΔ/Δ and control mice.
We found the upregulation of KDM4B in AML with 8;21 chromosomal translocation. Whentranscription factor AML1 is fused to ETO (a nuclear protein that interacts with co-factor molecules)
by the 8;21 translocation [t(8;21)], the fusion gene AML1-ETO activates or inactivates target gene
expression through recruitment of co-factors, resulting in the disease progression of AML. To
examine the role of Kdm4b in clonogenecity mediated by AML1-ETO, we retrovirally transduced the
AML1-ETO to c-Kit+ BM progenitor cells and subsequently performed serial hematopoietic colony
forming assay. As a result, the colony-replating capacity of Kdm4bΔ/Δ cells transduced with AML1ETO was significantly reduced compared to that of the AML1-ETO-transduced controls. A previous
study revealed that AE9a, a splice isoform of AML1-ETO exhibited more potent leukemogenic
activity in mice. Based on this finding, we also assessed the impact of Kdm4b on AE9a-mediatedclonogenecity by serially colony replating. Kdm4b deficiency in c-Kit+ BM cells transduced with
AE9a significantly decreased number of colonies at the successive plating rounds. Thus, these results
suggest that Kdm4b is required for robust clonogenic potential in t(8;21) leukemia.

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Next, we examined the effect of KDM4B suppression on human AML cells using SKNO1 cellline, a human t(8;21) leukemia cell line model. Two different lentiviral shRNA constructs with thepuromycin resistance gene were designed to effectively decrease the KDM4B protein in SKNO1
cells. Notably, stable knockdown of KDM4B altered cell morphology and led to markedly decreased
cell proliferation, coupled with increased cell death. We then performed RNA sequencing to identify
the molecular basis of the cellular impact by KDM4B knockdown. Previous reports showed that the
AML1-ETO fusion is crucial for proliferation of human t(8;21) AML cell lines including SKNO1.
We applied Gene Set Enrichment Analysis (GSEA) to our gene expression data based on reported
gene sets consisting of genes induced by AML1-ETO in human CD34+ hematopoietic stem cells.
As a result, we found that KDM4B knockdown cells showed a significant reduction of the AML1ETO-inducible gene expressions. Thus, a role of KDM4B promoting AML1-ETO-mediated gene
regulation is suggested, which may partly explain the mechanism underlying antiproliferative effectby KDM4B knockdown in SKNO1 cells.

Discussion & Conclusion
Our results highlight KDM4B-mediated epigenetic regulation contributing to the diseaseprogression of AML and suggest KDM4B as a potential therapeutic target for t(8;21) leukemia.

References
Epigenetic regulation by a histone demethylase in leukemia with 8;21 chromosomal translocation[abstract]. In: Consortium of Biological Sciences 2017; 2017 Dec 6–9; Kobe, Japan; Abstract 1P1020.

一般の皆様へ

急性骨髄性白血病で高発現を認めるエピゲノム制御因子が白血病の病態にどのように関与しているかを明らかにする研究を進めています。

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Treatment of metastatic tumors by systemic injection oftumor-retargeted oncolytic herpesviruses
Hiroaki Uchidahiuchida-tky@umin.net
Division of Bioengineering, Advanced Clinical Research Center,
The Institute of Medical Science, The University of Tokyo

Summary Abstract
The route of administration of oncolytic herpes simplex virus (HSV) in the majority of clinical trials
to date has been direct injection into the tumor. This appears to be one of the factors that may limit
the therapeutic potential of oncolytic HSV as a single agent against systemically metastatic tumors.
We have successfully established an HSV platform fully retargeted to tumor-associated antigens thatenable restriction of virus infection at the stage of entry. This achievement may prove to be a major
breakthrough that facilitates treatment of metastatic tumors via systemic injection of oncolytic HSV.
Key Words：Oncolytic virotherapy, Cancer, Herpes simplex virus

Introduction
Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective
viral replication in cancer cells but not in normal cells. Herpes simplex virus (HSV) is a promising
agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations thatfavor its replication in tumor cells. However, the route of administration of oncolytic HSV in the
majority of clinical trials to date has been direct injection into the tumor. This appears to be oneof the factors that may limit the therapeutic potential of oncolytic HSV as a single agent against
systemically metastatic tumors.

Results
Entry of HSV is initiated by the binding of glycoprotein D (gD) to one of its receptors, herpesvirus
entry mediator (HVEM) or nectin-1. This interaction results in a conformational change in gD,
triggering sequential activation of gH and gB to execute fusion between the viral envelope and cell
membranes. For virus retargeting to epidermal growth factor receptor (EGFR)-, carcinoembryonicantigen (CEA)-, or epithelial cell adhesion molecule (EpCAM)-expressing cells, we mutated ordeleted gD residues essential for binding to the natural entry receptors, inserted an EGFR-, CEA-, or
EpCAM-specific single-chain antibody (scFv), and observed specific virus entry into cells expressing
the cognate target antigen. We combined the retargeted gD alleles with hyperactive gB mutations
D285N/A549T identified through directional evolution experiments and found that these viruses
entered the target cells around 100-fold more efficiently than the wild-type-gB versions without
raising off-target entry. In addition, we found that not only initial entry but also subsequent cellto-cell spread of our retargeted viruses was stringently dependent on the cellular expression of the
cognate target antigens by carrying out experiments that distinguish between these two similar, but
distinct entry processes. One of the EGFR-retargeted viruses efficiently killed a panel of EGFRexpressing human tumor lines in vitro and reduced the growth of human tumors in nude mice.

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Toxicity experiments involving intracranial virus inoculation demonstrated symptom-free animal
survival at a dose that was 100,000-fold higher than a dose of wild-type virus that was fatal.
Then, we sought to determine whether the EGFR-retargeted virus would preferentially accumulate
in EGFR-positive human tumors in nude mice. Following the establishment of subcutaneous flanktumors, equal amounts of the wild-type virus and the EGFR-retargeted virs were administered bytail vein injection. The animals were sacrificed 2 days later and the biodistribution of each virus invarious organs including the tumor, brain, lung, heart, liver, spleen, and kidney, was determined
by qPCR for the viral genome. As a result, the number of the wild-type virus genomes per 100 ng
tissue DNA was comparable to or lower in the tumors than in most of the other tissues from the same
animals. In contrast, the EGFR-retargeted virus was detected at 100–1,000-fold higher levels in thetumors than in other tissues. These results indicated that the EGFR-retargeted virus preferentiallyhomed to the human EGFR-positive tumor tissue. Furthermore, we observed the intravenous
injection of the EGFR-retargeted virus reduced the growth of human EGFR-positive tumors in thesame mouse model.

Discussion & Conclusion
Our successful establishment of HSV platforms fully retargeted to tumor-associated antigens thatenable restriction of virus infection at the stage of entry may prove to be a major breakthrough thatfacilitates treatment of metastatic tumors via systemic injection of oncolytic HSV. In addition, thisnovel technology will allow researchers to pursue strategies of manipulating the HSV genes involvedin viral replication and/or cell-to-cell spread towards augmentation of oncolytic activity, instead of
its attenuation. Furthermore, the receptor-retargeting technology may prove to be useful to overcome
the issue that the oncolytic HSVs currently used are likely to be ineffective for tumors that do not
express gD receptors at sufficient levels.

References
1. Uchida H*, Hamada H, Nakano K, Kwon H, Tahara H, Cohen JB, Glorioso JC. (2018) Oncolytic
herpes simplex virus vectors fully retargeted to tumor-associated antigens. Current Cancer Drug
Targets. 18:162-170. *Corresponding author.
2. Okubo Y, Uchida H*, Wakata A, Suzuki T, Shibata T, Ikeda H, Yamaguchi M, Cohen JB,
Glorioso JC, Tagaya M, Hamada H, Tahara H. (2016) Syncytial mutations do not impair the
specificity of entry and spread of a glycoprotein D receptor-retargeted herpes simplex virus.
Journal of Virology. 90:11096-11105. *Corresponding author.
3. Shibata T, Uchida H*, Shiroyama T, Okubo Y, Suzuki T, Ikeda H, Yamaguchi M, Miyagawa Y,
Fukuhara T, Cohen JB, Glorioso JC, Watabe T, Hamada H, Tahara H. (2016) Development of an
oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-tocell spread. Gene Therapy. 23:479-488. *Corresponding author.
4. Uchida H, Marzulli M, Nakano K, Goins WF, Chan J, Hong CS, Mazzacurati L, Yoo JY, Haseley
A, Nakashima H, Baek H, Kwon H, Kumagai I, Kuroki M, Kaur B, Chiocca EA, Grandi P,
Cohen JB, Glorioso JC. (2013) Effective treatment of an orthotopic xenograft model of humanglioblastoma using an EGFR-retargeted oncolytic herpes simplex virus. Molecular Therapy.
21:561-569.
5. Uchida H, Chan J, Shrivastava I, Reinhart B, Grandi P, Glorioso JC, Cohen JB. (2013) Novelmutations in gB and gH circumvent the requirement for known gD receptors in HSV-1 entry andcell-to-cell spread. Journal of Virology. 87:1430-1442.

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6. Uchida H, Chan J, Goins WF, Grandi P, Kumagai I, Cohen JB, Glorioso JC. (2010) A double
mutation in glycoprotein gB compensates for ineffective gD-dependent initiation of herpes
simplex virus type 1 infection. Journal of Virology. 84:12200-12209.
7. Uchida H, Shah WA, Ozuer A, Frampton AR Jr, Goins WF, Grandi P, Cohen JB, Glorioso JC.
(2009) Generation of HVEM-restricted HSV-1 mutant viruses: Resistance of HVEM-expressing
cells and identification of mutations that rescue nectin-1 recognition. Journal of Virology.
83:2951-2961.

一般の皆様へがん細胞だけに特異的に侵入し、静脈内投与可能な標的化腫瘍溶解性ウイルス療法が開発できれば、原発巣のみならず全身の転移巣にも有効な治療法となりえる。私たちは、単純ヘルペス
ウイルス（HSV）にがん細胞表面抗原を認識する単鎖抗体を組み込むことにより、標的細胞のみに侵入可能な標的化 HSV の構築に独自に成功した。この標的化 HSV は、現在臨床開発が進められているウイルス複製の段階でがん選択性を発揮する HSV に比して、より静脈内投与に適していると考えられる。

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Revealing cell membrane functions through in-vitro analysis
by tip-enhanced Raman spectroscopy in liquid
Prabhat Verma
verma@ap.eng.osaka-u.ac.jp
Department of Applied Physics, Osaka University

Summary Abstract
In this research, we constructed tip-enhanced Raman spectroscopy (TERS) applicable in liquid
environment, which enables label-free imaging of membrane components through Raman analysis
at nanoscale. With the constructed system, we have obtained Raman spectrum of lipid bilayers withmetallic TERS tips. This would be a promising result to realize a powerful technique that visualizes
how membranes behave at nanoscale in a physiological condition without any labeling.
Key Words：Raman spectroscopy, Tip-enhanced Raman spectroscopy, cell membranes

Introduction
Membrane of a cell is not just a frame of cell but an important platform that possesses channel
proteins, receptors and catalytic areas to maintain our health. Also, membrane components work asa trigger for various medicines. It is therefore essential to understand how membranes function withtiny molecules. For this aim, it is required to develop an analytical technique that can analyze “real”
membranes at nanoscale in a physiological condition. TERS is a promising for this purpose becauseof label-free analysis through Raman spectroscopy and nanoscale spatial resolution through nearfield light localized at a metallic tip.

Results
We have constructed TERS applicable in liquid environments by combining Raman spectroscopy
with an atomic force microscopy available in liquid conditions. In the system, a z-polarizer was
installed to generate z polarization at a metallic tip so that near-field light is efficiently excited byoscillating plasmon along the tip axis. A pinhole was also located to drastically reduce scattering
background through confocal configurations.
In TERS, metallic tips also play a very important role because localization and intensity of nearfield light (spatial resolution and sensitivity) are strongly dependent on structure of metallic tips.
Slight differences in the metallic tip structures largely changes property of near-field light so that
it is crucial to develop a practical method to fabricate metallic tips that show strong enhancement
and localization of near-filed light with high reproducibility. For this aim, we utilized electron
beam deposition technique and sputtering technique for tip fabrication. We first deposited anamorphous carbon tip, which is extremely sharp with tip diameter of less than 10 nm, by electron
beam deposition under atmosphere of organic molecule precursors. We then coated it with silver
by sputtering technique. Through these processes, silver rod-like structures were obtained at the
tip apex. By changing exposure time of electron beam and sputtering, we can precisely control thelength and thickness of the rod structure, which means precise control of spatial resolution and nearfield light intensity. The dependence on the length and thickness was also confirmed through finite
difference time domain methods.

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To make liquid-TERS system more practical, we developed a technique to recycle tips. By dipping
silver-coated tips in HNO3 solution for a hour, and then treating them by plasma etcher over night,
silver coating was entirely removed and cleaned. We confirmed that we can re-fabricate silver rodlike structure on recycled tips.
Another issue in the liquid-TERS is contamination of metallic tips. Metallic tips are easily
contaminated with biological molecules floating in an observation pool, which gives unwanted
Raman signals and disrupt measurements. To avoid this problem, we applied sodium chloride in theobservation buffer. Since chloride ions have strong affinity to silver, they are strongly attached on thesurface of silver-coated tips by removing contaminations from the tips. We clearly observed decreaseof Raman signals from contaminations after dipping metallic tips in 100 mM sodium chloride
solution for 30 minutes. Since 100 mM of sodium chloride is quite similar with physiologicalenvironments, we can get another unique advantage that this method does not disturb cell membrane
activities.
With the constructed setups and developed techniques, we first tried fluorescent measurements.
We successfully imaged fluorescent beads over the diffraction limit of light. DNA labeled withfluorescent molecules was also imaged, which proved that this technique is applicable for biologicalsamples. Moreover, imaging rate was several seconds, which implies that this technique even has apotential for dynamic imaging of molecules in liquid.
Since we confirmed performance of the constructed TERS system, we then performed TERS
imaging with carbon nanotubes in liquid with nanoscale spatial resolution. Further, we demonstrated
TERS measurements of lipid bilayers. We successfully obtained Raman signals when metallic tip isin contact to the lipid bilayers. This result is highly promising to realize nano-analytical technique toobserve “real” cell membranes at work in physiological environments.

Discussion & Conclusion
We have constructed TERS system working in liquid environments, which was confirmed withsuper-resolution fluorescent imaging of several samples. Promising results on lipid bilayers withTERS was also obtained. Besides, we made huge progresses in metallic tip fabrication techniques,
which is highly important to make TERS system more practical. However, there are still some rooms
that we can work on to improve our TERS system. For instances, cell membrane has 3D structure sothat it will be required to track tip positions in z-directions by piezo control and feedback systems.
Metallic tips still did not give consistent enhancement for TERS measurements of cell membranes.
By optimizing these things, we believe that our liquid-TERS system contributes to reveal cell
membrane functions at nanoscale details in the future.

References
1) Satoshi Kawata, Yasushi Inouye, and Prabhat Verma “Plasmonics for near-field nano-imaging andsuperlensing”, Nature Photonics, 3, 388-394 (2009).
2) Prabhat Verma, “Tip-enhanced Raman Spectroscopy: Technique an Recent Advances”, Chemical
Reviews, 117, 6447 (2017).

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一般の皆様へ
細胞膜は細胞同士を隔てる単なる仕切りではなく、細胞外からの情報をやり取りしたり、必要な物質を細胞内に輸送したり、私たちの健康を維持するために必要な機能を沢山持っています。また、多くのウィルスや薬も細胞膜を足掛かりとして作用するので、細胞膜の様々な現象を単分子レベルで解明することは、創薬の発展にもとても重要です。しかし、ありのままの細胞膜を単分子レベルで観察する手法は非常に困難です。私たちは、これを実現すべく生体環境

で使える先端増強ラマン散乱顕微鏡という装置を開発しました。単分子レベルの分解能でありのまま細胞膜を観察できるため、今後細胞膜の研究にますます役立つことと期待しています。

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Elucidation of the immune-nerve-metabolic systems network
in cardiac homeostasis and failure
Ichiro Manabe
manabe-tky@umin.ac.jp
Chiba University

Summary Abstract
In the present study we identified a novel heart-brain-kidney network that control adaptive
response to pressure overload to the heart. In the heart cardiac resident macrophages are essential for
cardioprotection and amphiregulin is a key mediator produced by the cardiac macrophages. We havealso analyzed the mechanism that confers cardioprotection by amphiregulin.
Key Words：Heart failure, Macrophage

Introduction
Heart failure is a complex clinical syndrome characterized by cardiac function that is insufficient
to meet systemic demand. The prevalence of heart failure is rapidly increasing partly due to aging
society so that heart failure is now considered as a global pandemic. In addition to abnormalities
intrinsic to the heart, dysfunction in other organs and systemic factors greatly affect the development
and consequences of HF. For instance, nearly half of chronic HF (CHF) patients also have chronic
kidney disease (CKD), which increases their rate of cardiovascular mortality, suggesting cardiorenal
linkage via mechanisms still poorly understood. Moreover, previous studies have mainly focusedon the signaling mechanism that regulate cardiomyocyte hypertrophy, recent studies have been
unraveling crucial roles played by nonmyocytes in the response to cardiac stress. In the present study
we aim to elucidate the function of cardiac macrophages in the maintenance of cardiac homeostasis.

Results
We found that there are at least two subpopulations of macrophages in the myocardium. In response
to pressure overload induced by transverse aortic constriction (TAC) only cardiac resident Ly6Clo
macrophages were increased. In a series of experiments we found that cardiac Ly6Clo macrophageactivation and proliferation are regulated by a heart-brain-kidney network. Pressure overload in theheart activates renal collecting duct epithelial cells via the sympathetic nervous system. The renalcollecting duct epithelial activation triggers communication between collecting duct epithelial cells,
renal CD11b+F4/80hiLy6Clo macrophages and endothelial cells. Activated renal endothelial cells
then produce and secrete CSF2, which stimulates the cardiac Ly6Clo macrophages essential for theproper cardiac adaptive response to pressure overload. This dynamic heart-brain-kidney interplay
is essential for the body’s response to cardiac stress. Dysfunction of this network impaired proper
adaptive response to pressure overload and mice developed heart failure.
Our results indicate that cardiac Ly6Clo macrophages are necessary for a proper cardiac adaptive
response to pressure overload. We identified amphiregulin (AREG) as a key cardioprotective
mediator produced by cardiac Ly6Clo macrophages. In the heart cardiac Ly6Clo macrophages are themajor source of AREG and expression of AREG is upregulated by TAC. Ly6Clo macrophages that

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for the Promotion of Sciencelack Areg failed to protect the heart from heart failure induced by TAC when they were transplanted
into the heart. Inhibition of AREG signaling using a neutralizing antibody significantly reduced
cardiomyocyte hypertrophy, cardiac function and overall survival in wild-type mice after TAC
without affecting cardiac Ly6Clo macrophage numbers. Conversely, recombinant AREG rescued
collecting duct epithelial cell-specific Klf5 knockout mice that develop heart failure after TAC
from the heart failure phenotype. Collectively, these results demonstrate that AREG is importantfor cardioprotection. Mechanistically, AREG is the major mediator secreted from cardiac Ly6Clo
macrophages that induces a hypertrophic response in cardiomyocytes.
To further elucidate the cardioprotective function of cardiac Ly6C lo macrophages and theinteraction between cardiomyocyte, macrophages and other nonmyocytes, we analyzed transcriptome
and epigenome in cardiac macrophages by RNA-seq, ChIP-seq, and ATAC-seq. We found that
epigenome in cardiac macrophages are dynamically modulated by cardiac stress. Using in vitro
coculture systems and in vivo analyses we also found that the interactions between cardiomyocyte,
macrophages and other nonmyocytes, including neurons, shape the epigenome in cardiac
macrophages. Moreover, we identified candidates of transcription factors and histone modifiers thatare important for shaping the cardiac macrophage-specific epigenome.
We also elucidated additional mechanisms, by which AREG confers cardioprotection. In addition
to activate the hypertrophic program in nearby cardiomyocytes, our results indicate that AREG has
multiple functions that are essential for cardiac homeostasis in the steady state as well as cardiac
stress. Consequently, while Areg knockout mice did not show cardiac phenotype in the steadystate, cardiac homeostasis was already modulated. Upon various cardiac stress Areg knockout micedeveloped heart diseases, such as heart failure.

Discussion & Conclusion
Our results demonstrate that cardiac resident macrophages are a key player in the maintenance of
homeostasis and adaptive response to cardiac stress. In the present study we analyzed the effects of
AREG on cardiomyocytes and found that the cytokine crucially contributes to the maintenance of
cardiac homeostasis by multiple mechanisms. In addition, we found that local cell communication
with cardiomyocytes and nonmyocytes is essential for the generation of cardiac macrophage-specificepigenome. Moreover, cardiac macrophages are regulated by the heart-brain-kidney network. The
signaling mechanisms identified in the present study appear to be important as potential therapeutictargets for heart failure.

一般の皆様へ
心不全は急速に増加しており、新しい治療法を開発することは喫緊の課題です。私たちは、心臓に存在するマクロファージ（免疫細胞の一種）に注目して、その機能の解析を行いました。
心臓マクロファージは心臓へのストレスに対して心臓を守る機能を持っています。また、アンフィ

レグリンという分子を作ることが大事なことを見いだしました。心臓マクロファージの機能は、
心臓－脳－腎臓をつなぐネットワークによって制御されています。また、心臓の中では、心臓に存在する心筋細胞等の細胞との関連によって制御されています。本研究で同定したメカニズムは今後の心不全治療法の開発に役立てることができると考えています。

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Roles of synaptic plasticity and replay during sleep
in memory consolidation
Yasunori Hayashi
yhayashi-tky@umin.ac.jp
Department of Pharmacology
Kyoto University Graduate School of Medicine

Summary Abstract
To elucidate roles of synaptic plasticity and replay during sleep in memory consolidation, wedeveloped a new optical method for erasing hippocampal LTP and memory. In this system, cofilin
was fused with SuperNova (CFL-SN) and light was illuminated to inactivate cofilin. structural
LTP induced in the single dendritic spine in the hippocampal slice culture expressing CFL-SN wascancelled by light irradiation. Furthermore, we showed memory was erased by light irradiated on
CA1 neurons expressing CFL-SN through optic fiber. This method is a potential tool to know when
and where synaptic plasticity occur during sleep and memory consolidation.
Key Words：memory consolidation, Cofilin, CALI, synaptic plasticity

Introduction
Memory is retained in the hippocampus for up to one week after initial learning. It is thentransferred to the neo-cortex where it becomes permanently stored. This process is called memory
consolidation. Although it has been speculated that LTP-like mechanism during sleep is involved inthis process, there has been no information when and where it happens. We previously found thatin the initial phase of LTP, cofilin is transported to the spine and persistently accumulates at there.
Therefore, we assume that cofilin-dependent synaptic plasticity during sleep plays an important rolein memory consolidation.

Results
In order to inactivate cofilin, we employeda genetically encoded photosensitizer
protein, SuperNova. To validate thissystem, a persistent enlargement of thespine (structural LTP) was induced with
two-photo n ( 2 P ) u n cag in g o f M N Iglutamate in the single dendritic spinein the hippocampal slice culture, which
express SN-fused cofilin (CFL-SN), and559 nm laser was subsequently irradiated.
Followed by laser irradiation on the single
spine 10 min after LTP induction, the spine
volume gradually decreased. Decrease in
the spine volume was also observed up to

Erasure of sLTP by optically inactivating
cofilin(A) Structure of fusion proteins. (B)GFP image. Ten min after LTP induction byphotouncaging of glutamate, CALI was carriedout. (C)Summary.

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30 after respectively after LTP induction but not at 50 min after. Furthermore, the laser irradiation on
the spine 1 min before sLTP had no effect on the spine enlargement. The laser irradiation on the spine
without LTP induction had no effect on the spine volume. These results indicate cofilin is critical for
maintenance of LTP up to 30 min after induction, and the inactivation of cofilin had negligible effecton LTP induction and the volume of unstimulated spine .
We confirmed electrically recorded LTP
is canceled by the illumination. CofilinSN was overexpressed and field EPSP wasrecorded from stratum radiatum of CA1.
Light from Hg lamp (520-550 nm, 500 uW,
3 min) was illuminated 10 min after HFS.
After illumination, EPSP slope gradually
decreased. LTP could be induced again 40
min after CALI and potentiation of EPSP
was observed, indicating the cancellation
Electrically recorded LTP was canceled by CALI
of LTP by CALI is not due to non-specific(A)Field recording and CALI(B) The averaged
epsp.(C)Time course of slope of EPSP.
impairment in synaptic transmission.
To test this system in memory formation,
we expressed CFL-SN in hippocampal
neurons by injecting AAV-floxed-CFL-SN
into CA1 region of CaMKII-Cre mouse,
then implanted an optical fiber above
the virus injection region. Memory was
examined by Inhibitory Avoidance (IA)task. Memory was not impaired just by
overexpress of CFL-SN in CA1 neuron.
In contrast, memory was significantly
impaired when 593 nm laser light wasdelivered 2 min after an electrical shock at
day 1. On the other hand, memory was not
impaired when the light delivered 1 min
before the electrical shock. Illumination
2 hours after the shock had no effect on(A) IA test and CALI.(B)Distribution of cofilinmemory. These data are consistent with
SN expressed by AAV vector in CA1 pyramidal
those from slice culture. This new method
neurons (immunostaining). (C). Results of IA
test.
can specifically erase memory shortly (2min) after memory formation without any
effect on subsequent induction of memory.

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Discussion & Conclusion
We developed a new optical method for erasing hippocampal LTP and memory. By using thissystem, structural LTP induced in the single dendritic spine in the hippocampal slice culture
expressing CFL-SN can be cancelled by light irradiation. Furthermore, Memory in vivo can beerased by light irradiated on CA1 neurons expressing CFL-SN through optic fiber. By testing theeffect of CALI at different time points after the learning, we can identify the time window when
NMDAR-dependent LTP process takes place. We can test the involvement of LTP-like mechanism
in memory consolidation. By monitoring EEG, the timing of replay can be detected as ripple/sharp
wave. We can detect ripple/sharp wave (as both awake and sleep) and sleep status (REM or nonREM). Light can be delivered whenever the certain brain status are observed. For example, we can
induce CALI, when sharp wave is observed with delay (for example, 1 min) to test the involvementof replay in inducing LTP locally at hippocampus. In this experiment, the effect of LTP erasure will
be monitored by behavior and also EEG itself, such as frequency and amplitude of replay (bothawake and sleep).

References
Kim K, Lakhanpal G, Lu HE, Khan M, Suzuki A, Kato-Hayashi M, Narayanan R, Luyben TT,
Matsuda T, Nagai T, Blanpied TA, *Hayashi Y & *Okamoto K. (2015). A Temporary Gating of Actin
Remodeling during Synaptic Plasticity Consists of the Interplay between the Kinase and Structural
Functions of CaMKII. Neuron 87, 813-826. *: co-corresponding authors
Bosch M, Castro J, Saneyoshi T, Matsuno H, Sur M & Hayashi Y. (2014). Structural and molecularremodeling of dendritic spine substructures during long-term potentiation. Neuron 82, 444-459.

一般の皆様へ
記憶は学習後、海馬に短期的に保持されたのち皮質に移行し長期的に保存されます。これは記憶の固定化と呼ばれ、我々の日常において重要な機能です。しかしその詳細なメカニズムは分かっていません。我々は記憶の固定化において LTP（シナプス長期増強）が重要な役割を担っていると考えました。本研究では光照射によって LTP を解除する方法を開発しました。光照射によって LTP の解除することで、LTP が生じる詳細な場所と時間を知ることが可能になりました。
したがいまして本技術は、記憶の固定化の過程（特に睡眠中）のいつどこで LTP が生じて固定化が完了するかを知ることができる画期的な技術と言えます。

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Study on the Mechanism of Sex Determinationin Germline Stem Cells
Minoru Tanaka
mtanaka@bio.nagoya-u.ac.jp
Graduate School of Science, Nagoya University

Summary Abstract
Foxl3 is a switch gene acting in germline stem cells to determine the sexual fate of germ cells (tobecome eggs or sperm) in the teleost fish, medaka. The possible candidate genes downstream offoxl3 were isolated. In addition, we successfully showed that germ cells posses an innate character tofeminize the body. The character is independent of commitment of germ cells to gametogenesis.
Key Words：Germ Cells, Foxl3, Sex Determination

Introduction
Germline stem cells are the cells that produce eggs or sperm but they are sexually indifferent eventhough they are in mature ovary or testis. For the first time in vertebrates, we identified a switch
gene, foxl3, for the germline stem cells to commit to either eggs or sperm. Although understandingof the switch mechanism will definitely contribute a lot to promotion of the studies on regenerativeand reproductive medicine and animal husbandary, the detail of the switch mechanism is largely
unknown. For this purpose using teleost fish, medaka, we will focus on character of germline stem
cells and address the target genes by foxl3 in germ cells.

Results
Downstream Targets of Foxl3.
We have generated transgenic medaka that recapitulated the expression of foxl3 by fluorescence,
EGFP, and reared medaka foxl3 mutant with the transgene. The transgenic foxl3 mutant medaka does
not express the functional Foxl3 protein but the germ cells that would otherwise express foxl3 arelabeled with EGFP. With the transgenic and foxl3 mutant medaka, germ cells were isolated by a cell
sorter.
By comparing transcriptomes between germ cells with foxl3 expression and those without foxl3,
DEG(+) (transcripts upreguated in wildtype germ cells) and DEG(-)(transcripts upreguated in foxl3
mutant germ cells) genes were picked up as the candidates regulated by foxl3. The comparison
allowed us to obtain approximately, over 200 candidate genes.
To further screen the candidate genes, we have employed the iDamIDSeq method to identify thecis-element (binding sequence) of Foxl3. For this purpose, Dam-methylase gene was linked foxl3.
The RNA transcribed from the fused genes was injected into medaka fertilized eggs. One day after
injection, the methylated DNA was analyzed at the level of entire genome and compared with that incontrol embryos.
We have examined genome sequences of the candidate gene (DEG+ and DEG- genes) and selected
the genes with the cis-element. This process allowed us to obtain much smaller number of thecandidate genes.

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Next we examined if Foxl3 may bind to the cis-element in the candidate genes by a ChIP method.
Because many germ cells expressing foxl3 were required for this purpose, we used germ cells
isolated from meioC mutant. As a result, a specific binding to the cis-element could be confirmed.
Characterization of Germ Cells:
Previously we have been showing that germ cells have a feminizing effect on the body inmedaka. In other words, medaka cannot be female even if it does not have a Y chromosome. Germcells are prerequisite for developing female tissue structures. However, there are many different
developmental steps that germline stem cells have to go through (PGCs - germline stem cells – cystic
germ cells – meiotic germ cells (follicles)) to become sperm or eggs. Then the question arose which
step of germ cells are responsible for feminizing the body.
To address this question, we have generated several medaka mutants in which one of thedevelopmental steps was impaired. Initially we expected follicles to be responsible for the feminizing
power of germ cells. Unlike our expectation, medaka mutants that cannot develop follicles (figlamutant) or cystic germ cells (meioC mutant) can become female. Then we generated the mutant thatshows the defect in germ cells at an earlier step (dazl mutant). PGCs of this mutant do not develop
beyond the step of germline stem cells. Approximately a half of the XX mutant still shows normal
female.
We have also examined the feminizing role of germ cells on the process of male to female sex
reversal. Gsdf mutant exhibits male to female sex reversal. The XY medaka with mutations in bothgsdf and meioC displayed female phenotype. This indicates that germ cells are important for theprocess of male to female sex reversal.

Discussion & Conclusion
The switch mechanism that drives germline stem cells to develop into oogenesis or spermatogenesis
is critically important to promote studies on reproduction and regenerative medices. However,the details on the mechanism are not in mystery. In this study, the candidate genes regulated by
Foxl3 protein have been successfully obtained. These genes are expected to have a critical roleon the switch mechanism. In additions, we have revealed an innate character of germ cells. The
conventional view on germ cells was that germ cells were very passive cells regulated by somatic
cells and specialized cells that only develop into gametogenesis. Our results are providing a novel
view that germ cells have a positive role on feminization and, hence, germ cells are more thangametogenesis. .

References
Nishimura, T., Yamada, K., Fujimori, C., Kikuchi, M., Kawasaki, T., Siegfried, K.R., Sakai, N. andTanaka, M. Germ cells in the teleost fish medaka have an inherent feminizing effect. PLoS Genetics(2018) 14(3) e1007259 doi.org/10.1371/journal.pgen.1007259.

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一般の皆様へ
生殖幹細胞が卵になるか精子になるかの仕組み（生殖細胞の性決定）を理解することは、

基礎生物学のみならず、生殖医学や再生医学、さらには育種分野においても極めて重要である。
にもかかわらず、この仕組みはほとんどわかっていない。今回の研究により、この生殖細胞の性決定を司る遺伝子の候補を絞り込むことができた。さらには今まで知られていない生殖細胞の特性も明らかにできた。生殖細胞は単に卵や精子になるだけではなく、メスになるためには必要不可欠の細胞であり、この特性は生殖細胞が本来もつ特性（すなわち自身が卵になったり精子になったりすることとは関係ない）であることを示すことができた。

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ROR1-mediated caveolae formation and caveolar endocytosis
in lung cancer
Tomoya Yamaguchi
tyamaguchi@kumamoto-u.ac.jp
Kumamoto University

Summary Abstract
The receptor tyrosine kinase-like orphan receptor 1 (ROR1), a transcriptional target of the lineagesurvival oncogene NKX2-1/TTF-1, sustains pro-survival signaling from multiple receptor tyrosine
kinases (RTKs) in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions asa scaffold protein to facilitate interaction between CAV1 and CAVIN1, and consequently maintains
caveolae formation. Here, we report that ROR1 possesses a novel scaffold function indispensablefor efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site
distinct from those for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular
localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore,evidence for a mechanistic link of ROR1-CAVIN3 interaction as well as consequential caveolae
trafficking with RTK-mediated pro-survival signaling towards AKT in early endosomes was found inlung adenocarcinoma cells.
Key Words：Caveolae, Endocytosis, Survival signaling, ROR1, CAVIN3, Lung cancer

Introduction
We previously reported that ROR1 is a transcriptional target of the lineage-survival oncogene
NKX2-1/TTF-1 that shows frequent gene amplification and overexpression in lung adenocarcinomas,
where it sustains a favorable balance between pro-survival PI3K-AKT and pro-apoptotic ASK1-p38
signaling axes in both ROR1 kinase-dependent and independent manners. In addition, an unexpected
finding obtained in our recent study revealed that this RTK plays a role as a scaffold protein for
CAV1 and CAVIN1, facilitating their association at the plasma membrane. This kinase-independentfunction of ROR1 maintains CAV1 expression by preventing its lysosome-dependent degradation as
well as consequential caveolae formation, which in turn sustains pro-survival signaling toward AKTfrom multiple RTKs, including EGFR, MET, and IGF-IR.
In the present study, we found that ROR1 interacts with CAVIN3 and sustains caveolae-mediatedendocytosis. Furthermore, we provide evidence of a mechanistic link of the ROR1-CAVIN3
interaction with PI3K-AKT pro-survival signaling downstream of EGFR and IGF-IR in EEA1positive endosomes in lung adenocarcinoma cells.

Results
We initially analyzed the effects of siROR1 treatment on caveolae-dependent endocytosis bytreating NCI-H1975 cells with cholera toxin. In contrast to marked internalization of AlexaFluor
488-conjugated cholera toxin B in siControl-treated cells, ROR1 knockdown significantly increased
the number of cells with membrane staining and conversely decreased that of those with intracellular
staining, reflecting inhibited internalization. Recent studies have shown that CAVIN3 is localized

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for the Promotion of Sciencein both surface-associated caveolae and internalized caveolae, and plays a role in trafficking of
caveolae vesicles inside cells [1, 2]. We speculated that ROR1 may cooperate with CAVIN3 incaveolae-dependent endocytosis. Two-color immunofluorescence staining revealed the co-existenceof punctate signals of ROR1 and CAVIN3. These findings demonstrated that ROR1 and CAVIN3 colocalize in caveolae structures in lung adenocarcinoma cells, and play a role in caveolae-dependentendocytosis.
We investigated whether the CAVIN3-binding region of ROR1 is required to sustain the caveolae
structure and/or CAVIN3 subcellular compartmentalization. NCI-H1975 cells were stably
introduced with either siROR1-resistant wild-type ROR1 (WTm) or that lacking the CAVIN3binding region (ΔTK1m), then treated with siROR1. WB analysis combined with sucrose densitygradient centrifugation demonstrated a marked difference in terms of CAVIN3 subcellularcompartmentalization between siROR1-treated WTm and ΔTK1m transfectants. We then analyzed
the caveolae structure using electron microscopic observations of ultrathin sections and foundthat siROR1 treatment did not appreciably affect the number of those structures in ROR1ΔTK1m-expressing cells as compared to ROR1-WTm-expressing cells. The present findings thus
indicated that an interaction of ROR1 with CAVIN3 is required to sustain CAVIN3 subcellularcompartmentalization in caveolae, while it is not necessary for caveolae formation per se in lungadenocarcinoma cells.
It has been shown that CAVIN3 plays a role in caveolae dynamics through its interactions with
CAVIN1 and CAV1, while the association between caveolae and actin may involve the motor protein
MYO1C [3, 4, 5]. We then investigated whether deletion of the CAVIN3 binding region of ROR1
affects complex formation among CAVIN1, CAVIN3, and MYO1C. We found that unlike ROR1WTm, CAVIN3-binding-deficient ROR1-ΔTK1m could not sustain the interaction of CAVIN3 witheither CAVIN1 or MYO1C. These results suggest that the CAVIN3-binding region of ROR1 isrequired for interactions of CAVIN3 with CAVIN1 and MYO1C in lung adenocarcinoma cells.
We therefore examined which binding region(s) of ROR1 are required for caveolae-dependentendocytosis. To this end, NCI-H1975 cells were reconstituted with various mutant forms of siRNAresistant ROR1. ROR1-ΔTK1m-reconsituted cells exhibited significantly impaired uptake of CTB488 into cytoplasm, which was similarly observed in ROR1-ΔTK2+3m (the lacking the CAVIN1binding region) and ROR1-ΔS/T2m (the lacking the CAV1-binding region) cells. We also quantified
the internalization of transferrin-488 and found lack of any significant difference between ROR1WTm- and -ΔTK1m-reconstituted NCI-H1975 cells. The present findings thus clearly indicated that
ROR1 plays a role as a scaffold protein for caveolae components, including CAV1, CAVIN1, andCAVIN3, which are crucially involved in caveolae formation and caveolae-dependent endocytosis inlung adenocarcinoma cells.
Together with our previous notion of ROR1-mediated sustainment of pro-survival signaling in lungadenocarcinoma cells, the present findings led us to speculate that the CAVIN3 interacting functionof ROR1 may also be involved in this regard. NCI-H1975 and PC-9 cells were accordingly treatedwith siRNAs against either ROR1 or CAVIN3, which resulted in inhibition of proliferation to asimilar extent. We also observed that CAVIN3 knockdown significantly decreased EGF- and IGFI-induced AKT phosphorylation in NCI-H1975 cells to a degree similar to that seen with siROR1
treatment.

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Notably, reconstitution with ROR1-ΔTK1m did not rescue EGF- or IGF-I-induced AKT
phosphorylation, in contrast to that with ROR1-WTm in NCI-H1975 cells. Accumulated evidence
strongly suggests that membrane receptor signaling pathways elicit signals from various endosome
compartments [6, 7]. In proximity ligation assay (PLA), we observed that both EGF- and IGF-Istimulation resulted in readily detectable fluorescence signals with a punctate-like distribution in
ROR1-WTm-reconsituted NCI-H1975 cells, indicating robust interaction of a phosphorylated formof AKT with EEA1, a specific marker involved in signaling in early endosomes. In marked contrast,
neither EGF- nor IGF-I-stimulated interaction between EEA1 and phosphorylated AKT was elicitedin ROR1-ΔTK1m-reconsituted NCI-H1975 cells. EEA1 knockdown significantly decreased AKT
phosphorylation in a manner similar to that seen with siROR1 treatment.

Discussion & Conclusion
Recent studies have shown that CAVIN3 couples caveolae to the intracellular transport machineryby linking the CAVIN1/CAVIN3 complex to actin filaments with MYO1C. Co-localization of ROR1
and CAVIN3 on actin filaments was observed by super-resolution structured illumination microscopy,
thus the CAVIN3-binding region of ROR1 appears to be necessary for the interaction of CAVIN3
with not only CAVIN1 but also MYO1C. These findings indicate that ROR1 is a key scaffold protein
for at least 3 essential components in the caveolae complex, i.e., CAV1, CAVIN1, and CAVIN3, formaintaining caveolae formation and dynamics. However, we think that it is unlikely that ROR1 isinvariably involved in all tissues, based on its negligible expression in normal human adult tissues.
It is possible that there may be other yet unidentified molecule with similar functions in a given cellstate and lineage.
In summary, the present study revealed a novel function of ROR1 in lung adenocarcinoma cells.
ROR1 interacts with CAVIN3 and MYO1C, and has a crucial role as a scaffold protein to sustain
caveolae-dependent endocytosis and RTK-mediated pro-survival signaling towards AKT in earlyendosomes. Future studies for development of novel therapeutic strategies focused on inhibition of
the multifaceted scaffold functions of ROR1 are thus warranted, which may ultimately help to reducethe intolerable number of deaths due to this devastating cancer.

References[1] McMahon KA, et al., SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae
function. EMBO J, 28:1001-15 (2009).
[2] Hansen CG, et al., SDPR induces membrane curvature and functions in the formation of caveolae.
Nat Cell Biol, 11:807-14 (2009).
[3] Hernandez VJ, et al., Cavin-3 dictates the balance between ERK and Akt signaling. eLife, 2:e00905(2013).
[4] Echarri A, et al., Caveolae-mechanosensitive membrane invaginations linked to actin filaments. J
Cell Sci, 128:2747-58 (2015).
[5] Mohan J, et al., Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of
caveolae. J Cell Sci, 128:979-91 (2015).
[6] Le Roy C, et al., Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat
Rev Mol Cell Biol, 6:112-26 (2005).
[7] Palfy M, et al., Endosomal crosstalk: meeting points for signaling pathways. Trends Cell Biol,
22:447-56 (2012).

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一般の皆様へ
これまで私達は、リネジ生存癌遺伝子である TTF-1によって転写活性化される ROR1が、EGFR や MET、IGF-IR 等の多様な受容体の活性化の維持に寄与することで、肺腺がんにとっての重要な生存シグナルを担うことを明らかにしてきた。

本研究では、受容体としての ROR1の新たな機能に着目し、肺腺がん細胞におけるカベオラの詳細な生理機能の解明を目的とした。

本研究により、ROR1はカベオラ依存的なエンドサイトーシスによって生じたエンドソームにおいて様々なアダプター蛋白質をリクルートし、肺腺がん細胞の生存シグナルを経時的、及び空間的に制御していることが判明した。

本研究の成果は、ROR1とエンドサイトーシスに関わる CAVIN3との相互作用を標的とした、

これまでにない肺腺がんの生存シグナルを特異的に抑える独自性の高い革新的な阻害剤の開発につながると期待される。

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Regulation of energy metabolism by environmental cueand epigenome
Yoshihiro Matsumura
matsumura-y@lsbm.org
Research Center for Advanced Science and Technology, The University of Tokyo

Summary Abstract
The mechanisms by which environmental cues regulate energy metabolism in adipocytes via
epigenome are not fully understood. Genome-wide analyses of transcriptome and epigenome inadipocytes revealed that chronic cold stimuli facilitates induction of genes involved in thermogenesis
and lipid metabolism in beige adipocytes via demethylation of histone H3 lysine 9 (H3K9). On theother hand, excess glucose facilitates induction of genes involved in glucose metabolism in white
adipocytes via demethylation of H3K9 and promotes lipid accumulation.
Key Words：Metabolic syndrome, epigenome, adipocytes, metabolism

Introduction
Adipose tissues play a central role in the regulation of whole-body energy homeostasis. Dysfunctionof adipose tissues is associated with onset of metabolic syndrome and diabetes (1). White adipocytes
are specialized for fat storage as an energy source, while brown adipocytes and beige adipocytes
are specialized for fat burning and thermogenesis upon acute and chronic cold stress, respectively.
The epigenome is acquired genetic information consisted of DNA methylation and histone posttranslational modifications. Environmental cues could alter epigenome via as yet poorly understoodmechanisms and regulate adipocytes fate and function (2).

Results
To reveal the epigenetic mechanisms by which chronic cold stimuli facilitates formation ofbeige adipocytes, immortalized preadipocytes derived from stromal vascular fraction of inguinal
subcutaneous white adipose tissue from 3- to 6-week-old mice were induced for beige adipocytes
and subjected to genome-wide analysis of transcriptome by RNA-sequencing (RNA-seq). RNAseq analysis upon differentiation of beige adipocytes revealed that genes involved in thermogenesis
and lipid metabolism (e.g. Ucp1, Cidea, Cpt1b, Elovl3) are upregulated by β-adrenergic signaling
and PPARγ (Peroxisome Proliferator-Activated Receptor γ) signaling. Inhibition of β-adrenergic
signaling by propranolol or removal of PPARγ agonist prevented induction of genes involvedin thermogenesis and lipid metabolism. Epigenome analysis in beige adipocytes by chromatin
immunoprecipitation (ChIP)-PCR showed that repressive histone H3 lysine 9 (H3K9) di-methylation
on Ucp1 and Cidea is removed during beige adipocytes differentiation by pro-longed β-adrenergic
signaling and the action of histone H3K9 demethylase JMJD1A. Functional analysis of JMJD1A
revealed that β-adrenergic signaling and PPARγ signaling are integrated by phosphorylation of
JMJD1A at Ser265 via protein kinase A (PKA) to induce beige adipocyte formation. JMJD1A wasrecruited to beige-selective genes during differentiation of beige adipocytes upon activation of
β-adrenergic signaling. Phosphorylation-resistant S265A mutation prevented JMJD1A interaction

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with PPARγ, PGC-1α (PPARγ coactivator 1α), and PRDM16 (PRD1-BF1-RIZ1 homologous domain
containing 16), recruitment to beige-selective genes, histone H3K9 demethylation, induction ofbeige-selective genes, as well as beige adipocyte formation in vivo. On the other hand, phosphomimetic S265D mutation facilitated JMJD1A interaction with PPARγ, PGC1-α, and PRDM16,
induction of beige-selective genes, and beige adipocyte formation. Interestingly, in brown adipocytes,
which are responsible for acute response to cold exposure, rapid induction of genes involved in fat
burning and thermogenesis depends on PKA-phosphorylation of JMJD1A but does not depend on
epigenome alteration (i.e. histone demethylation by JMJD1A). These results indicate that pro-longed
cold stress is transmitted to adipocytes epigenome via phosphorylation of and histone demethylation
by JMJD1A to induce the formation of fat-burning beige adipocyte for chronic adaptation to cold (3).
To understand the epigenetic mechanism how extracellular glucose regulates epigenome, fate, andfunction of adipocytes, genome-wide analysis of transcriptome was performed using 3T3-L1 white
adipocyte cell line. RNA-seq analysis revealed that genes involved in glucose metabolism (e.g.
Slc2a4, Hk2, Gpi1) are upregulated in the presence of excess extracellular glucose. Furthermore,genes involved in citrate cycle enzymes as well as citrate cycle metabolites are elevated during
white adipocytes differentiation. Upregulation of genes involved in glucose metabolism depends
on elevation of key metabolites for epigenome regulation (i.e. acetyl-CoA and α-ketoglutarate).
Acetyl-CoA is required for histone acetylation reaction, while α-ketoglutarate is required forhistone demethylation reaction. Depletion of glucose or inhibition of citrate cycle by knockdownof isocitrate dehydrogenase 3β (IDH3β) prevented upregulation of genes involved in glucose
metabolism. Genome-wide analysis of epigenome in white adipocytes cell line by ChIP-sequencing
showed that repressive H3K9 di-methylation on Slc2a4 and Hk2 is removed in the presence ofexcess extracellular glucose. Inhibition of citrate cycle by knockdown of IDH3β inhibited elevationof α-ketoglutarate and prevented demethylation of H3K9 on Slc2a4 and Hk2 during differentiationof white adipocytes. These results suggest that glucose availability is transmitted to epigenome via
cellular metabolism to characterize fat-storing white adipocytes.

Discussion & Conclusion
Environmental cues alter cellular epigenome in adipose tissue and promote formation of either
beige adipocytes responsible for fat burning or white adipocytes responsible for fat accumulation
through poorly understood mechanisms. This study revealed that histone H3K9 demethylase
JMJD1A senses pro-longed cold stress via PKA-phosphorylation at Ser265 to induce the formationof beige adipocytes by integrating β-adrenergic signaling and PPARγ signaling. This study also
revealed that excess extracellular glucose alters epigenome of white adipocytes to induce genes
involved in glucose metabolism and facilitate fat accumulation. Thus, therapeutic target of Ser265phosphorylation of JMJD1A might prove to be a novel target for the treatment of metabolic
syndrome via promoting beige adipocytes formation.

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References
1) Guilherme A, Virbasius JV, Puri V, Czech MP (2008) Adipocyte dysfunctions linking obesity toinsulin resistance and type 2 diabetes. Nat Rev Mol Cell Biol 9, 367-377.
2) Inagaki T, Sakai J, Kajimura S (2016) Transcriptional and epigenetic control of brown and beige
adipose cell fate and function. Nat Rev Mol Cell Biol 17, 480-495.
3) Abe Y, Fujiwara Y, Takahashi H, Matsumura Y et al. (2018) Histone demethylase JMJD1A
coordinates acute and chronic adaptation to cold stress via thermogenic phosphor switch. Nat
Commun, in press.

一般の皆様へ
脂肪組織は全身のエネルギー代謝を担う重要な組織で、脂肪組織の機能破綻はメタボリック

シンドロームの発症と関係しています。白色脂肪細胞は脂肪を蓄える一方で、ベージュ脂肪細胞は脂肪を燃焼し熱を産生します。環境は後天的なゲノム修飾であるエピゲノムを介して、脂肪細胞の性質を決めると考えられています。本研究により、寒さの刺激は脂肪を燃焼するベージュ脂肪細胞を作り、過剰なエネルギーは脂肪を蓄える白色脂肪細胞を作ることが分かりました。

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Mitochondrial dynamics and antiviral innate immunity
Takumi Koshiba
koshiba.takumi.688@m.kyushu-u.ac.jp
Department of Biology, Faculty of Science, Kyushu University

Summary Abstract
Mitochondria act as a platform for antiviral innate immunity, and the immune system depends on
activation of the retinoic acid-inducible gene I (RIG-I) dependent signaling pathway via an adaptor
molecule, mitochondrial antiviral signaling (MAVS). In this study, we revealed that RIG-I-mediatedantiviral innate immunity requires mitochondrial dynamics.
Key Words：Mitochondria, RNA virus, innate immunity, cellular function

Introduction
Mitochondria are crucially involved in numerous cellular processes, including apoptosis andcalcium homeostasis. Mitochondria also have a unique role in innate immunity against RNA viruses.
Mitochondrial-mediated antiviral immunity depends on activation of the RIG-I signaling pathway
and MAVS, a downstream adaptor of RIG-I at the mitochondrial outer membrane (MOM), has a
key role in the signal transduction. Upon viral infection, MAVS recruits various types of effectors at
the MOM, and the orchestrated “MAVS signalosome” is the primary unit governing antiviral innate
immunity. Although the role of the MAVS signalosome in mitochondria to provide a molecular
platform that facilitates signal transduction is well characterized, insight into how the organelle
functions to facilitate antiviral immunity has remained unclear.

Results
To elucidate the molecular basis of mitochondrial dynamics coupled with the RIG-I signaling
pathway, we speculated that optic atrophy 1 (OPA1), a mediator of mitochondrial fusion, has a rolein antiviral innate immunity because of our previous evidences on Mitofusin (Koshiba et al. Sci.
Signal. 2011). As reported previously, we observed that OPA1-/- cells contained less mitochondrial
DNA (mtDNA) with completely lacked mitochondrial fusion, and had a disrupted cristae
morphology (Yoshizumi et al. Sci. Rep. 2017). These phenotypic defects were restored when a WT
OPA1 isoform gene (L-OPA1; variant 1), but not an inactive mutant K301A or short isoform (S-OPA1),
was re-introduced into null cells. Strikingly, although the OPA1-/- cells exhibited severely impaired
SeV-induced production of IFN-β and IL-6, the immune responses were substantially restored byrecovery of the mtDNA level in rescued cells (Yoshizumi et al. Sci. Rep. 2017). Given that OPA1
is important for mtDNA stability and the antiviral immune response, we used histochemical staining
for cytochrome c oxidase (complex IV) activity to directly assess the OPA1 function involved in
mitochondrial respiration. As expected, the COX staining pattern of the V1(WT)-rescued OPA1-/- cells
showed full recovery of the respiration defect, whereas K301A and S-OPA1 failed to rescue the COX
activities (Yoshizumi et al. Sci. Rep. 2017). Taken together, these results indicate that functional
actions of OPA1 are linked to the improvement of mitochondrial dynamics and the induction of
mitochondrial-mediated antiviral innate immunity.

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Discussion & Conclusion
The findings of our study using cell biological approaches provided new insight into how
mitochondria facilitate antiviral immunity through its dynamic properties. Mitochondria are
believed to have evolved from organisms such as α-proteobacterium, and the discovery of
their role in host-cell defense was unexpected. It is not surprising, however, that cells gaining
respiratory function through a symbion could greatly advance not only through enhanced energy
production, but also through safeguarded host defense against other infectious pathogens,
especially in vertebrates.

References1). Yoshizumi, T., Imamura, H., Taku, T., Kuroki, T., Kawaguchi, A., Ishikawa, K., Nakada, K., andKoshiba, T. (2017) RLR-mediated antiviral innate immunity requires oxidative phosphorylation
activity. Sci. Rep., 7, 5379.
2). 小柴琢己、今村博臣（2017）蛍光 ATP プローブによるミトコンドリア呼吸活性の評価 . 生物
物理 , 57, 268-270.

一般の皆様へ近年の研究から、ミトコンドリアには今までに知られていない新たな生理機能として、RNAウイルスに対する自然免疫に密接に関わる仕組みが備わっていることが明らかになってきた。
しかしながら、このミトコンドリアを介した自然免疫の作動はどのような機構により調節されているのか？不明な点が多く残されている。本研究では、ミトコンドリアの形態的な特徴に着目し、ダイナミックな構造特性が自然免疫応答に必要であることを明らかにした。

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Analysis of epigenetic regulation toward establishment ofa stable culture method of neural progenitor cells
Noriaki Sasai
noriakisasai@bs.naist.jp
Nara Institute of Science and Technology

Summary Abstract
During development, uncommitted progenitor cells continuously change their characteristics andgradually acquire their specific functions. The purpose of this research is to reveal the molecularmechanisms of the transition of the progenitor characters from the epigenetic point of view. We
particularly focused on a histone modification enzyme, a DNA methyltransferase and an ubiquitin
ligase, and analysed their functions by means of forced expression in chick embryos.
Key Words：Neural tube, sonic hedgehog (Shh), chromatin modification

Introduction
Any functional cells originate from the uncommitted naïve cells. The uncommitted stem cells areconverted into the progenitor state by the inductive signals and then further differentiate into thepostmitotic cells that have their unique characteristics. Importantly, the transition from uncommitted
to postmitotic state is irreversible; the postmitotic functional cells cannot revert to the lessdifferentiated state. We postulated that this irreversibility is due to the chromatin modification of
the specific regions of the genome. Based on this hypothesis, we focused on a histone modification
enzyme, DNA methyltransferase, and an ubiquitin ligase that are highly expressed during the neuraldevelopment.

Results
The neural tube, formed as a narrow tissue along the rostral-caudal axis in the dorsal part of theembryo, is the embryonic organ of the central nervous system, and is comprised of a number of typesof neural progenitor and postmitotic neurons. These cells originated from the uncommitted cells
generated in the neuroepithelium during gastrula stages, and gradually acquire unique characteristics
by signal molecules emanating from the specific regions of the neural tube. Sonic hedgehog (Shh),produced in the floor plate region, is one of such signal molecules, and forms a gradient from ventral
to dorsal. The different types of neural progenitor cells are generated depending on the concentrationof Shh. However, we have shown that the timing of the signal – when the progenitor cells receive thesignal molecule – is also important to determine the cell fate (Sasai et al., 2014). This theory is called“competence”, or how the progenitor cells respond to a signal, and is assumed as one of the criticaltheory in the determination of the cell fate as well as another theory “induction’ which represents theimportance of the kind of the signal molecules.
We hypothesized that the competence is regulated by the activities of chromatin modifying
enzymes, including DNA methyltransferases, histone modifiers and ubiquitin ligases.
First, we focused on the DNA methyl transferase DNMT3. DNMT3 per se is expressed in thewhole neural tube, but has been shown to regulate the expression of the Arx gene, expressed in the

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floor plate region of the neural tube. We inferred that DNMT3 is essential for limiting the expression
area of Arx in the floor plate, and electroporated the siRNA targeting DNMT3 in the neural tubeof the chick embryos. As the result we found the Arx gene expression aberrantly in the dorsal area,
which is supposed to express the interneuron progenitor genes.
Next we analysed the importance of a polycome-type of the transcription factor. This factor,
which we call vPTF after ventral polycome transcription factor, is expressed only in the progenitor
regions of the neural tube, and we hypothesized that this factor is involed in the maintenance ofthe progenitor state. To address this question, we performed the forced expression of vPTF in theneural tube, and consequently found the progenitor region is expanded in the postmitotic neuronalarea. These two results suggest that the chromatin modifying enzymes play critical roles in thedetermination of the differentiate state during the neural diufferentiation.
Further analysis will reveal the mechanisms by which the expression and activities of thesechromatin modifying enzymes are regulated. The possible upstream factors include kinases andscaffold proteins. We will perform a systematic screening searching for such interacting proteins
and will elucidate the whole picture of the epigenetic regulation that controls the temporal shift
in the response to the signal molecules, and consequently makes the differentiation irreversible.
Furthermore, the maintenance of the progenitor state by manipulating the chromatin state will makeit possible to culture the progenitor cells stably. The ultimate goal of this research is to develop thetechnology to maintain the progenitor state, which will generate the specific functional neurons more
efficiently.

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Elucidating the regulatory mechanism of histone modification status
in colorectal cancer stem cells to maintain stemness
and its application for drug development
Ryuji Hamamoto
rhamamot@ncc.go.jo
Division of Molecular Modification and Cancer Biology,
National Cancer Center Research Institute

Summary Abstract
In this study, we demonstrated that the histone methyltransferase SUV39H1 was significantly
overexpressed in colorectal cancer cells. Given that inhibition of SUV39H1 resulted in the growth
inhibition of cancer cells, SUV39H1 appears to be an ideal molecular target to develop anti-cancer
agents against colorectal cancer stem cells.
Key Words：Cancer stem cell, Histone modification

Introduction
In late years, the importance of epigenetics in human tumorigenesis has been attracting attention,and it is revealed that histone modifications play a central role in epigenetic alteration of cancer cells.
On the other hand, the significance of cancer stem cells in human cancer recently becomes clear; itis suggested that development of anti-cancer drugs targeting cancer stem cells is a useful tool to treat
cancer effectively. In the present study, we tried to clarify the importance of histone modifications tomaintain the stemness of cancer stem cells, and to develop anti-cancer drugs targeting cancer stem
cells,

Results
The difficulty in expanding cancer-initiating cells in vitro is one of the major obstacles for theirbiochemical characterization. Dr. Okamoto in National Cancer Center Research Institute, who
is a collaborator of this project, previously found that Rho kinase (ROCK) inhibitors greatly
facilitated the establishment of spheroids from primary colon cancer (1). Following the procedure
established, we dissected and cultivated primary cancer cells under spheroid culture conditions toisolate and expand cancer cancer-initiating cells from primary colon cancer in this study. Then we
purified cancer stem cells from operatively extracted specimens of colorectal cancer using a Rho
kinase inhibitor; Subsequently, we conducted subculture of those cells to clarify their physiologicalcharacteristics. To identify the histone modification enzyme that is specifically up-regulated incancer stem cells, we performed transcriptome analyses using the microarray. In this case, we
compared expression levels of histone modification enzymes between CD44-positive cells (cancer
stem cell group) and CD44-negative or low expressing cells (control cells), and identified that thehistone lysine methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39H1) and Protein
arginine N-methyltransferase 5 (PRMT5) were significantly overexpressed in colorectal cancer
stem cells. Since overexpression of SUV39H1 was observed in colorectal cancer stem cells from 4
different patients and we observed big differences of SUV39H1 expression levels between CD44-

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positive cells and CD44-negatibe cells, we validated its expression by quantitative real-time PCR.
Consequently, elevated expression of SUV39H1 was observed in all cases of cancer stem cells.
The Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain-containing proteins constitute themajor group of histone lysine methyltransferases. SET domain-containing proteins are characterized
by the SET domain consisting an approximately 130-140 amino acid, evolutionary well conserved
sequence motif, which was initially discovered in the Drosophila proteins Su(var)3-9, Enhancerof-zeste and Trithorax (2). In addition to the SET domain, SUV39 family proteins possess preSET (N-SET) and post-SET (C-SET) domains at the amino and carboxyl boundaries of the SET
domain respectively (3, 4). Unlike the pre-SET and SET domain, the post-SET domain does nothave some specific structure, but is proximal to the active site (5). The post-SET domain includes
three conserved cysteine residues that are reported to be crucial for histone lysine methyltransferase
activity in the SUV39 family proteins. Substitution of these cysteine residues completely abolishedthe enzymatic activity of DIM-5 and SETDB1 (6, 7). SUV39H1 is a member of the suppressor ofvariegation 3-9 homolog family and the first histone lysine methyltransferase identified. This enzyme
specifically methylates lysine 9 of histone H3 (H3K9). Trimethylation of H3K9 represents a specific
tag for epigenetic transcriptional repression by recruiting heterochromatin protein 1 (HP1) proteins
to methylated histones. Mainly functions in heterochromatin regions, thereby playing a critical rolein the establishment of constitutive heterochromatin at pericentric and telomere regions. Although
it is known that SUV39H1 functions as the organization of heterochromatin and the segregation ofchromosome, functions of the enzyme in cancer cells are still unknown. Therefore, we tried to clarify
the significance of SUV39H1 in cancer cells.
We designed small interfering RNA (siRNA) targeting SUV39H1 in order to specifically suppress
the expression of SUV39H1, and transfected the siRNA into two independent cancer cell lines withtwo control siRNAs (siEGFP and siLuciferase). Subsequently we measured viability of cancer
cell lines using Cell Counting Kit-8 (CCK8), which allows sensitivity colorimetric assays for thedetermination of cell viability in cell proliferation and cytotoxicity assays. As a result, we confirmedthat SUV39H1 inhibition repressed the proliferation of cancer cells.

Discussion & Conclusion
Our expression profile analysis clearly indicated that the histone lysine methyltransferase
SUV39H1 was significantly overexpressed in colorectal cancer stem cells. In addition, knockdownof SUV39H1 resulted in the proliferation of cancer cells. These result imply that SUV39H1 appears
to be an ideal target for the treatment of colorectal cancer, targeting on colorectal cancer stem cells.
Indeed, it is known that histone methyltransefrases and demtehylases have been attracting attention
as a novel type of target to develop anti-cancer drugs (8). Moreover, some chemical compound,which may inhibit SUV39H1, has already been developed (9).In the present study, we identified thatthe inhibition of SUV39H1 is likely to be a novel way to cure colorectal cancer, targeting colorectal
cancer stem cells. In this case, still detailed molecular mechanisms of how SUV39H1 contributesof stemness functions of colorectal cancer stem cells are unknown; therefore, we need to clarify thispoints. Further detailed molecular analysis may explore the significance of SUV39H1 in colorectal
cancer stem cells, and its importance as a target to cure colorectal cancer.

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References
1. Ohata H, Ishiguro T, Aihara Y, Sato A, Sakai H, Sekine S, et al. Induction of the stem-like cell
regulator CD44 by Rho kinase inhibition contributes to the maintenance of colon cancer-initiating
cells. Cancer research. 2012;72:5101-10.
2. Cui X, De Vivo I, Slany R, Miyamoto A, Firestein R, Cleary ML. Association of SET domain andmyotubularin-related proteins modulates growth control. Nature genetics. 1998;18:331-7.
3. Wu H, Min J, Lunin VV, Antoshenko T, Dombrovski L, Zeng H, et al. Structural biology ofhuman H3K9 methyltransferases. PloS one. 2010;5:e8570.
4. Dillon SC, Zhang X, Trievel RC, Cheng X. The SET-domain protein superfamily: protein lysine
methyltransferases. Genome biology. 2005;6:227.
5. Min J, Zhang X, Cheng X, Grewal SI, Xu RM. Structure of the SET domain histone lysine
methyltransferase Clr4. Nature structural biology. 2002;9:828-32.
6. Zhang X, Tamaru H, Khan SI, Horton JR, Keefe LJ, Selker EU, et al. Structure of the Neurospora
SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell. 2002;111:117-27.
7. Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ, 3rd. SETDB1: a novel KAP1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediatedsilencing of euchromatic genes by KRAB zinc-finger proteins. Genes & development.
2002;16:919-32.
8. Hamamoto R, Saloura V, Nakamura Y. Critical roles of non-histone protein lysine methylation inhuman tumorigenesis. Nature reviews Cancer. 2015;15:110-24.
9. Lai YS, Chen JY, Tsai HJ, Chen TY, Hung WC. The SUV39H1 inhibitor chaetocin induces
differentiation and shows synergistic cytotoxicity with other epigenetic drugs in acute myeloid
leukemia cells. Blood Cancer J. 2015;5:e313.

一般の皆様へ本研究代表者は、ヒストンメチル化異常の細胞がん化における重要性を世界に先駆けて明らかにし、その後も常に世界最先端の研究を行ってきた結果、ヒストンを含めたタンパク質メチル化は非常に多様性のある現象であることを解明してきた。一方、最近ヒトがんにおけるがん幹細胞の重要性が明らかになってきており、がん幹細胞を標的とした分子標的治療薬を開発することにより、効果的ながん治療を行うことができる可能性が示唆されている。本研究は、大腸がん幹細胞における、タンパク質メチル化の重要性を明らかにし、ヒストンメチル化関連酵素を標的とした、革新的な新規分子標的治療薬を創生することを目的として施行した。

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Lymphoma progression promoted by the breakdown of the systemfor convergence of inflammation
Yoshio Katayama
katayama@med.kobe-u.ac.jp
Hematology, Kobe University Hospital

Summary Abstract
We have identified TFL, a critical modulator for the convergence of inflammation, from a patientwith Follicular Lymphoma. In this proposed study, we found that the prognosis of murine lymphoma
model is modulated by the TFL deficiency and identified a candidate inflammatory molecule that is
critically regulated by TFL in this process.
Key Words：TFL, lymphoma, inflammation, convergence

Introduction
Malignant Lymphoma is a kind of hematologic malignancies and is characterized by the features ofinflammation such as fever, body weight loss and night sweat. The aim of this study was to examine
whether the impaired convergence of inflammation could cause lymphoma progression, particularly
by focusing on the molecule TFL.

Results
We have previously reported the translocation of the immunoglobulin light chain κ locus gene witha possible tumor suppressor gene, TFL, in transformed follicular lymphoma (1, 2). Subsequently,
we generated TFL knockout (TFL-/-) mice. Experimental autoimmune encephalitis (EAE)
induced in TFL-/- mice demonstrated persistent severe paralysis. Central nervous system (CNS)infiltrated CD4+ T cells in TFL-/- mice contained a higher proportion of IL-17 producing T cells
than TFL+/+ mice during the resolution phase of EAE. Thus, TFL is a novel gradual and persistent
posttranscriptional regulator of the inflammatory cytokines and attenuates the excessive inflammation(3, 4).
In this proposed study, we first crossed TFL-/- mice with murine lymphoma model vavP-Bcl2transgenic (Bcl2Tg) mice to generate double mutant mice. The survival of TFL-/-Bcl2Tg mice wassignificantly altered compared with TFL+/+Bcl2Tg littermate mice. We could confirm that TFL
is a critical regulator of the lymphoma progression. We are currently evaluating the pathologicalinvolvement of lymphoma in the organs of these mice.
We noticed that B lymphoid lineage cells with abnormal surface markers existed in the bonemarrow of the Bcl2Tg mice. We sorted this particular cell population and compared the gene
expression profiles between TFL+/+Bcl2Tg and TFL-/-Bcl2Tg mice. This analysis picked up
several genes which drastically changed in the absence of TFL. Among these, we noticed that thefactor X, which is well known as an inflammatory soluble factor, was included. The protein levelof factor X was extremely high in the blood of TFL-/-Bcl2Tg compared to TFL+/+Bcl2Tg mice atapproximately one year of age. We are now trying to confirm that B lymphoid lineage cells with

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The Novartis Foundation(Japan)
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To clarify whether TFL could degrade factor X via the posttranscriptional pathways, we generated
dual-luciferase reporter vectors with 3’UTR of factor X and IL-2 as a control. This vector wastransfected into the several hematopoietic cell lines including lymphoid and myeloid lineages. In thissystem, IL-2 was degraded in most cell lines by TFL. Whereas TFL degraded factor X preferentiallyin lymphoid cells.
These results suggest that TFL is a key regulator of the inflammatory feature of lymphoma, which
may influence the disease progression.
In the study of gene profiles of B lymphoid lineage cells with abnormal surface markers in thebone marrow as mentioned above, the levels of some microRNA were found to be altered as well
as factor X in the absence of TFL. TFL is known to be located at P-bodies where many microRNAs
are processed in the cell body (2). We would like to expand our research to identify the regulatory
mechanism of microRNA by TFL in the control of lymphoma.

Discussion & Conclusion
The usual treatments for malignancies are traditional cytotoxic drugs and recent molecular targeted
agents. The prognosis of lymphoma is getting better because of the combination of these therapies.
But in case of high aged patients, current therapeutic strategy is sometimes not enough or intolerable.
Targeting the inflammatory mechanism could be a novel and patient friendly therapy. Based on our
results, TFL may play a critical role to control the production of inflammatory factors, which may
compromise the patient status. We believe that TFL is a good candidate molecule as a target for anovel therapy.

References
1) Minagawa K, Yamamoto K, Nishikawa S, Ito M, Sada A, Yakushijin K, Okamura A, Shimoyama
M, Katayama Y, Matsui T. Deregulation of a possible tumour suppressor gene, ZC3H12D,
by translocation of IGK@ in transformed follicular lymphoma with t(2;6)(p12;q25). Brit J
Haematol 139: 159-167, 2007
2) Minagawa K, Katayama Y, Nishikawa S, Yamamoto K, Sada A, Okamura A, Shimoyama M,
Matsui T. Inhibition of G1 to S phase progression by a novel zinc finger protein P58TFL at
P-bodies. Mol Cancer Res 7:880-889, 20093) Minagawa K, Wakahashi K, Kawano H, Nishikawa S, Fukui C, Kawano Y, Asada N, Sato M,
Sada A, Katayama Y, Matsui T. Posttranscriptional modulation of cytokine production in T cells
for the regulation of excessive inflammation by TFL. J Immunol. 192: 1512-1524, 2014
4) Minagawa K, Katayama Y, Matsui T. TFL, a hidden post-transcriptional modulator behind
inflammation (Review article). Inflammation & Cell Signaling 1: e244 (article number), 2014

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一般の皆様へ
我々がリンパ腫の患者さんから独自に発見した TFL という分子は、炎症の収束に必須の分子でした。この分子がないと、一旦起こった炎症が収束せずに続いてしまうのです。悪性リンパ腫

は血液悪性腫瘍の一種ですが、発熱や体重減少、寝汗など全身性炎症症状が特徴です。マウス
モデルを用いた本研究で、我々はマウスリンパ腫モデルの予後が TFL 分子の有る無しで変化する
ことと、その変化に重要な TFL によってコントロールされている可能性の高い分子を見いだしました。成因のわかっていない悪性リンパ腫の進展を癌という観点ではなく炎症収束機構の破綻と捉える考え方ができる可能性を示唆しており、通常用いられている抗がん剤ではなく炎症を標的としたリンパ腫治療の可能性を探る糸口になるかもしれません。

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Role of CRIMI as a novel regulator for osteoblast functions revealed
sing osteoporotic mutant mice
Tatsuya Furuichi
m-kanda@med.nagoya-u.ac.jp
Co-Department of Veterinary Medicine, Faculty of Agriculture, Iwate University

Summary Abstract
imla mice are novel mutants that exhibit reduced bone mass. By whole exome sequencing andlinkage analysis, we identified the causative gene, Crim1, for an osteoporosis-like phenotype in imla
mice. Furthermore, we showed that Crim1 mRNA was expressed in an osteoblast cell line. There
results strongly suggest that Crim1 is involved in the regulation of osteoblast differentiation and/or
function.
Key Words：osteoporosis, mutant mice, whole exome sequencing, Crim1

Introduction
We identified a mouse mutant that exhibits microphthalmia, hindlimb syndactyly, and osteoporosis
from an ENU mouse mutagenesis screening (Fig. 1). These phenotypes were shown to be inherited
as an autosomal recessive trait. We named this mutant allele as imla (Iwate microphthalmia withlimb anomalies) and tried to identify it by whole exome sequencing.

Results
We identified 71 mutations that might change the coded protein functions by whole exome
sequencing. By searching for MGI mouse database, we knew that the phonotypes of Crim1 knockout
mice were very similar to those of imla homozygotes, suggesting that Crim1 was the causative
gene. The identified Crim1 mutation, c.418T> A, was a missense mutation that causes a Cys-to-Ser
substitution at position 140 (p.Cys140Ser). Linkage analysis showed that the two markers locatednear Crim1 region completely linked to imla phenotypes. These results strongly suggest that imla is
the Crim1 mutation, p.Cys140Ser.
Imla mice exhibited an osteoporosis-like phenotype, suggesting that Crim1 is involved in bonehomeostasis. Indeed, Ctim1 mRNA was modestly expressed in osteolastic cell line MC3TC-E1,
and Its expression gradually increased during differentiation (Fig. 2). The secretion of p.Cys140Ser
mutant protein that were over-expressed in COS7 cells was severely inhibited compared to the wildtype CRIM1 protein.

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Figures

Discussion & Conclusion
Crim1 is the causative gene for an osteoporosis-like phenotype in imla mice, and its mRNA isexpressed in osteoblast cell line. There results strongly suggest that Crim1 is involved in bonehomeostasis by regulating osteoblast differentiation and/or function.

一般の皆様へ
私たちは骨粗鬆症を発症する新規変異マウスを同定し、imla マウスと命名しました。本研究では全エクソーム解析と連鎖解析という方法を用いて、imla マウスにおける骨粗鬆症の原因遺伝子として Crim1を同定しました。Cim1は骨を作る骨芽細胞に発現しており、骨芽細胞の機能を調整することによって、骨量を制御している可能性が示されました。今後は骨量制御におけるCRIM1の役割を明らかにし、CRIM1を標的とした骨粗鬆症治療薬の創薬研究基盤の確立を目指
して、研究を展開します。

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Peroxisome biogenesis disorders: Molecular mechanism of athogenesis
in the cerebellum
Yukio Fujiki
yfujiki@kyudai.jp
Medical Institute of Bioregulation, Kyushu University

Summary Abstract
A model mouse for peroxisome biogenesis disorders (PBD) shows upregulation of a neurotrophin(NT) and a dominant-negative isoform of NT receptor, TrkB-T1, in cerebellum, where abnormal
dendritic arborization and axonal swelling of Purkinje cells are evident. In this study, we addressed
involvement of peroxisomal metabolic abnormality in upregulation of NT. We found that abnormal
cytosolic reductive state in peroxisome-deficient cells is likely responsible for the upregulation of
NT. In addition, primary culture of cerebellar neurons suggested that the malformation of Purkinjecells in PBD model mouse is caused by a combination of the elevated NT and prominent expressionof TrkB-T1.
Key Words：Peroxisome, peroxisome biogenesis disorder, neurotrophin, TrkB, cerebellum

Introduction
Peroxisome is an organelle participating in various anabolism and catabolism such as β-oxidationof very-long-chain fatty acids and biosynthesis of ether phospholipids. Patients with peroxisomebiogenesis disorders (PBDs) manifest dysmorphogenesis of central nervous system (CNS) such asabnormal arborization of Purkinje cells in cerebellum. We found that secretion of a neurotrophin (NT)was increased in peroxisome-deficient astrocytic cell line. The expression of NT and a dominantnegative isoform of NT receptor, TrkB-T1, were also elevated in cerebellum of our PBD model
mouse. We investigated how and why peroxisome deficiency leads to the elevation of NT andsubsequent dysmorphogenesis of cerebellum. (99 words)

Results
Secretion of NT is elevated in the peroxisome-deficient RCR-1 cell, an astrocyte-like cultured
cell line derived from rat embryonic cerebellum. To investigate how deficiency of peroxisomebiogenesis in RCR-1 cells enhances the NT expression, several peroxisome metabolisms including
synthesis of plasmalogens and fatty acid β-oxidation were assessed. Restoration of plasmalogens inperoxisome-deficient RCR-1 cells by the treatment with hexadecylglycerol did not alter the elevated
NT expression. Similarly, the deficiency of peroxisomal fatty acid β-oxidation generated in RCR1 cells by knockdown of acyl-CoA oxidase 1 catalyzing the first step of β-oxidization showed noincrement of the NT mRNA level. Taken together, these results suggested that the upregulation of
NT mRNA level is not induced by the depletion of plasmalogens and the impaired peroxisomal
fatty acid β-oxidation. Cytosolic redox potential in the peroxisome-deficient cells is more reductive
than normal cells (Yano et al., 2010). Interestingly, NT expression was lowered by treatment with
3-amino-1,2,4-triazole, a catalase inhibitor, of the peroxisome-deficient RCR-1 cells but not control
cells. Accordingly, these results suggested that abnormal cytosolic redox state is likely involved in
the elevation of NT expression.

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Our PBD model mouse, Pex14ΔC/ΔC BL/ICR mouse established here, manifests the malformation of
cerebellum including abnormal arborization and axonal swelling of Purkinje cells. NT expression iselevated around Purkinje cell layers of cerebellum in the Pex14ΔC/ΔC BL/ICR mouse. To investigate
whether the malformation of the cerebellum in Pex14ΔC/ΔC BL/ICR mice is a consequence of theelevated NT protein level, we performed primary culture of cerebellar neurons in the absence orpresence of NT. Purkinje cells with swollen axons were more frequently detected upon treatment
with NT of the cells from Pex14ΔC/ΔC BL/ICR mouse. Moreover, the development of dendrites in
the Pex14-defective Purkinje cells was affected in the presence of NT. In the wild-type Purkinjecells, the axonal formation and dendritic arborization were not altered by the NT treatment. Suchdysmorphologies of Purkinje cells in vitro were in good agreement with those observed in the
cerebellum of Pex14ΔC/ΔC BL/ICR mouse. Taken together, these results suggested that an elevatedlevel of NT induces the dysregulation of NT signaling pathway only in the peroxisome-deficient
Purkinje cells, giving rise to the axonal swelling and the abnormal dendritic arborization. In the
cerebellum of Pex14ΔC/ΔC BL/ICR mice, TrkB-T1 is significantly elevated relative to that in the wildtype mice. Therefore, the malformation of Purkinje cells in Pex14ΔC/ΔC BL/ICR mouse is most likely
due to a combination of elevated NT and prominent expression of TrkB-T1 (Fig.1).
We investigated how the protein level of NT is elevated in cerebellum of Pex14ΔC/ΔC BL/ICR mouse.
NT mRNA level was not altered in the cerebellum, suggesting that cerebellar cells are less likely
involved in the upregulation of NT in Pex14ΔC/ΔC BL/ICR mouse. In contrast, mRNA and protein
levels of NT were upregulated in inferior olivary nucleus (ION) of Pex14ΔC/ΔC mutant mice. Given
these results together with the fact that climbing fiber projects from ION to Purkinje cells, we suggestthat the elevated NT protein in the Purkinje cell layer is most likely delivered via climbing fibers (Abeet al., 2018).

Fig.1 Schematic model of malformation of Purkinje cells caused by peroxisome biogenesis
deficiency. Peroxisome biogenesis deficiency leads to upregulation of TrkB-T1 in Purkinje cells
and elevation of NT in inferior olivary nucleus under reductive condition of cytosol. Excess amountof NT is secreted via climbing fiber, followed by abnormal development of dendrites and axonal
swelling of Purkinje cells.

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Discussion & Conclusion
In the developmental stage of the cerebellum, NT immunoreactivity is evident around Purkinjecells, whereas expression of NT mRNA is undetectable. We, therefore, suggest that the NT proteinin the Purkinje cell layer is delivered via climbing fibers from the ION, where NT mRNA is strongly
expressed. Indeed, the expression of NT mRNA is elevated in the ION neurons of Pex14ΔC/ΔC BL/
ICR mouse. On the other hand, Purkinje cells in Pex14ΔC/ΔC BL/ICR mouse predominantly express
TrkB-T1, which induces dominant-negative inhibition of NT signaling. In the primary culture of
cerebellar neurons, the dendritic development of Purkinje cells from Pex14ΔC/ΔC BL/ICR mouse iscompromised and swollen axons were increased by the NT treatment. Therefore, a combination ofelevated NT from ION neurons via climbing fiber and prominent expression of TrkB-T1 in Purkinjecells is likely responsible for the malformation of Purkinje cells (Fig.1). Further studies would be
required to elucidate the molecular mechanisms underlying the transcriptional upregulation of the NT
and TrkB-T1 in the ION and the Purkinje cells, respectively, of Pex14ΔC/ΔC BL/ICR mouse.

References
Yano, T., M. Oku, N. Akeyama, A. Itoyama, H. Yurimoto, S. Kuge, Y. Fujiki, and Y. Sakai.
2010. A novel fluorescent sensor protein for visualization of redox states in the cytoplasm and inperoxisomes. Mol. Cell. Biol. 30:3758-3766.
Abe, Y., M. Honsho, R. Kawaguchi, R. Itoh, M. Fujitani, K. Fujiwara, M. Hirokane, T. Matsuzaki,
K. Nakayama, R. Ohgi, T. Marutani, K.I. Nakayama, T. Yamashita, and Y. Fujiki. 2018. Peroxisomebiogenesis deficiency attenuates the neurotrophin-TrkB pathway-mediated development of
cerebellum. “Submitted for publication”.

一般の皆様へ
細胞小器官ペルオキシソームの形成障害は、大脳や小脳における神経発生・分化障害・神経変性を特徴とする先天性致死疾患ペルオキシソーム欠損症をもたらすが、その発症メカニズムは不明である。我々は病態発症の分子機構解明を目的として、ペルオキシソーム欠損症モデル

マウスを作出し、検証を進めるなかで小脳における神経栄養因子 NT の発現量増加を見出している。また、このモデルマウス小脳の神経初代培養実験により、過剰な NT が小脳プルキンエ細胞の形態異常を導くことやサイトゾルの還元化がその発現量増加に関与することを明らかにした。これらの研究成果は、未だ治療法が確立していないペルオキシソーム欠損症の治療法開発にも繋がるものと期待される。

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Optically transparent bamboo-cellulose/collagen nanocomposite
having antibacterial action
Yoko Okahisa-Kobayashi
okahisa@kit.ac.jp
Kyoto Institute of Technology

Summary Abstract
The potential of bacterial (Staphylococcus aureus) and fungal attacks (Corious versicolar andTyromyces palustris) against moso bamboo shoot skin (Phyllostachys pubescens Mazel) extracts
were evaluated. Dichloromethane and hexane extracts from bamboo shoot skin had antibacterial
activity, and Dichloromethane extracts shown growth inhibitory effect against Tyromyces palustris.
Cellulose nanofibers could be obtained from bamboo shoot skin. These results suggest the possibilityof production of nanocomposite having antibacterial action from bamboo shoot skins that are mainly
discarded at present.
Key Words：Bamboo shoot skin, cellulose, nanofiber, antibacterial activity

Introduction
Bamboo grows widely in zones with a wet season ranging from the tropics to temperate areas andan attractive material due to their rapidity of growth. They are fast-growing renewable biomass
and some bamboo species may provide higher biomass yield than dedicated energy crops such as
corn stover, switchgrass or miscanthus. This study focused on moso bamboo shoot skins for the
production of cellulose nanofiber having antibacterial activity.

Results
In this experiment, we evaluated theantibacterial and anti-fungal activity of extracts
from moso bamboo shoot skin. Moso bamboo
shoot skins were serially extracted withn-hexane, dichloromethane, and methanol for 24
h at 180 rpm.
The results of antibacterial assay are shownin fig. 1. The cfu of dichloromethane extract
was <10, n-hexane was 1.0 x 10, methanol
was 9.0 x 10, and control was 1.2 x 106. The
dichloromethane and n-hexane extract showedstrong antibacterial activity.
Fig.1. Antibacterial assay of bamboo
The results of anti-fungal assay are shown
shoot extract of control(A), n-hexane(B),dichloromethane(C),in Fig.2. The dichloromethane extracts shown
methanol (D).
growth inhibitory effect against Tyromyces
palustris. However, there was no effect on
Corious versicolar.
Finally, we successfully obtained cellulose nanofiber from bamboo shoot skin.

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for the Promotion of ScienceFig.2. Antifungal assay (growth rate) of bamboo shoot extract against
Tyromyces palustris (left) and Corious versicolor (right)

Discussion & Conclusion
It was reported that 2,6-dimethoxy-pbenzoquinone was isolated from the skin of bamboo trees as
an antibacterial compound (1). It was also reported that bamboo shoot skin has antibacterial activity
against S. aureus (2). However, 2,6-dimethoxy-p-benzoquinone was not detected in extracts ofbamboo shoot skin by high-performance liquid chromatography analysis (2). It was suggested that
antibacterial compounds other than 2,6-dimethoxy-pbenzoquinone are present in bamboo shoot skin.
In this study, we found that bamboo shoot skin has anti-fungal activity against T. palustris not only
S. aureus. Moreover, we could obtain cellulose nanofiber from residues after extracting the active
compounds of bamboo shoot skin. Currently, bamboo shoot skins are either burned or composted.
This study indicates the possibility of bamboo shoot skins as antibacterial nanofiber materials.

References(1) Nishina A, Hasegawa K, Uchibori T, Seino H, Osawa T (1991) 2,6-Dimethoxy-p-benzoquinone
as an antibacterial substance in the bark of Phyllostachys heterocycla var. Pubescens, a species ofthick-stemmed bamboo. J Agric Food Chem 39:266–269(2) Tanaka A, Hyo Jung Kim H, Oda S, Shimizu K, Kondo R (2011) Antibacterial activity of moso
bamboo shoot skin (Phyllostachys pubescens) against Staphylococcus aureus. J Wood Sci 57:
542-544

一般の皆様へ
竹材の表皮や筍皮の細胞には抗菌性があることが経験的に知られており、古来より食品の保存や薬品などに用いられてきました。本研究では、このような竹の持つ抗菌・抗酸化成分を利用した抗菌ナノセルロース医療用材料の創製を目的に、抗菌成分の抽出、活性試験、および抽出残渣からのセルロースナノファイバー製造を行いました。その結果、抽出した成分からは黄色ブドウ球菌のようなバクテリアのみならず、真菌類への阻害効果が示唆されるなど、新たな効果が発見され、また抽出残渣からはセルロースナノファイバー製造に成功しました。高い生体適合性を有しながらも機械的特性の低さからこれまで利用が制限されてきたコラーゲンなどの医療用ゲル材料補強に、今回得られた筍皮由来抗菌成分およびナノファイバーを用いることで、幅広い用途展開につなげて行きたいと考えています。

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Establishment of therapeutic genome editing techniques applicablefor post-mitotic neurons
Izumi Oinuma
izumi@sci.u-hyogo.ac.jp
Graduate School of Life Science, University of Hyogo

Summary Abstract
The goal of our research is to develop a novel therapeutic strategy to activate the potential ofneurons to elongate their nerve fibers and to re-build a new neural network in the injured central
nervous system (CNS). In the adult mammalian CNS, it is well known that injured nerve fibers
exhibit very limited regeneration ability. The evidence is now emerging that such inability ofregeneration is not simply due to poor intrinsic regeneration ability of adult neurons, but rather due
to the inhibitory environment. If such inhibition can be overcome, nerve regeneration would begreatly enhanced. In this research project, we have established genome editing techniques applicablefor activation of intrinsic nerve growth-promoting factors of post-mitotic neurons.
Key Words：nerve regeneration, genome editing, gene therapy, central nervous system

Introduction
Precise wiring patterns are one of the remarkable features of neurons, and establishment of theproper neuronal network requires control of synapse formation. Fluorescence imaging of spatiotemporal localization of synaptic proteins at single-cell resolution in highly complicated neuralnetworks is prerequisite for analyzing the cell biological mechanisms of synaptogenesis. Ectopic
expression of fluorescent-tagged proteins is a commonly used, simple and easy way. However,
it is unsuitable for observing synaptic proteins, because it tends to cause alterations in synaptic
morphology and plasticity, due to aberrant localization and amount of the expressed proteins. Several
methods other than ectopic expression, including generation of recombinant antibody-like probes
or conditional knock-in mice sparsely expressing fluorescently tagged proteins, have been recently
developed. However, there has been no method that provides high-throughput tools for examinationof various endogenous synaptic proteins in primary cultured post-mitotic neurons.

Results
In tis study, by combining the CRISPR/Cas9-mediated genome editing technology withelectroporation techniques, we tried to modify endogenous proteins of interest in vivo in themouse retina and brain and in vitro in the rat neuron primary cultures by inserting the reportertagged cDNAs into the target gene loci. Unlike cultured cell lines which can proliferate infinitely,or fertilized eggs which give rise to adult bodies after many cell divisions, neurons in vivo do notclonally expand. Therefore, it is almost impossible to use the correctly targeted neurons for further
studies after genotyping. To overcome this problem, we planed to develop a method to identify
the correctly targeted neurons without genotyping. We focused on the fact that there is often no
promoter activity around the exons encoding the C-termini of proteins. We expected that the knockin targeting vectors, which correspond to the genomic DNAs encoding the C-termini of proteins, do

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not express fluorescent reporters by themselves, but that the reporter genes are turned on when thetargeting vectors are correctly integrated into the genome by CRISPR/Cas9-mediated homologous
recombination.
Through several optimizations, our results show that the efficiency of CRISPR/Cas9-mediated geneknock-in in the rodent neural tissues is unexpectedly high, reaching up to ~10% of the transfected
cells at some gene loci, and that the “C-terminal fusion approach” worked well with few falsepositive signals.
Optimization of in vivo gene targeting technology in the mouse brain
To examine if the genome-editing technology can be optimized to be applicable to neurons in the
mammalian brain, we performed optimization of gene targeting technology to modify endogenous
proteins in vivo in the mouse brain. A post-synaptic protein, PSD-95 was chosen as a target, and the
CRISPR gRNA was designed to target the genome sequence around the stop codon of the PSD-95
gene. The targeting vector was designed to express a PSD-95-mClover C-terminal fusion proteinfrom the endogenous locus. Embryonic day 14 (E14) mouse brains were co-electroporated in utero
with PSD-95 targeting vector (mClover), CAG-tdTomato, and the CRISPR construct (CAG-Cas9/
U6-gRNA (PSD-95)). When the transfected brains were harvested and examined at adult (Postnatal
day 65), a PSD-95-mClover fusion protein were detected only in the presence of the CRISPR
construct, showing that endogenous PSD-95 was correctly tagged with mClover in the mouse brain
by in vivo gene targeting.
Optimization of in vitro gene targeting technology in the rat neuron primary cultures
To examine if the genome-editing technology can be optimized to be applicable to in vitro
differentiated neurons prepared from the mammalian brain, we challenged to modify endogenous
synaptic proteins in neuron primary cultures prepared from E18 rat cortex and hippocampus. PSD95 and a pre-synaptic protein, Synaptophysin (Syp), were chosen as targets, and the CRISPR gRNAs
were designed to target the exons containing the stop codons of the PSD-95 and Syp genes. The
targeting vectors were designed to express PSD-95-mCherry and Syp-EGFP fusion proteins fromthe endogenous loci. We observed CRISPR/Cas9-dependent gene knock-in at both gene loci inthe neuron primary cultures prepared from rat cortexes and hippocampi. Fluorescent signals weredetected only in the presence of the CRISPR construct, and their expression patterns were identicalto those of endogenous PSD-95 and Syp detected with the antibodies.

Discussion & Conclusion
At present, there have been no reports demonstrating the regeneration of nerve fibers from theinjured mature neurons in the central nervous system (CNS) by direct gene therapy. If the strategy
developed in this research project works and planned nerve fiber regeneration successfully occurs, itwill overturn the old dogma that CNS neurons cannot be regenerated in the adult mammalian brain.
It will further provide new insight into a non-invasive strategy in regenerative medicine without theneed for cell transplantation.

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References
Daiki Ohama, Takahiko Matsuda, and Izumi Oinuma (2018)
Differential regional and subcellular localization patterns of afadin splice variants in the mouse
central nervous system. Brain Research ( 印刷中 )一般の皆様へもし、本研究期間に至適化に成功した成熟後神経細胞へのノックイン手法により中枢神経回路の可塑性ともいえる再生現象が引き起こされれば、「中枢神経自らは再生しない」という科学的常識を覆し、生物学の教科書にある「常識」を書き換えるほどのインパクトを与えるともに、幹細胞移植に依らない非侵襲的再生治療法の実現のための基盤研究として、iPS 細胞開発以来の大きく、新たな視点が与えられる。

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Vascular intraluminal pressure-dependent switching machineryof ngiogenic mode
Koichi Nishiyama
nkanako@kumamoto-u.ac.jp
International Research Center for Medical Sciences, Kumamoto University

Summary Abstract
This research aimed to clarify a mechanobiological machinery for regulating angiogenesis in a
vascular intraluminal pressure-dependent way. Through the research, 1) we found that perivascular
pericytes might potently switch on and off angiogenesis by controlling vascular intraluminal pressureload, and 2) identified a mechanism of how vascular intraluminal pressure inhibits angiogenic branchelongation by endothelial cells.
Key Words：Angiogenesis, Mechanobiology, Intraluminal pressure

Introduction
Angiogenesis is a vital reaction to increase new blood vessels. It potently contributes not only
to tissue construction and homeostasis but also to the pathogenesis of various diseases. Bloodvessels have a lumen structure, and thereby the vascular walls are constantly exposed to mechanicalstimuli by blood flow. However, it still remains unclear whether or not and how blood flow affects
angiogenesis. Just recently, we identified that vascular intraluminal pressure load could cause
inhibition of angiogenesis at a certain condition. This research aimed to clarify a mechanobiologicalmachinery which regulates angiogenesis in a vascular intraluminal pressure-dependent way.

Results
We identified that vascular intraluminal pressure load could inhibit branch elongation during
endothelial cell driven angiogenesis so far. On the other hand, a recent report showed that bloodflow enhanced lumen formation (Ref. 1). From two findings, we hypothesized that there is astutemachinery that can switch two different angiogenic modes, branch elongation and lumen formation,by spatiotemporally controlling vascular intraluminal pressure. Especially, this research focused toclarify how exposure to vascular intraluminal pressure load is controlled in angiogenic endothelial
cell sprouts during the branch elongation mode.
1) Analyses using an in vivo murine retinal angiogenesis model
We assumed a possibility that emerging vessels structurally avoid excessive exposure to vascular
intraluminal pressure. First we checked the pattern of vascular lumen formation in emerging vesselsof P4 mice retina by immunohistochemically detecting lumen with ICAM2 and endothelial sprouts
with CD31. We found that lumen was not formed continuously from the proximal lumen. Instead,once vascular lumen in the angiogenic front was formed isolated from the proximal lumen where
blood flows, and it then fused to the proximal one. The result suggests that a possible mechanism to
avoid excessive intraluminal pressure load in just emerged branch.

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Next we checked whether or not pericytes contribute to the characteristic pattern formation ofvascular lumen in the emerged branch. We inhibited pericyte coverage over endothelial sprouts of P4
mice retina using anti-PDGFRbeta neutralizing antibody as previously reported (Ref. 2). We foundthat vascular lumen formation was not significantly changed in the absence of pericyte coverage.
However, vascular outgrowth was delayed and the vasculature was dilated, suggesting that pericyte
might contribute to control of vascular intraluminal pressure load in other ways.
2) Analyses using an in vitro angiogenesis model using microfluidic device
One of the reasons why blood flow-dependent mechanisma of angiogenesis regulation remained not
addressed was due to lack of useful methods. Therefore, we firstly developed an in vitro analyzing
system to assess impacts of vascular intraluminal pressure load on angiogenesis by applying a
recently established original assay system with a microfluidic technology (Ref. 3, 4). Using this new
system, we have ever identified that hydrostatic pressure load inhibits angiogenic branch elongationof endothelial cells.
From in vivo results, pericytes are supposed to contribute to control of vascular intraluminalpressure load. Therefore, we checked the effect of pericyte addition on in vitro angiogenesis upon thehydrostatic pressure load. We found that pericyte addition could mostly cancel hydrostatic pressureload-dependent inhibition of branch elongation, suggesting that pericyte regulates angiogenesis atleast via control of vascular intraluminal pressure load.
Finally, we addressed to dissect molecular and cellular mechanisms governing vascular intraluminalpressure-dependent inhibition of angiogenic branch elongation by endothelial cells. We found that
vascular intraluminal pressure generates vascular wall tension, which induces adequate F-actin
formation failure and subsequent antero-posterior polarity loss in angiogenic endothelial cells.
Eventually, endothelial cells failed to elongate the branch due to loss of directed migration. We also
checked contribution of YAP/TAZ, a well known molecular transducer of mechanical stimuli, but theexpression pattern in angiogenic sprouts was not significantly affected by hydrostatic pressure load.

Discussion & Conclusion
In this research, we found pericyte to be a possible controller, which switches angiogenic modeby regulating vascular intraluminal pressure load to endothelial cell vascular wall. This seems atleast not due to spatiotemporal regulation of vascular lumen formation. Furthermore, we found an
inhibitory role of vascular intraluminal pressure on angiogenesis, in which vascular wall tension
causes failure of directed collective endothelial cell migration. Although in the present research
it remains uncovered the mechanism of how pericyte controls vascular intraluminal pressureload, the present findings provide new insights to angiogenic mechanism from the point of viewmechanobiology and also provide a basis to develop epochal diagnostic tools and therapeuticstrategies those target angiogenesis in various diseases.

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References
1. Gebata V., et al. Nat Cell Biol 18(4):443, 2016
2. Ogura S., et al. JCI insight 2(3):e90905, 2017
3. Nashimoto Y., et al. J Vis Exp 134:e57242, 2018
4. Nashimoto Y., et al. Integr Biol 7(6):e007800, 2018

一般の皆様へ
血管新生は、もともとある血管から新しく血管をつくりだす現象である。我々の体が作られる

時のみでなく、その後の修復過程や様々な病気の発症や進行時に見られる極めて重要な現象

であり、その全貌を十分に理解する必要がある。今回の研究では、血管内腔を流れる血液（血流）により生じる内腔圧が血管新生を抑制するメカニズムを明らかにした。また、血管をとりまく細胞ペリサイトが血管内腔圧の作用を調節することで血管新生を制御する新しい血管新生メカ
ニズムが示唆された。本研究は、血管新生の理解に貢献すると共に、血管新生を標的とする
新たな治療法創生に資する。

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Novel therapeutic strategy for heart failure targeting PKG/TORC
signaling and its intracellular localization.
Taishi Nakamura
taishin@kumamoto-u.ac.jp
Kumamoto University Hospital

Summary Abstract
PKG1α can inhibit mTOR complex in redox manner via new phosphorylation site on TSC2, bywhich preventing PKG1α oxidation blunts pathological hypertrophy.
Key Words：cyclic GMP, redox regulation, cardiac hypertrophy, mTOR

Introduction
Cyclic GMP-dependent protein kinase (PKG1α) transduces NO- or natriuretic peptide-related
signaling to regulate physiological processes in cardiovascular system 1,2. The primary enzyme
activation occurs when cGMP binds to the regulatory domain 3. However, it’s recently reported that
PKG can be also stimulated by oxidation at C42, forming an intramolecular disulfide bond 4,5. We sought
to examine the role of PKG redox modulation on cardiac hypertrophy and heart failure, focusing on
TSC2 (tuberin), a new candidate of PKG substrate, which negatively regulates mechanistic target of
rapamycin (mTOR) signaling.

Results
We have shown that preventing C42 oxidation in PKG1α can blunt adverse cardiac remodeling
induced by cardiac pressure overload 6 . Our recent phospho-proteome analyses with rat
cardiomyocytes revealed a new PKG phosphorylation site on TSC2, so we decided to elucidate theunknown function in the development of pathological cardiac remodeling. TSC2, an upstream master
repressor of TORC1, is known to be enhanced by AMP-activated protein kinase (AMPK) or glycogensynthase kinase-3β(GSK-3β), whereas can be suppressed by Akt or ERK1/2 activation 7. We thereby
hypothesize that the PKG phospho-site potently activates TSC2 nearby GSK-3β phosphorylationsite. To test the hypothesis, we first checked if the downstream of mTORC1 signaling was modulated
in neonatal rat cardiomyocytes expressing either redox insensitive PKG1α (PKG1αC42S) or wild-type
PKG1α (PKG1αWT). The results showed endothelin-1 (ET1) stimulation increased phosphorylationlevels in p70S6K and elF4E binding protein-1(4EBP1) in cells expressing PKG1αWT, while thelevels were significantly blunted in ones expressing PKG1αC42S despite no remarkable changes
in the upstream signaling such as Akt, ERK1/2, AMPK, or GSK-3β. These results indicate thatsome unknown upstream factors including TSC2 play an important role to substantially mediate
mTORC1 signaling pathways to regulate balance of protein synthesis and the recycling. We also
made sure ET1-induced fetal gene upregulations such as Nppb and regulator of calcineurin-1 werealso attenuated in cells expressing PKG1αC42S compared to PKG1αWT, which findings corroborate
the critical role of PKG/TSC2/mTORC1 to blunt pathological cardiac hypertrophy in redox manner.
We produced the mouse model harboring TSC2 phosphorylation mutation at PKG site in order tocheck the reproducibility in vivo as well. We suppose that examining in vivo study with the valuable

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mice will provide the significance of phosphorylation site on mTORC1 regulation and the antihypertrophic effects against cardiac pressure overload.
We have shown PKG redox modulation can control its localization and the targeting. PKG normally
distributes throughout the cell at rest. However, it acutely moves to outer plasma membrane after
stimulation upon hormone or mechanical stress. Preventing PKG oxidation enables to remain there
after the translocation, whereas oxidized PKG stays in the cytosol. We tested if PKG localization
can modulate autophagy in neonatal rat cardiomyocytes expressing PKGWT or PKGC42S. The confocal
immunofluorescence so far showed cells expressing PKG1αWT substantially inhibits autophagyproteolysis followed by mTOR hyper-activation and thus more hypertrophy, recapitulating our invitro results.

Discussion & Conclusion
In cell model, we demonstrated that pathological hypertrophy signaling was enhanced with ET1
stimulation and that the responses were accompanied by mTORC1 activation along with an enhanced
protein synthesis and the decreased degradation. We also found the consequent responses were
attributed to the inhibition of negative regulator TSC2 which was the novel PKG target. Moreover,
PKG appeared to activate TSC2 by phosphorylation at unknown site S1365 in redox-dependent
manner. These results should be finally confirmed in vivo mouse model such as phospho-mimeticor phospho-silenced TSC2 mutants exposed to sustained cardiac pressure overload. Our study andthe findings would provide helpful therapeutic strategy for heart failure through novel PKG/TSC2/mTORC1 signaling pathway.

References
1. Frantz S, et al. Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted
inactivation of cyclic GMP-dependent protein kinase I. Eur Heart J. 2013;34(16):1233–1244.
2. Kukreja RC, Salloum FN, Das A. Cyclic guanosine monophosphate signaling andphosphodiesterase-5 inhibitors in cardioprotection. J Am Coll Cardiol. 2012;59(22):1921–1927.
3. Kim JJ, Lorenz R, Arold ST, Reger AS, Sankaran B, Casteel DE, Herberg FW, Kim C. Crystal
structure of PKG1: cGMP complex reveals a cGMP-mediated dimeric interface that facilitatescGMP-induced activation. Structure. 2016;24(5):710-720
4. Burgoyne JR, et al. Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science.
2007;317(5843):1393–1397.
5. Prysyazhna O, Rudyk O, Eaton P. Single atom substitution in mouse protein kinase G eliminates
oxidant sensing to cause hypertension. Nat Med. 2012;18(2):286–290.
6. Nakamura T, Ranek MJ, Lee DI, Shalkey Hahn V, Kim C, Eaton P, Kass DA. Prevention of
PKG1α oxidation augments cadioprotection in the stressed heart. J Clin Invest. 2015;125(6):246872
7. Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, Cantley LC, Manning BD.
Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at thelysosome. Cell. 2014;156(4):771-85

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一般の皆様へサイクリック GMP 依存性プロテインキナーゼ (PKG) の活性は、サイクリック GMP の結合により惹起される従来の調節機構以外に、酸化還元 ( レドックス ) によるサイクリック GMP 非依存性の調節機構がわかり、心血管における意義が注目されている。本研究は、細胞の成長や増殖、生存を調節する哺乳類ラパマイシン標的蛋白質 (mTOR) のシグナルを抑制するチュベリン(TSC2) 上に、新規の PKG リン酸化部位を見出し、病的心肥大における同部位のリン酸化の意義を明確にすることを目的にした先駆的な研究です。本研究成果を契機に、心不全治療における新たな分子標的戦略の発展が期待されます。

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Virus-mediated suppression of host innate immune response
Hiroyuki Oshiumi
oshiumi@kumamoto-u.ac.jp
Department of Immunology, Faculty of Life Sciences, Kumamoto University

Summary Abstract
The innate immune system is a first line of defense against viral infection. On the other hand,
many viruses have evolved to escape host innate immune response. In this study, we focused on thehost and virus interaction, and revealed new molecular mechanism of how virus escape host innate
immune response.
Key Words：Innate immunity; virus

Introduction
The innate immune system is essential for protection against viral infection especially at earlyphase of viral infection (Oshiumi H et al 2016). However, many viruses have evolved to escapehost innate immune response. Previously, we have revealed that extracellular vesicles (EV) released
from hepatocytes infected with hepatitis B virus (HBV) suppressed the innate immune responseof macrophages (Kouwaki T et al 2016). But, underlying mechanism of EV-mediated suppression
remains elusive. In this study, we focused on the molecular mechanism and found that microRNAs
within EVs were important for the suppression.

Results
Extracellular vesicles (EVs) were collected from cell culture medium of hepatocytes infected withor without hepatitis B virus (HBV). We determined microRNA expression levels in collected EVs
and found that miR-21 and miR-29a expression levels in EVs were increased by HBV infection.
miR-21 and miR-29a are known to suppress IL-12 expression. Thus, we investigated whether
EVs released from HBV-infected hepatocytes could reduce IL-12 expression in macrophages.
Interestingly, we found that IL-12 expression of macrophages was reduced by adding EVs into cell
culture medium.
To reveal the underlying mechanism, we examined whether EVs delivered those microRNAs into
macrophages. RNAs within EVs were stained with SYTO RNA select dye, which is a membranepermeable dye and emits green fluorescence. EVs stained with the dye were added to cell culture
medium of macrophages, and then cells were incubated for 6 hrs. Interestingly, we observed green
fluorescence at the cytoplasm of macrophages, suggesting that RNAs within EVs were transferred tothe macrophage cytoplasm.
Considering that EVs deliver RNAs into the cytoplasm of macrophages, we reasoned that
intracellular microRNA levels would be increased after incubation of macrophages with EVs.
As expected, we observed significant increase of intracellular miR-21 and miR-29a levels after
incubation of macrophages with EVs. Collectively, these data suggest that EVs deliver miR-21 and

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for the Promotion of SciencemiR-29a from HBV-infected hepatocytes to macrophages, thereby suppressing IL-12 expression in
macrophages.
Considering that HBV increased miR-21 and miR-29a levels in EVs, it is expected that viralproteins increased miR-21 and miR-29a levels. To test this hypothesis, viral proteins were overexpressed in hepatocytes, and microRNA levels in EVs were subsequently determined. As expected,
we could determine viral proteins that increased miR-21 and miR-29a levels in EVs.
To test the effect of host innate immune response on miR-21 and miR-29a expression in EVs, weinvestigated whether stimulation with pathogen-associated molecular patterns (PAMPs) increased
miR-21 and miR-29a expression in EVs. PolyI:C, CL097, and non-methylated CpG DNA are PAMPs
and are recognized by TLR3, TLR7, and TLR9, respectively. We have previously shown that TLR3,
TLR7, and TLR9 are involved in the recognition of HBV. We found that TLR3 could increase miR21 levels. But, TLR7 and TLR9 failed to increase the expression. Although TLR3 increased miR-21
levels in EVs, the viral protein-mediated increase of miR-21 and miR-29a expression in EVs weremore significant. Taken together, our data suggest that viral proteins play a major role in the increaseof miR-21 and miR-29a expression levels in EVs released from HBV-infected hepatocytes.
Sorting of microRNA into EVs requires several proteins. hnRNPA2B1 is a protein required forsorting of microRNAs with EXO-motif into EVs. To investigate whether hnRNPA2B1 is involved in
the sorting of miR-21 and miR-29a into EVs after HBV infection, we performed knockdown assay
using siRNA for hnRNP2AB1 and control. siRNA for hnRNPA2B1 and control were transfected into
HBV-infected hepatocytes. 4 days after transfection, EVs were collected from cell culture medium,and microRNA levels were subsequently determined. Interestingly, miR-21 and miR-29a levels weredecreased by siRNA for hnRNPA2B1. Taken together, our data suggest that viral proteins increased
the sorting of miR-21 and miR-29a into EVs via hnRNPA2B1 and that released EVs deliver miR21 and miR--29a into the cytoplasm of macrophages, thereby attenuating the production of IL-12
required for the activation of NK and Th1 cells.

Discussion & Conclusion
An accumulating body of evidence has shown that EVs deliver functional RNAs, suchas microRNA and mRNA, from donor to recipient cells, thereby mediating intercellularcommunications. However, the role of EVs in viral escape from host innate immune response has
not been fully elucidated. In this study, we revealed a novel molecular mechanism of EV-mediatedsuppression of host innate immune response by viral proteins. Our observations suggest that HBVutilizes EVs to suppress host innate immune response.
HBV persistently infects human hepatocytes. It is still unclear whether EVs plays a major role in
the persistent infection. Further studies using patient samples are required to assess the in vivo roleof EVs in pathogenesis.

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References
Kouwaki T, Fukushima Y, Daito T, Sanada T, Yamamoto N, Mifsud EJ, Leong CR, TsukiyamaKohara K, Kohara M, Matsumoto M, Seya T, Oshiumi H. Extracellular Vesicles Including Exosomes
Regulate Innate Immune Responses to Hepatitis B virus infection. Frontiers in Immunology 7: 335,
2016
Oshiumi H, Kouwaki T, Seya T. Accessory Factors of Cytoplasmic Viral RNA Sensors Required
for Antiviral Innate Immune Response. Frontiers in Immunology 7: 200, 2016 doi: 10.3389/
fimmu.2016.00200

一般の皆様へ
自然免疫応答は、ウイルス感染初期の生体防御に必須の役割を果たす。しかし、多くのウイルス

はこの自然免疫応答を抑制する能力を進化の過程で獲得しているため、ヒトに感染し病気を引き起こす。Ｂ型肝炎ウイルスは、ヒトの肝がんの主要な原因の一つである。本研究では、Ｂ型肝炎

ウイルスが細胞外小胞を利用して自然免疫応答を抑制するメカニズムを解明することで、Ｂ型

肝炎ウイルスが持続感染するメカニズムの一端を解明した。今後、この過程を阻害するような、
新たなＢ型肝炎治療薬開発につながると期待される。

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Treg Related Exosomal microRNA Involving to the Spatial andChronologic Evolution of CRC
Koshi Mimori
kmimori@beppu.kyushu-u.ac.jp
Kyushu University Beppu Hospital

Summary Abstract
In recent years, the immune response ton cancer has attracted attention. Regardless of the presenceof an immune response mechanism, we can’t overcome colorectal cancer yet. It is suggested thatthe local immune response of colorectal cancer is not functioning normally, so the elucidation of theimmune response and the reaction of tumor cells in colorectal cancer is an important issue.
We are focusing on regulatory T cell and microRNA, and try to elucidate these problems.
Key Words：microRNA, regulatory T cell, colorectal cancer

Introduction
Regulatory T cell (Treg) cells maintain the balance of immune self-tolerance and homeostasis via
limiting aberrant or excessive inflammation1. MicroRNAs (miRNAs) have emerged as important
regulators in many physiological and pathological processes including development, differentiation,
metabolism, immunity, cell proliferation and apoptosis2. Previous studies suggest that miRNAs play
important roles in the function of Treg cells3, 4.

Results
We collected 58 samples (11 cases: multi-region, 3cases: single-region) from the primary site ofcolorectal cancer.
Samples were stored at -4℃ and separated to tumor infiltrating lymphocyte (TIL). TIL included
5.0x10^5 – 3.5x10^7 cells, so we were able to enforce cell sorting analysis. We tried to separate
effector Treg and non-Treg from TIL, but it was difficult to determine the cut off line of CD25. So
we’ll add to fluorescent antibody and determine the cut off line of CD25. As we mentioned above,
our study is amid at the experiments.

Discussion & Conclusion
By referring to miRNA group of high expression in effector Treg and non-Treg, we implement
an enrichment analysis using online miRNA tool miRAA to identify pathway related to the Treg
function. Then, we disclose the prognostic significance of isolated serum miRs preoperatively.
Regulatory T cell might play a crucial role in cancer progression via the immune evading system. We
expect the revelation of data about epigenetic regulators, such as micro RNA, which should be themost powerful diagnostic and therapeutic tool in the future.

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References
1) Sakaguchi, S., Yamaguchi, T., Nomura, T. & Ono, M.Regulatory T cells and immune tolerance.
Cell 133, 775–787, 10.1016/j.cell.2008.05.009 (2008).
2) He, L. & Hannon, G. J. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev
Genet 5, 522–531, 10.1038/nrg1379 (2004).
3) Lu, L. F. et al. Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cellsby targeting SOCS1 protein. Immunity 30, 80–91, 10.1016/j.immuni.2008.11.010 (2009).
4) Kohlhaas, S. et al. Cutting edge: the Foxp3 target miR-155 contributes to the development ofregulatory T cells. J Immunol 182, 2578–2582, 10.4049/jimmunol.0803162 (2009).

一般の皆様へ近年、癌に対する免疫応答が注目されるようになり、悪性黒色腫等では、抗 PD-1抗体である

ニボルマブが臨床応用されています。しかし、適応は一部の癌腫に限定されており、大腸癌では臨床試験の段階です。

免疫応答機構の存在に関わらず、大腸癌の克服に至っていないのは、大腸癌の局所での免疫応答が正常に機能していないことが示唆せれ、大腸癌における免疫応答と腫瘍細胞の反応機序の解明は重要な課題です。

そこで、我々は、がん抗原に対する免疫応答を制御する制御性 T 細胞に注目し、研究を進めています。

さらに、免疫機構で、重要な役割を担う遺伝子・タンパク群の中で、情報伝達分子の役割を担う microRNA に注目し、制御性 T 細胞と microRNA の関連性を解明しようと考えています。

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Elucidation of the sialyl-Tn antigen-mediated survival mechanismof hypoxic tumor cells
Kazuaki Ohtsubo
kohtsubo@kumamoto-u.ac.jp
Department of Analytical Biochemistry, Faculty of Life Sciences, Kumamoto University

Summary Abstract
Intratumoral hypoxia induces Sialyl-Tn (sTn) antigen production, which activates integrin signal
leading to the enhanced tumor invasion and metastasis. sTn antigen elongates the lifetime of
integrin molecules that increased protein abundance of integrins and resulted in the enhancementof the integrin signal. Moreover, the signal activated the NF-kB, which elevated transcription ofsuperoxide dismutase (SOD) 2. This allowed tumor cells to avoid the ischemia-reperfusion stress andconsequently apoptosis. These findings indicated that the hypoxia induced sTn antigen is a functional
molecule important for the survival of tumor cells in tumor tissues and metastasis.
Key Words：Cancer, hypoxia, glycosylation, Sialyl-Tn antigen

Introduction
In the process of tumor development, extensive tumor cell growth and insufficient blood circulation
cause hypoxic conditions, which activate a transcription factor HIF-1. This regulates various gene
expressions regarding tumor growth, metastasis, angiogenesis, anti-apoptosis and so on. However,details of molecular mechanism of the hypoxia induced tumor metastasis and survival have notbeen well understood. Recently, we revealed that a glycan tumor marker molecule, Sialyl-Tn (sTn)antigen, is synthesized under hypoxic conditions and facilitates metastasis and survival. The present
study address to reveal the molecular mechanism underlying the sTn antigen-mediated tumor
development under hypoxic conditions.

Results
We addressed to reveal the mechanism of the enhanced tumor invasion. Immunoblot analyses
revealed that sTn antigen was highly expressed on α2 and β1 integrins, which work as a collagen
receptor to stimulate cellular motility. sTn antigen expressing cells exhibited facilitation ofcollagen-mediated cell adhesion and invasion, which were mediated by activation of FAK (Focal
adhesion kinase) downstream of integrin signal. The up-regulation of FAK activity was attributed
to the elevation of protein abundance of α2 integrin without transcriptional augmentation in sTn
antigen expressing cells. Cell surface protein dynamics analyses revealed that the lifetime of α2
integrin carrying sTn antigen was elongated on the cell surface that resulted in the increase ofprotein abundance of α2 integrin. Moreover, the hypoxia-induced tumor invasion was suppressedby genetic attenuation of ST6GalNAc-I (enzyme synthesizing sTn antigen) or α2 integrin. These
results rationally explain that sTn antigen evokes the facilitation of tumor invasion and metastasis
through integrin signal in the presence of collagen. Analyses of gene expression profile of sTn
antigen expressing cells revealed that expression level of some antioxidant enzymes, encompassing
HO-1 (Heme oxygenase 1) and SOD2 (Superoxide dismutase 2), were increased. Consistent with

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this, sTn antigen expressing tumor cells acquired resistance to oxidative stress and avoids theoxidative stress-induced apoptosis. Inhibition of HO-1 by using SnPP-IX or inhibition of SOD2
by genetic attenuation increased intracellular oxidative stress levels in sTn antigen expressing
cells at steady state. These results indicate that sTn antigen induces anti-oxidative enzymes, HO-1
and SOD2 and reduces intracellular oxidative stress levels. This may contribute to the survival oftumor cells in tumor microenvironments. Analyses of the mechanism of the sTn antigen-induced
expression of HO-1 revealed that sTn antigen activates integrin and the down stream signal, AKT.
AKT activation stimulates nuclear translocation of a transcription factor NRF2. The nuclear NRF2
binds to the promoter region of HO-1 gene and activates transcription of HO-1. Indeed, α2 integrinknock down attenuated the activation levels of AKT and the binding of NRF2 to HO-1 promoter.
Besides, analyses of the mechanism of the sTn antigen-induced expression of SOD2 revealed thatsTn antigen activates NF-κB, which binds to the enhancer region of SOD2 gene and transactivates
SOD2 expression in sTn antigen expressing cells. In vitro ischemia-reperfusion experiments revealed
that the cellular superoxide production by ischemia-reperfusion challenge was significantly reduced
and the resulting apoptosis were mostly abolished in sTn antigen expressing cells. Challenge of an
ST6GalNAc-I inhibitor compound abolished sTn antigen expression. This resulted in the reductionof SOD2 expression and elevation of superoxide production under ischemia- reperfusion challenge
conditions. Indeed, levels of oxidative stress and number of apoptotic cells were significantly reduced
in sTn antigen positive regions in tumor tissues. These overall results indicate that the hypoxiarelated sTn antigen production induces expression of anti-oxidative enzymes to enable tumor cells tosurvive in high oxidative stress environments in tumor tissues. This must contributes to increase therate of the hypoxia-induced tumor metastasis. The ST6GalNAc-I inhibitor effectively attenuates thesTn antigen-induced SOD2 expression and survival of tumor cells in tumor tissues that suggests it
can be the lead of the next generation of antitumor drugs.

Discussion & Conclusion
It has been well recognized that the cellular anti-oxidative activity is important for formation of
distant tumor metastasis, since tumor cells are exposed to high oxidative stress in blood stream.
Consistent with this, metastatic tumor cells acquires intracellular metabolic changes and antioxidative enzymes to reduce cellular oxidative stress levels. However, the molecular mechanism ofthese alterations has not been well understood. Our study clearly indicates that intratumoral hypoxia
induces sTn antigen production in tumor cells that facilitates autonomous cellular motility, leading tometastasis. Moreover, sTn antigen induces anti-oxidative enzymes, which eliminate reactive oxygen
species generated in ischemia-reperfusion cycles in tumor tissues that allows tumor cells survive intumor microenvironments. These sTn antigen-mediated cellular alterations facilitates tumor survival
and metastasis in the process of tumor development. Pharmacological attenuation of the sTn antigen
production can be a rational target to suppress tumor metastasis that may contribute to develop thenext generation of anti-tumor drugs in the future.

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一般の皆様へがんが転移する過程においてがん細胞は酸化ストレスに曝されるため死滅します。しかし、

この酸化ストレスへの耐性を獲得した一部のがん細胞は生存し転移を果たします。これまで、
この酸化ストレスへの耐性獲得のメカニズムは殆ど明らかになっていませんでした。私たちはがん細胞が作り出すシアリル Tn 糖鎖抗原ががんの浸潤能を活性化するとともに抗酸化酵素の産生を誘導することで、がん細胞の生存と転移を直接促進することを明らかにしました。この発見はがん細胞の転移の仕組みを明らかにするのみならず、新しい抗がん剤の開発にとって重要な知見であります。

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III.
Reports from the Recipients of Grants
for International Meetings

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Report on Research Meeting
1. Name of Research Meeting / Conference
The 6th Symposium on ATP1A3 in Disease
2. Representative
Masayuki Sasaki
3. Opening period and Place
Opening period: September 21, 22. 2017
Place: The Palace Hotel Tachikawa, Tachikawa city, Tokyo
4. Number of participants / Number of participating countries and areas
Number of participants: 120 (48 from overseas, 72 from Japan)
Number of participating countries (14; USA, UK, France, Germany, Netherlands, Denmark,Belgium, Norway, Sweden, Italy, Spain, Australia, India, Japan)
5. Total cost
¥7,225,000
6. Main use of subsidy
Part of Meeting room: ¥301,180
Cotton Tote bag print: ¥98,820
7. Result and Impression
The 6th Symposium on ATP1A3 in Disease was held at the Palace Hotel Tachikawa in
Tachikawa, Tokyo. Forty eight participants were welcomed from oversea countries, and72 participants were from Japan. Participants consisted of medical doctors (neurologists orpediatricians), basic scientists (pharmacology, molecular biochemistry, or molecular genetics),
and family members of patients with ATP1A3-related neurological disorders.
Fifteen speakers were invited from Europe, the USA, and Australia. These speakers were the
top class specialists for ATP1A3-related neurological disorders, basic biochemistry, or molecular
genetics. These speakers gave participants the up-to-date information about ATP1A3-related
neurological disorders, for example, Alternating Hemiplegia of Childhood (AHC), Rapidonset Dystonia-Parkinsonism (RDP), Cerebellar ataxia, areflexia, pes cavus, optic atrophy, andsensorineural hearing loss (CAPOS) or recurrent encephalopathy with cerebellar ataxia (RECA),
especially in the fields of clinical diagnosis (semiology and classification) and treatments, andbasic science of molecular biochemistry of the gene and pump protein (Na/K transporting pump
ATPase).
Seven speakers were invited from Japan. They gave participants from all over the world the latest
topics on the new treatment methods using iPS cells and the mechanism of hereditary Parkinson
disease which is related to autophagy.
In this Symposium, participants discussed how we were able to utilize these advanced techniqueeffectively in the development of the treatment methods for ATP1A3-related neurologicaldisorders.

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The social gatherings were held in lunch time and dinner time in this Symposium. All participants
became acquainted with each other, and some international collaborative studies were started
since this Symposium. Most attendants expressed that they were satisfied with the contents of thisSymposium. And they gave kind applause to the host members when the Symposium was closed.
Most attendants expressed that they would like to participate in the next Symposium which would
be held at Chicago, the USA in 2018.
8. Additional description

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Report on Research Meeting
1. Name of Research Meeting / Conference
29th International Symposium on Chirality [Chirality 2017; ISCD-29]
2. Representative
Chair: Toshikazu Takata (Tokyo Institute of Technology, Japan)
Co-Chair: Toru Asahi (Waseda University, Japan)
Honorary Chair: Yoshio Okamoto (Nagoya University, Japan, Harbin Engineering University)
3. Opening period and Place
July 9–12, 2017, Waseda University International Conference Center, Tokyo, Japan
4. Number of participants / Number of participating countries and areas
355 person from 24 countries and areas
5. Total cost
JPY 30,000,000
6. Main use of subsidy
The subsidy was allocated for a part of the traveling costs and accomodation fee of Invited
speakers and Lecturer of Short Couse:
Expense item
travel expenses &accomodation fee

Printing costs

JPY
Prof. Wojciech Dzwolak (University of Warsaw, Poland)

150,000

Prof. Nina Berova (Columbia University, USA)130,000

Prof. Takeo Kawabata (Kyoto University, Japan)

52,020Prof. Hiroaki Sasai (Osaka University, Japan)

53,700Program & Abstract book printing costs(as part of 972,000 yen)

14,280

7. Results and Impression
International symposium on chirality have been held since 1988. This symposium has been
focused on “Exploring the Chirality of molecule or molecular aseembly” and provides a forum
for broad scientific and technological exchange among researchers from all over the world.
The intention is to emphasize the significance and multidisciplinary character of cutting-edge
research on important chirality aspects, and accordingly to put together researchers and students
from various disciplines such as chemistry, physics, and life science in academic, industrial, andgovernmental communities for discussing research findings and promoting research networking
among them. At this meeting, 82 lectures including plenary, keynote, invited, and general ones,
and 184 excellent poster presentations were programmed. As the plenary speakers, we invited
Prof. Hisashi Yamamoto, President of the Chemical Society of Japan, he is a representative
organic chemist of Japan and Prof. Andrew J. Gellman, Carnegie Mellon University, he is apioneer of chiral metal surface science. As the invited speakers, we invited Prof. W. Dzwolok(biophysical scientists), Prof. T. Kawabata (organic chemist), Prof. H. Sasai (organic chemist),and other 31 speakers of various scientific and technological fields. In addition to these

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presentations, chirality 2017 provided Short Course lectures as technical seminar for participants
for free, and we invited Prof. Nina Berova (analytical scientists) as one of the lecturer of shortcourse by this fundation. On the opening ceremony, winner of the 2017 Chirality Medal, Prof.
Takuzo Aida (the university of tokyo) presented the Award Lecture. Despite the hot weather
everyday, a lot of participants visited the venue from early morning, and active discussion wasseen in each lectures. Social programs to promote the personal contact among the participants
were provided for three days during this symposium and we got favorable reviews. On theclosing ceremony, 10 awardees of student poster presentation competision were hornored bychairman, subsequently Chiraity 2018 was introduced by the next chairman of the meeting. As
the related meetings, domestic meeting of chirality, MC 2018, and asia area meeting, MC Asia
2018, were also preannounced. (333 words)
8. Additional description

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Report on Research Meeting
1. Name of Research Meeting / Conference
The 15th International Conference on Na,K-ATPase and Related Transport ATPases
2. Representative
Hiroshi Suzuki, Haruo Ogawa, and Chikashi Toyoshima
3. Opening period and Place September 24th to 30th, 2017
Lake Biwa Otsu Prince Hotel (Otsu city, Shiga prefecture)
4. Number of participants / Number of participating countries and areas 139 people from 20
countries and areas
5. Total cost 30,060,000 yen
6. Main use of subsidy
Financial assistance for transportation to students and young post-doc researchers
7. Result and Impression
We are delighted to report that “The 15th International Conference on Na,K-ATPase and Related
Transport ATPases”, held from September 24th to 30th, 2017 in Otsu, has been a great success.
The conference is a historic international meeting, concerning molecules belonging to the P
type ATPase family represented by sodium pump. The P-type ATPase family is essential for lifeand extremely important in medicine. It consists of an ion pump group that actively transports
ions such as calcium, sodium, potassium, proton, copper, manganese and the like, and a flippasegroup that produces asymmetric distribution of membrane phospholipids. All these couple thechemical energy of ATP and the transport. It is an important research topic, as P-type ATPases
are deeply implicated in serious diseases such as muscle/myocardial disorder, cancer, diabetes,
immune abnormality, neurological abnormality etc. This conference has been hold every three
years in various countries of the world since 1973, and this is the second time the conference isheld in Japan. The last time was at Sapporo in 1999. There are 139 participants from 20 countries,
mainly from Europe and the North America. We invited Professor Toshio Ando (Kanazawa
University) as a special lecturer, who is the leading expert of developing the high speed AFM in
Japan. Professor Poul Nissen (Aarhus university, Denmark) and professor Chikashi Toyoshima(The university of Tokyo) gave keynote lectures on structural biology of P-type ATPases. There
are 8 sessions in the conference, and thirty leading scientists in this field gave presentationsof their latest findings as invited speakers. Sixteen authors were selected from their abstracts
through a peer review by committee members, and they had opportunities to present their works
as oral presentations. There are 160 posters, and active discussions were taken place during theconference. We introduced “1 min flash talk” as a new attempt, so that every poster presenters,
including young researchers and students, had a chance to give a one-minute oral presentation.
This attempt was highly appreciated by many researchers.

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8. Additional description
We sincerely appreciate your understanding of our conference and support. Thanks to yoursupport, we could offer 40,000 yen as a travel assistance for ten excellent poster presenters (all ofthem are students).

会場案内看板

講演会場の様子

ポスター会場の様子

記念撮影

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Report on Research Meeting
1. Name of Research Meeting / Conference
31st Annual Meeting of the Japanese Society for Biological Sciences in Space (JSBSS)
2. Representative
Akihisa Takahashi, ph.D.
Gunma University Heavy Ion Medical Center
3. Opening period and Place
September 20th to 22nd, 2017
“Gunma Kaikan” (2-1-1 Oote-machi, Maebashi, Gunma, 371-0026, Japan)
4. Number of participants / Number of participating countries and areas
200 (1-USA / 2-China / 1-Indonesia / 1-Iran)
5. Total cost
2,500,000 JPY
6. Main use of subsidy
Venue rental fee
7. Result and Impression
The year of 2017 is the 60th anniversary of The “Sputnik 1” orbited the Earth, the first major
milestone in space exploration. Forty-eight years following the first moonwalk, now it is possible
for humankind to spend more than six months in the Outer Space, which accommodated insideof the “International Space Station.” Next, we have challenging dreams that we will get backto the Moon, going to the Mars, and will be able to live in the Space permanently with a highquality of life. However, the plans still have so many obstacles which are needed to be anticipatedand settled biologically. We believe that by investigating a life from outer Space may lead us
to the discovery of unknown biological functions which are hidden in the global environment.
Therefore, our “Biological Sciences in Space” is expected to solve these obstacles. Thisconference aimed to continue to provide a forum for discussion about fundamental and timely
issues and trends as well as for developing collaborations through the exchange of research
results and ideas in the field of space life sciences.
In this meeting, we invited Dr. M. Hada (Senior Research Scientist /Preview A & M University)from USA. She is specialist of bio-dosimetry using by chromosome aberration assay for risk
evaluation of the space radiation in the Astronaut, and gives a speech to us about “Chromosome
aberrations induced by acute and chronic dose rate GCR simulated exposure”. In addition, we
asked Dr. S. Furukawa (JAXA) to give a special lecture about “The International Space Station(ISS) and space medical biology”. As a doctor and astronaut, he has personally experienced
extreme stresses during a five-and-a-half month stay in Space in 2011. He suffered, for example,
from skeletal muscle atrophy due to zero gravity, a dull headache induced by body fluid shifts,
biological rhythm dysfunction triggered by the enclosed environment, space radiation exposure,microbial risks, etc. He sensed the synergistic interactions among these factors and suspected that

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they might also be relevant challenges on Earth. We were able to know these valuable experienceand seeds of science. We have special session about “lunar farm, space experiment, and manned
space program” and 4 symposiums about (1) gravity-response in plants, (2) gravity-response inanimals, (3) space radiation effect, and (4) astrobiology. There were total 74 presentations.
We enjoyed all sessions and also discussion. I think we had a fruitful discussion for our futurecollaborative research.
8. Additional description
Thank you for your assistance. I think the 31st JSNSS meeting succeeded.

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Report on Research Meeting
1. Name of Research Meeting / Conference
International Conference on“The Origin of Life –Synergy among the RNA, Protein and Lipid Worlds –“2. Representative
Prof. Masayuki Imai (Tohoku University)
Prof. Tadashi Sugawara (Kanagawa University)
3. Opening period and Place
May 29 -30, 2017
The main auditorium, Graduate School of Mathematical Sciences,
The University of Tokyo
4. Number of participants / Number of participating countries and areas
172/ 4 countries (India, Japan, UK, USA)
5. Total cost
Facility Fee: 217,700Abstract: 76,986Part-time job fee: 94,640Invitation Fee: 1,791,987Overseas
Prof. Szostak 1,007,060Prof. Deamer 421,349Prof. Chen 203,182Prof. Rajamani* 19,700Domestic
Prof. Ichikawa (Osaka Univ.) 60,840Coffee Break: 79,856Total:

2,181,313-*Prof. Rajamani (Indian Institute of Science, Education, and Research) was invited by ELSI
(Earth-Life Science Institute, Tokyo Institute of Technology) from 5/22 to 6/9 and ELSI supported
her travel expense. We supported her accommodation fee from 5/28 to 5/30.
6. Main use of subsidy
Invitation Fee: 80,540Prof. Rajamani and Prof. Ichikawa
Abstract: 76,986Part-time job fee: 94,640Total: 252,166-*

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The Novartis Foundation(Japan)
for the Promotion of Science*The difference from the original estimation (400,000-) originated from the invitation fee ofProf.
Rajamani, which was supported by ELSI.
7. Result and Impression
This international conference was organized to encourage communications among synthetic
biologists, synthetic chemists and non-equilibrium physicists to reveal the mystery of the originof life. For this purpose we invited representative researchers at the forefront of this topic. The
conference was started by a keynote talk of Prof. Sugawara on a vesicle based model protocell,that is a most advanced synthetic protocell including self-reproduction, self-maintenance, andevolution abilities. In the second keynote talk, Prof. Deamer, who is a pioneer of the astrobiology,
showed a most plausible scenario of the origin of life in the prebiotic era, which is synthesisof biopolymer at hydrothermal fields and encapsulation of the biopolymer within membranouscompartments. In the third keynote talk Prof. Szostak, who is winner of Nobel Prize in 2009,
discussed plausible pathway for the transition from chemical evolution toDarwinian evolution,
where the main unsolved problems are the chemistry of nucleotide activation and a means ofcarrying out multiple sequential rounds of RNA replication. In the final keynote lecture, Prof.
Kaneko addressed theoretical approach toward protocells, which includes i) Thermodynamic
realizability of an autonomously reproducing protocells, ii) Waste problem, iii) Diversity, iv)
Kinetic origin of information, and v) Compartmentalization. From these keynote lectures one
can extract the essence of the origin of life based on his/her own background. In the conference
participants had a lot of discussions to exchange their research results and polish up their ideas,
which suggests activity of this research field. In fact, at first, we estimated that the participants ofthis conference will be around 50 people, but the actual number of participants was 172 people,
which indicates a great success of this international conference. Finally I show impressions of
this conference presented by foreign invited speakers;
Prof. Deamer: “Thank you for all the work you did that made this such a successful conference.
It was a pleasure to meet you and the other Japanese scientists, and to see how we are using
parallel approaches to answer one of the great remaining questions of biology: How can RNAbased life begin on habitable planets? I hope you enjoyed the talks as much as I did.”Prof. Szostak: “Thanks again for inviting us all to this very nice conference.”
Prof. Chen: “Thank you very much for the invitation to the conference and for your hospitality inTokyo. It was a wonderful program of talks and I learned a lot during the symposium. It was also
very nice to meet both of you after reading much about your work.”
Prof. Rajamani: “I wish to convey my most sincere thanks for having invited me to thewonddddderful conference that you both organized. It was a truly rejuvenating meeting and it
indeed is an honour to be a part of the wonderful group of speakers that you both brought together
for the conference...:):)”
8. Additional description

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31st Grant Report（FY2017）
The foundation has been conducting public interest activities such as research grant, meeting grant
and international exchange programs since its establishment on Sep. 4, 1987 in Japan underauthorization of the Ministry of Education, Science, Sports and Culture, followed by a transition to a
public interest incorporated foundation on Apr. 1, 2012. The grants conducted in FY 2017 are as
follows.
31st Novartis Research Grant: 41Researchers (JPY 1 mil.), Subtotal JPY 41 mil.
Research Meeting Grant: 5 Meetings (JPY 0.4 mil.),
Subtotal JPY 2.0 mil.
Total
JPY 43.0 mil.

31st Novartis Research Grant(FY2017)

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

Name

Institution

Title

Research Project
Epigenetic and Metabolic Changes ontransdifferentiation of white adipocytes

1 Inagaki Takeshi

Institute for Molecular andCellular Regulation,
Gunma University

professor

2 Takahashi Yasufumi

Kanazawa University

Associate Development of super resolution chemical
Professor imaging microscopy
Elucidation of molecular bauplan todetermine the positioning of the posterior
body structure along the anteroposterioraxis

3 Suzuki Takayuki

Nagoya University

Lecturer

4 Kondoh Kunio

National Institute forPhysiological Sciences

Neural circuits that regulate glucose
Assistantmetabolism between hypothalamus andProfessor
peripheral organs.

5 SiewKee Low

Japanese Foundation forCancer ResearchGroup
Leader

6 Matsumoto Yoshinori

Okayama University

Assistant Investigation of the new signaling
Professor pathway in osteoblastogenesis

7 Nomura Seitaro

The University of Tokyo
Hospital

Heart Cell Molecular Atlas Project toAssistantdissect the regulatory mechanisms ofProfessor
circulatory homeostasis

8 Isotani Ayako

Nara Institute of Science andTechnology

Associate
Development of the new organ lack model
Professor

9 Yoshiba Satoko

The Univarsity of Tokyo,
Faculty of Pharmaceutical
Sciences

Assistant Molecular mechanism for the initial
Professor assembly of human procentriole

10 Kanai Ryuta

The University of Tokyo

Research Structural basis for inactivation of
Associate Na+,K+-ATPase by β-amyloid

Graduate School of Science
11 Matsumura Shigeyoshi and Engineering, Universityof Toyama
12 Shimada Midori

Yamaguchi University,
Joint Faculty of Veterinary
Medicine

Exploratory analysis to evaluate mutation
spectrum of circulating tumor DNA frommetastatic breast cancer treated witharomatase inhibitors

Assistant Evolutionary development of fluorescentprofessor RNA sensors by droplet technology
Professor

Functional Analysis of Chk1 Targets for
Breast Cancer Treatment

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#

Name

Institution

Title

Research Project

13 Matsuo Takuya

Nagoya University, Center for
Gene Research

Lecturer

Revealing a new red light signaling
pathway for resetting the circadian clockin green algae

14 Matsuzaka Takashi

University of Tsukuba

Associate Elucidation of the role of fatty acid
Professor elongase Elovl6 in the pancreatic β-cell

15 Osafune Kenji

Center for Osafune lab, CiRA,
Kyoto University

Development of regenerative therapy
and novel therapeutic drugs for renal
Professor
anemia using human iPS cell-derived
erythropoietin-producing cells

16 Nakajima Kazunori

Keio University

Professor

17 Nakao Shu

Ritsumeikan University

Direct reprogramming into cardiac
Assistantpacemaker cells for a novel regenerative
Professor
medicine for arrhythmias

18 Urabe Daisuke

Structure determination of formosalide A
Toyama Prefectural University Professor by the combined technology of the NMR
calculation and total synthesis

19 Watanabe Kazunori

Okayama University

Development of apoptosis induction
Assistantmethod with spatiotemporal control using
Professor
microRNAs.

20 Nemoto Tetsuhiro

Graduate School of
Pharmaceutical Sciences,
Chiba University

Study on the enantioselective total
synthesis of dragmacidin E: a natural
Professor
product with high potential for medicinal
chemistry researches

21 Kumano Hironori

University of Yamanashi

Understanding the neural mechanism
Research underlying flexible decision making by
Associate the combination of neurophysiology anddeep neural networks

22 Asai Masashi

Tokyo University of Science,
Faculty of Pharmaceutical
Sciences

Drug development of DYRK1A inhibitors:
Assistanttargeting excess phosphorylation in
Professor
Alzheimer’s disease

23 Murata Takahisa

The University of Tokyo

Associate Development of combination therapy
Professor targeting on tumor endothelial cells

24 Baba Yoshihiro

Medical Institute of
Bioregulation, Kyushu
University

Molecular mechanism of store-operated
Professor Ca2+ entry signalosome and its
physiological significance25 Suzuki Hiromu

Sapporo Medical University

Professor

26 Kondo Shigetada

Osaka Prefecture University

Elucidation of long non-coding RNA
Professor family induced by molecular targetinganti-cancer drugs

27 Shindo Takayuki

Shinshu University Graduate
School of Medicine

Development of anti-metastatic therapy
Professor based on the regulation of vascular
integrity

28 Akiyama Taishin

RIKEN Center for Integrative
Medical ScienceTeam
leader

29 Kimura Keiji

University of Tsukuba

Associate Regulation of cellular functions by theProfessor nuclelous as a stress sensor

30 Nakagawa Osamu

National Cerebral andCardiovascular Center
Research Institute

DirectorTras-omics approach to elucidate
cellular signaling pathways in vasculardevelopment, morphogenesis and disease

31 Saitoh Hisato

Kumamoto University

Professor

Studies on formation and function of
extracellular DNA fibers

32 Ibi Daisuke

Meijo University

Assistant 5-HT2A receptor agonists in treatment toProfessor psychiatric disorders

Mechanism that controls the neuronalsurvival/death in the developing brain

Decoding the epigenome program which
defines normal and tumor endothelial cells

Mechanisms underlying commitment tothymic epithelial cells preventing onset of
autoimmune diseases

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#

Name

Institution

Title

Research Project

33 Teh OoiKock

Hokkaido Univerity Institute
Assistant RopGEF in the regulation of planar cellfor International Collaboration Professor polarity in Plants

34 Muroi Yoshikage

Obihiro University ofAgriculture and Veterinary
Medicine

Associated A novel oxytocin neuronal system forProfessor regulating lactating behavior in mice

35 Ohigashi Izumi

Institute of Advanced Medical
Science, University ofTokushima

Associate Molecular mechanism for the bifurcation
Professor of thymic epithelial cells

36 Nakatsuka Atsuko

Okayama University Hospital

Assistant The role of Heat shock protein 40 Member
Professor C1 in hepatic lipid metabolism

37 Kawada Kenji

Kyoto University

Lecturer

38 Takehana Yusuke

Nagahama Institute of BioScience and Technology

Associate
Evolution of sex determination systems
Professor

39 Tainaka Kazuki

Brain Research Institute,
Niigata University

Specially
Design principle for specific chemical
Appointed
probes toward 3D neuropathology
Professor

40 Miyanishi Hiroshi

University of Miyazaki

Assistant Molecular basis of osmoregulatory
Professor ionocytes

41 Hayashi Shinichiro

National Center of Neurology
and Psychiatry

Section
Chief

Novel treatment strategy targeting CCR1positive myeloid cells in colorectal cancer

Clarification of molecular mechanisms
underlying the maintenance of satellite
cell stemness and its therapeutic
application for Muscular Dystrophy

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FY2017 Research Meeting Grant(JPY 400 thousand x5 = 2.0 million)#

Meeting

Date(Place)Institution / Title

Name

1

6th Symposium on ATP1A3
in Disease2017.9.21-22(Tokyo)National Center of
Neurology / Director of theDep. of Child Neurology

Sasaki Masayuki

2

The 29th International Symposium
on Chirality, Chirality2017; ISCD-29

2017.7.9-12(Tokyo)Tokyo Institute ofTechnology / Professor

Takata Toshikazu

The 15th International Conference
3 on Na,K-ATPase andRelated Transport ATPases

2017.9.24-30(Ohtsu)

IMCB, The Univ. of Tokyo /Associate Professor

Ogawa Haruo

31st Annual Meeting of Japanese
4 Society for Biological Sciencesin Space (JSBSS31st)2017.9.20-22(Gunma)Gunma University Heavy Ion
Medical Center / Professor

Takahashi Akihisa

2017.5.29-30(Tokyo)Tohoku University / Professor

Imai Masayuki

5International Symposiumon The Origin of Life

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第31期（2017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。2017年度は、下記の総額4,300万円の助成事業を実施しました。
第31回ノバルティス研究奨励金

41件（1件100万円）

4,100万円

研究集会助成5件（1件 40万円）

200万円
4,300万円

総額

第31回ノバルティス研究奨励金（2017年度）
この事業は、生物・生命科学および関連する化学の領域において、我が国で行われる創造的な研究の助成を目的としています。

（受付順、敬称略、所属職位は申請時、贈呈額：１件 100万円）

No.

氏

名

所

1

稲垣

毅

2

高橋

康史

属

群馬大学
生体調節研究所

金沢大学
ナノ生命科学研究所

3

鈴木

孝幸

名古屋大学大学院
生命農学研究科
附属鳥類バイオ
サイエンス研究センター

4

近藤

邦生

生理学研究所5

ロー シューキー

6

松本

佳則

7

野村征太郎

8

磯谷

綾子

9

吉場

聡子

10

金井

11

公益財団法人がん研究会
がん研究所
岡山大学

職

位

教授

研究課題

脂肪細胞の形質制御における代謝エピ
ジェネティクス

准教授

超解像度ケミカルイメージング顕微鏡の創成

准教授

体の前後軸形成における下半身の位置を決めるバウプランの分子実体の解明

助教

視床下部と末梢臓器を結ぶ、糖代謝を制御する神経回路の解明

アロマターゼ阻害薬治療を受けた転移性グループ
乳がん患者における血中循環腫瘍ゲノム
リーダー
の解析
助教

骨粗鬆症克服を目指した新たな骨芽細胞
シグナルネットワークの解明

特任助教

心臓細胞アトラスによる循環恒常性制御
機構の解明

准教授

新規臓器欠損モデルの開発

東京大学大学院
薬学系研究科・薬学部
生理化学教室（北川研究室）

助教

中心小体におけるカートホイール構造構
築の分子機構の解明

隆太

東京大学

助教

松村

茂祥

富山大学
大学院理工学研究部（理学）

βアミロイドによる Na+,K+-ATPase の不活化機構の構造的解明

助教

12

島田

緑

13

松尾

拓哉

名古屋大学
遺伝子実験施設

講師

14

松坂

賢

筑波大学

准教授

15

長船

健二

京都大学 iPS 細胞研究所
長船研究室

教授

東京大学
医学部附属病院

国立大学法人奈良先端
科学技術大学院大学

山口大学
共同獣医学部

教授

ドロップ・テクノロジーを用いた RNA
蛍光センサーの進化的創出

がんの病態形成に関わる Chk1 標的因子の機能解明と革新的治療法の確立

緑藻の概日時計をリセットする未知の赤色光受容伝達経路の解明

膵β細胞における脂肪酸伸長酵素 Elovl6の役割の解明ヒト iPS 細胞由来エリスロポエチン産生細胞を用いた腎性貧血に対する再生医療と新規治療薬の開発

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No.

氏

16

仲嶋

17

名

所

属

職

位

一範

慶應義塾大学

教授

中尾

周

立命館大学

助教

18

占部

大介

公立大学法人
富山県立大学

教授

19

渡邉

和則

岡山大学

助教

20

根本

哲宏

千葉大学大学院薬学研究院

教授

21

熊野

弘紀

山梨大学

22

浅井

将

23

村田

24

特任助教

研究課題

発生期脳神経細胞の生死を決める機構
リプログラミング技術を応用した心臓ペースメーカー細胞の誘導法の確立

NMR 計算と全合成を基盤とした稀少ポ
リケチドホルモサリド A の構造決定
マイクロ RNA を用いた時空間制御可能なアポトーシス誘導法の開発

未開拓創薬有用天然物：ドラグマシジン
E の不斉全合成研究
神経生理学と深層学習の組み合わせによる柔軟な判断の神経基盤の解明

ダウン症関連遺伝子 DYRK1A を標的とした新規アルツハイマー病治療薬の開発

東京理科大学薬学部

助教

幸久

東京大学

准教授

馬場

義裕

九州大学
生体防御医学研究所

教授

25

鈴木

拓

札幌医科大学

教授

26

近藤

茂忠

大阪府立大学

教授

27

新藤

隆行

信州大学
大学院医学系研究科

教授

28

秋山

泰身

理研
チーム 自己免疫疾患の発症を抑制する胸腺上皮
統合生命医科学研究センター リーダー 細胞への分化決定機構の同定

29

木村

圭志

筑波大学

准教授

30

中川

修

国立循環器病研究センター
研究所 分子生理部

部長

31

斉藤

寿仁

熊本大学
大学院先端科学研究部

教授

32

衣斐

大祐

名城大学

助教

33

鄭

恵国

北海道大学
国際連携機構5-HT2A 受容体刺激薬の精神疾患治療薬としての有用性

助教

Developmental Biology in Plants

34

室井

喜景

帯広畜産大学

准教授

35

大東いずみ

徳島大学
先端酵素学研究所

エネルギー状態に基づき授乳行動を制御する新規オキシトシン神経機構の解明

准教授

胸腺上皮細胞分岐分子機構の解析

36

中司

敦子

岡山大学病院
腎臓・糖尿病・内分泌内科

助教

肝脂質代謝における Heat shock protein
40 Member C1 の意義

37

河田

健二

京都大学

講師

38

竹花

佑介

長浜バイオ大学

准教授

39

田井中一貴

新潟大学脳研究所

特任教授

40

宮西

宮崎大学

助教

国立研究開発法人
国立精神・神経医療研究
センター
神経研究所

浸透圧調節を担う塩類細胞における分子
基盤の構築

室長

筋衛星細胞の未分化性維持機構の解明と筋ジストロフィー治療法への応用

41

林

弘
晋一郎

癌内皮細胞を標的とした抗癌剤感受性増大法の開発
ストア作動性 Ca2+ 流入シグナルソームの分子機序と生理的意義

正常と腫瘍血管内皮を規定するエピゲノ
ムプログラムの解読

がん分子標的薬によって誘導される悪性
腫瘍化 RNA ファミリーの解明
血管の統合性制御に基づく、転移抑制法の開発

ストレスセンサーとしての核小体を介した細胞機能の制御

血管発生・形態形成とヒト血管病におけるシグナル伝達機構：多階層オミックス
解析による研究
細胞外 DNA の形成機序とその生理作用に関する研究CCR1 陽性骨髄由来細胞をターゲットにした新たな大腸癌治療
性決定システムの進化機構の解明

3 次元神経病理学に向けた特異的ケミカ
ルプローブの設計戦略

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for the Promotion of Science

2017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。2017年度は５件の助成を行いました。

（受付順、敬称略、所属・職位は申請時、贈呈額：１件40万円）

No.

研究集会名

開催日
（開催地）

1

第６回 ATP1A3遺伝子関連疾患に関する国際シンポジウム

2017.9.21-22（東京）

2

第29回不斉に関する国際会議

3

第15回ナトリウムポンプと関連
イオンポンプに関する国際会議

2017.7.9-12（東京）

4

日本宇宙生物科学会第31回大会

5

国際シンポジウム 「生命の起源」

2017.9.24-30（大津）2017.9.20-22（群馬）

2017.5.29-30
（東京）

所属・職位

氏

名

国立精神・神経医療研究センター病院／
小児神経診療部長

佐々木征行

東京工業大学／教授

高田十志和

東京大学 分子細胞生物学
研究所／准教授

小川治夫

群馬大学重粒子線医学研究センター／教授高橋昭久

東北大学／教授

今井正幸

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The Novartis Foundation(Japan)
for the Promotion of Science

31st Financial Report

Balance Sheet

Statement of Net Assets

As of March 31, 2018

From April 1, 2017 to March 31, 2018
（Unit：JP Yen）

Account（Unit：JP Yen）

AmountⅠAssets

Ⅰ General Net Assets Changes

1.Current Assets

1.Ordinary income & Expenditure

Current Assets Total

19,859,149

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

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

634,194(3) Other Long - term Assets
Other Long - term Assets Total

79,971,399

AmountAccount(1) Ordinary income
Donation

40,108,000

Ordinary Income Total

55,420,100(2)Ordinaty Expenditure
Project Expense

53,492,407

Grant Expense

43,000,000

Novartis Research Grant

41,000,000

Research Meeting Grant

2,000,000

Fixed Assets Total

1,180,605,593

Administrative Expense

14,748,656

Assets Total

1,200,464,742

Ordinary Expenditure Total

68,241,063

Ⅱ Liabilities

Ordinary Balance without Appraisal Profit or Loss △ 12,820,963

1.Current Liabilities

2.Nonrecurring Profit & Loss

Current Liabilities Total

52,245,214

Nonrecurring Balance of Current Period

Liabilities Total

52,245,214

General Net Assets Ending Balance
Ⅱ Designated Net Assets Changes

Ⅲ Equity(Net Assets)

Designated Net Assets Change

1.Designated Net Assets
Designated Net Assets Total(Amount Appropriating to basic Fund)
2.General Net Assets(Amount Appropriating to)147,585,334△ 108,000

1,000,634,194

Designated Net Assets Ending Balance

1,000,634,194

1,000,000,000

Ⅲ Net Assets Balance Ending Balance

1,148,219,528

147,585,334
100,000,000

Equity Total(Net Assets)

1,148,219,528

Liabilities & Equity Total

1,200,464,742

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The Novartis Foundation(Japan)
for the Promotion of Science

第31期（2017年度）財務報告

貸借対照表

正味財産増減計算書

2018 年 3 月 31 日現在

2017 年 4 月 1 日から 2018 年 3 月 31 日まで（単位：円）

科

目

金 額

Ⅰ資産の部

科

19,859,149

⑴

経常収益
受取寄付金

(1) 基本財産

経常収益
1,100,000,000(2) 特定資産
特定資産合計634,194(3) その他固定資産
その他固定資産合計
固定資産合計
資産合計

79,971,399

43,000,000

ノバルティス研究奨励金

41,000,000

研究集会助成金

2,000,000
14,748,656

計68,241,063-12,820,963

２．経常外増減の部
52,245,214

当期形状外増減額

52,245,214

一般正味財産期末残高

147,585,334

Ⅱ指定正味財産増減の部
当期指定正味財産増減額
1,000,634,194
1,000,000,000
147,585,334（うち基本財産への充当額）

100,000,000

負債及び正味財産合計

支払助成金

当期経常増減額

２．一般正味財産
正味財産合計55,420,100
53,492,407

経常費用

１．指定正味財産（うち基本財産への充当額）

計

事業費

1,200,464,742

Ⅲ正味財産の部
指定正味財産合計

40,108,000

経常費用

管理費

１．流動負債
負債合計⑵

1,180,605,593

Ⅱ負債の部
流動負債合計金 額

１．経常増減の部

２．固定資産基本財産合計目

Ⅰ一般正味財産増減の部

１．流動資産流動資産合計（単位：円）

指定正味財産期末残高
Ⅲ正味財産期末残高-108,000
1,000,634,194
1,148,219,528

1,148,219,528
1,200,464,742

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The Novartis Foundation(Japan)
for the Promotion of Science

List of Board Members[Board of Trustees] 5 trustees, 2 auditors

As of Oct 1, 2017

Post

Name

Chairman

Akimichi KANEKO

Kio University; Emeritus Professor, Keio University

Shigetaka ASANO

Visiting Professor, MD, School of Medicine, Kobe University;
Emeritus Professor, University of Tokyo

Masao ENDOH

Emeritus Professor, MD, Yamagata University

Kuniaki TAKATA

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

Trustee

Masao TORII
AuditorTokuzo NAKAJIMA
Masanori FUSE

Title

President, Novartis Holding Japan K.K.
Certified Public Accountant
Financial Advisor, Finance & Administration Div., Novartis Pharma K.K.

[Board of Councilors] 10 councilors
Post

Name

Chairman

Tsuneyoshi KUROIWA

Councilor

As of Oct 1, 2018
Title

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

Norio AKAIKE

Head, Kumamoto Kinoh Hospital Clinical Research Center
Visiting Professor, Kumamoto University, Graduate School of Medicine
and Pharmaceutical Research
Emeritus Professor, Kyushu University

Hiroyuki KAWASHIMA

Former Professor, Graduate School of Medical & Dental Sciences, Niigata
University

Masakatsu SHIBASAKI

Director, Microbial Chemistry Research Center, Microbial Chemistry
Research Foundation

Akihiko NAKANO

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

Tadanori MAYUMIEmeritus Professor, Osaka University

Miwako MORI
Toyoshi FUJIMOTO

Emeritus Professor, Hokkaido University
Professor, MD, Graduate School of Med, Nagoya University

Masamitsu IINO

Specially appointed Professor, MD, Nihon University
Emeritus Professor, University of Tokyo

Tohru HIROSE

Director, Division Head Japan Development, Novartis Pharma K.K.

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for the Promotion of Science

130

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

Name

As of Oct 1, 2018
Title

Masafumi TAKIGUCHI Professor, MD, Center for AIDS Research, Kumamoto University
Koichiro KUWAHARA Professor, MD, School of Medicine, Shinshu University

MemberMasanobu OHSHIMA

Professor, Cancer Research Institute, Kanazawa University

Yosuke TAKAHAMA

Professor, Institute for Genome Research, Tokushima University

Eisuke NISHIDA

Director, RIKEN Center for Biosystems Dynamics Research

Mitsuyasu HASEBE

Professor, National Institute for Basic Biology

Haruhiko BITO

Professor, MD, Graduate School of Medicine, University of Tokyo

Jun YAMASHITA

Professor, MD, Center for iPS Cell Research and Application, Kyoto
University

Motoko YANAGITA

Professor, MD, Graduate School of Medicine, Kyoto University

Masafumi YANO

Professor, MD, School of Medicine, Yamaguchi University

Hirokazu ARIMOTO

Professor, MD, Graduate School of Life Sciences, Tohoku University

Hiroyuki TAKEDA

Professor, Graduate School of Science, University of Tokyo

Eiji HARA

Professor, Research Institute for Microbial Diseases,
Osaka University

Makoto HAYASHITeam Leader, Plant Symbiosis Reseach Team, RIKEN Center for
Sustainable Resource Science

Hirokazu HIRAIProfessor, MD, Head of Department of Neurophysiology and Neural
Repair, Gunma University

Kouichi FUKASE

Professor, Graduate School of Science, Osaka University

Michisuke YUZAKI

Professor, School of Medicine, Keio University

Akihiko YOSHIMURA

Professor, Department of Microbiology and Immunology,
Graduate School of Medicine, Keio University

Hiroshi Ito

Professor, Department of Graduate School of Medicine, Keio University

Takashi Ohshima

Professor, Drug Discovery and Development Center, Graduate Schoolof Pharmaceutical Sciences affiliated Company-Academia-Government
collaboration, Kyushu University

Yoko Hamazaki

Professor, Graduate School of Medicine, Kyoto University

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for the Promotion of Science

公益財団法人ノバルティス科学振興財団
役 員 名 簿2018 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

代表理事

理事

監事

氏

名

金子

章道

浅野

茂隆

遠藤

政夫

高田

邦昭

鳥居

正男

中嶋

德三

布施

正則

現

慶應義塾大学名誉教授

職

神戸大学大学院医学系研究科客員教授
東京大学名誉教授

山形大学名誉教授

群馬県立県民健康科学大学学長
群馬県公立大学法人理事長

ノバルティスホールディングジャパン ( 株 )代表取締役社長
中嶋德三公認会計士事務所
公認会計士

ノバルティスファーマ ( 株 ) 企画管理本部
ファイナンシャルアドバイザー

就任年月日

常勤 ･ 非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2018年6月25日

非常勤

2016年6月10日

非常勤

2016年6月10日

非常勤

評 議 員 名 簿2018 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

氏

名

評議員長

黒岩

常祥

赤池

評議員

現

職

就任年月日

常勤 ･ 非常勤日本学士院会員
東京大学名誉教授

2016年6月10日

非常勤

紀扶

医療法人社団寿量会熊本機能病院
臨床研究センター所長、学術顧問
熊本大学大学院医学薬学研究部客員教授
九州大学名誉教授

2016年6月10日

非常勤

川島

博行

元新潟大学大学院医歯学総合研究科教授

2016年6月10日

非常勤

柴崎

正勝

公益財団法人微生物化学研究会
微生物化学研究所長

2016年6月10日

非常勤

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
生細胞超解像イメージング研究チーム
チームリーダー2016年6月10日

非常勤

眞弓

忠範

大阪大学名誉教授

2016年6月10日

非常勤

森

美和

北海道医療大学客員教授
北海道大学名誉教授

2016年6月10日

非常勤

藤本

豊士

名古屋大学大学院研究科教授

2016年6月10日

非常勤

飯野

正光

日本大学医学部特任教授
東京大学名誉教授

2016年6月10日

非常勤

廣瀬

徹

ﾉﾊﾞﾙﾃｨｽ ﾌｧｰﾏ ( 株 ) 取締役開発本部長

2016年6月10日

非常勤

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The Novartis Foundation(Japan)
for the Promotion of Science

選 考 委 員 名 簿
2018 年 10 月 1 日現在 ( 順不同、敬称略 )
職

名

選考委員長

氏

現

職

就任年月日

常勤 ･ 非常勤

2015年6月26日

非常勤

桑原宏一郎

信州大学医学部教授

2015年6月26日

非常勤

正伸

金沢大学がん進展制御研究所教授

2015年6月26日

非常勤

西田

栄介

理化学研究所生命機能科学研究センター
センター長

2015年6月26日

非常勤

長谷部光泰

自然科学研究機構基礎生物学研究所教授

2015年6月26日

非常勤

東京大学大学院医学系研究科教授

2015年6月26日

非常勤

山下

潤

京都大学 iPS 細胞研究所教授

2016年6月10日

非常勤

素子

京都大学大学院医学研究科教授

2016年6月10日

非常勤

大島

尾藤

柳田

選考委員

名

熊本大学エイズ学研究センター教授

滝口

雅文

晴彦

矢野

雅文

山口大学医学部医学科教授

2016年6月10日

非常勤

有本

博一

東北大学生命科学研究所教授

2016年6月10日

非常勤

武田

洋幸

東京大学大学院理学系研究科教授

2017年6月19日

非常勤

原

英二

大阪大学微生物病研究所教授

2017年6月19日

非常勤

理化学研究所環境資源科学センター
チームリーダー2017年6月19日

非常勤

林

誠

平井

宏和

群馬大学大学院医科学系研究科教授

2017年6月19日

非常勤

深瀬

浩一

大阪大学大学院理学研究科教授

2017年6月19日

非常勤

柚﨑

通介

慶應義塾大学医学部教授

2017年6月19日

非常勤

吉村

昭彦

伊藤

裕

慶應義塾大学大学院医学研究科教授

2017年6月19日

非常勤

慶應義塾大学大学院医学部教授

2018年6月25日

非常勤

大嶋

孝志

九州大学大学院薬学研究院附属産学官連携創薬
育薬センター教授

2018年6月25日

非常勤

濵﨑

洋子

京都大学大学院医学研究科教授

2018年6月25日

非常勤

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The Novartis Foundation(Japan)
for the Promotion of Science

事 務 局 便 り

ご寄附のお願い
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：支出した寄附金は、通常一般の寄附金の損金算入限度額と同額まで、別枠で損金に算入できます。
ご寄附は、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下さい。
（電話：03-6899-2100、E-メール：foundation.japan@novartis.com）

事務局より
本年度もお陰様で、財団年報を発行できることとなりました。これも偏に、助成を受けられた皆様および財団関係者の皆様のご尽力の賜物と心より感謝
申し上げます。
1987年9月の財団設立以来、助成件数は総数で1,722件、総額20億円を超えるものとなりました。
2018年3月に財団設立30周年記念式典を開催し、また8月には30周年記念誌も刊行できました。前事務局長の田中基晴氏の貢献に感謝します。
事務局は、今後とも財団の設立目的である学術の進展に寄与するべく、研究助成を中心とした公益事業に邁進して参ります。
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。
2018年10月吉日
事務局長 岩見 文明〒105-6333 東京都港区虎ノ門1-23-1
虎ノ門ヒルズ森タワー 29F
TEL：03-6899-2100 FAX：03-6899-2101
E-メール：foundation.japan@novartis.com
ホームページ：http://japanfoundation.novartis.org/

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