Rice Farming and Chinese Civilization: Renovation of Integrated Studies of Rice-based Civilizations

金沢大学学際科学実験センター本年度は、中国の田螺山遺跡等で出土した植物遺体及び土器付着物について、イネ果実（炭化米）１粒程度のサンプル量で代謝物の分析を行う、メタボロミクスの実験系の構築を目指した。前年度から進めていた代謝物の抽出溶媒とUPLCによる分析条件の検討をさらに進め、中国で出土したイネ、ヒシ、ドングリの果実を用いて、破砕後にメタノールを溶媒として抽出を行い、親水性化合物も同定可能なHILICカラムを用いて、LC-MSによる分析を行った。分析の結果、ヒシやドングリでは、200種以上の代謝物が同定されたが、微生物由来と思われる代謝物も複数見られた。一方、イネで検出された代謝物は100種以下で、イネと他の2種で共通して検出された代謝物も少なかったが、イネのみで検出された代謝物には、脂肪酸や芳香族化合物等が見られた。ドングリでは、フィトセラミドとその分解産物などが特異的に検出された。ヒシのみに見られた代謝物には、アミノ酸類縁体や芳香族化合物等が含まれていた。出土したドングリについては、種同定に至っていないという問題もあるが、今後は現生果実との比較解析や出土場所の異なる試料での解析を行うことで、マーカーとなる代謝物の探索を進めたい。一方、４つの異なる土器付着物についても、植物遺体で用いた方法を一部改変して分析を行ったところ、複数の土器付着物で共通して見られる代謝物には脂肪酸が多く見られたが、魚類に多く含まれる一価不飽和脂肪酸であるイコセン酸も検出された。今後、多くの土器付着物を分析し、再現性よく検出される代謝物の中から食性復元のマーカーになるような代謝物の探索を進めて行きたい。研究課題/領域番号:18H04176, 研究期間(年度):2018-04-01 – 2020-03-31出典：研究課題「高感度質量分析計を用いた遺跡出土品のメタボローム解析による多様な食品利用の復元」課題番号18H04176（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PUBLICLY-18H04176/）を加工して作

Deciphering Origin and Establishment of Japonesians mainly based on genome sequence data

金沢大学学際科学実験センター多様な年代や地域で出土する動植物遺体中に残存する微量ペプチドの同定により、ヤポネシア人の動植物利用の復元を試みる。さらに歯石や土器付着物等のより複雑な試料についてもプロテオミクス解析を行うことで、ヤポネシア人の食生活や疾患を復元し、ヤポネシアへの渡来と定着の過程における食生活、疾病や動植物利用の変遷を明らかにする。遺跡から出土する動物骨中のコラーゲンタンパク質は非常に安定であり、歯石中に残されたタンパク質も古代人の疾患や食生活の復元について有用な知見をもたらす。一方で、植物遺存体は、イネ果実のように経年により炭化することも多く、タンパク質の分析については実験系が確立されていない。本研究では、動物骨や歯石のプロテオーム解析に加えて、炭化米等の植物遺存体からタンパク質を抽出・精製する方法について検討を進め、質量分析計を用いたショットガンプロテオミクスを行っている。前年度に開発したアセトン沈殿によるタンパク質精製を改良した方法を用いることで、弥生時代の福岡の2遺跡と韓国の1遺跡の3種のイネ果実（炭化米）試料から、多数のペプチドを検出し、多くのタンパク質を同定することができた。同定されたタンパク質の多くは、イネ種子に特異的に蓄積していると報告されていたタンパク質であり、中でも、63kDグロブリン様タンパク質は、3遺跡全ての試料で同定され、いずれも多数のペプチド(4-12本)が検出された。イネ科には、この63kDグロブリン様タンパク質と類似なタンパク質が存在し、キビ属とは70％弱の類似性であり、異なるアミノ酸配列が多数見出されたことから、イネとキビなどの雑穀を分別できるマーカータンパク質の候補と考えられた。また、ジャポニカとインディカにおいても複数のアミノ酸が異なっており、多数のペプチドを検出できれば、判別が可能であると考えられた。また、土器付着物についても、植物遺存体を用いた上記に類似した方法を適応して解析を進めており、植物だけではなく、動物や微生物由来のタンパク質も検出されており、土器付着物のプロテオーム解析による食生活の復元に向けた実験基盤を確立することができた。研究課題/領域番号:19H05345, 研究期間(年度):2019-04-01 – 2021-03-31出典：研究課題「プロテオミクスで紐解くヤポネシア人の食生活の復元」課題番号19H05345（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PUBLICLY-19H05345/）を加工して作

葉と根の間でのシステミックな傷応答遺伝子発現の解析

金沢大学学際科学実験センター局所的及び葉と葉の間の傷ストレス応答に関わるシグナルとして、植物ホルモンであるジャスモン酸(JA)が中心的な役割を担うことが知られている。JAはオクタデカノイド経路によってリノレン酸からOPDAなどの中間産物を経て産生される。シロイヌナズナのマイクロアレイを用いた解析から、根を傷処理し30分後に地上部で発現量が増加した遺伝子群にも、JA合成経路(オクタデカノイド経路)に関わる遺伝子やJA応答性遺伝子が多く含まれることを明らかにしていた。JAに加えてOPDAも傷応答遺伝子の発現に関与していることが示唆されているが、根を傷つけたときの地上部におけるJA量とOPDA量の経時的な変化を調べたところ、傷処理後30分にJA量が約6倍に上昇し、OPDA量は6時間後に約2倍に上昇していた。さらに、葉に傷をつけた植物の根での応答についてアレイ解析を行った結果、葉から根への器官間傷応答性を示す遺伝子は比較的少なく、顕著な応答性を示すものも少なかったが、根から葉への期間傷応答遺伝子として同定されていたJAZファミリー遺伝子がこちらにも共通して含まれていた。また、根から葉への器官間傷応答遺伝子の1つであるエチレン応答性転写因子のAtERF13について、AtERF13promoter-Luciferase形質転換体を用いて解析したところ、根を傷つけた時の地上部では約5倍以上にルシフェラーゼ活性が上昇するのに対して、葉を傷つけた時の根での応答は2倍にも満たなかったことから、AtERF13は双方向には器官商傷応答性は示さないことが明らかになった。今後、双方向の器官間傷応答性を示した遺伝子についても同様の解析を進めて行きたい。研究課題/領域番号:17770031, 研究期間(年度):2005 – 2007出典：「葉と根の間でのシステミックな傷応答遺伝子発現の解析」研究成果報告書　課題番号17770031（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-17770031/)を加工して作

Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition

Comparative anatomy of embryogenesis in three species of Podostemaceae and evolution of the loss of embryonic shoot and root meristems

During embryogenesis in angiosperms, the embryonic shoot and root meristems are created at opposite poles of the embryo, establishing a vertical body plan. However, the aquatic eudicot family Podostemaceae exhibits an unusual horizontal body plan, which is attributed to the loss of embryonic shoot and root meristems. To infer the embryogenetic changes responsible for the loss of these meristems, we examined the embryogenesis of three podostemads with different meristem characters, that is, Terniopsis brevis with distinct shoot and root meristems, Zeylanidium lichenoides with reduced shoot and no root meristems, and Hydrobryum japonicum with no shoot and no root meristems. In T. brevis, as in other eudicots, the putative organizing center (OC) and L1 layer (= the epidermal cell layer) arose to generate a distinct shoot meristem initial, and the hypophysis formed the putative quiescent center (QC) of a root meristem. Z. lichenoides had a morphologically unrecognizable shoot meristem, because a distinct L1 layer did not develop, whereas the putative OC precursor arose normally. In H. japonicum, the vertical divisions of the apical cells of eight-cell embryo prevented putative OC initiation. In Z. lichenoides and H. japonicum, the putative QC failed to initiate because the hypophysis repeated longitudinal divisions during early embryogenesis. Based on their phylogenetic relationships, we infer that the conventional embryonic shoot meristem was lost in Podostemaceae via two steps, that is, the loss of a distinct L1 layer and the loss of the OC, whereas the loss of the embryonic root meristem occurred once by misspecification of the hypophysis. © 2011 Wiley Periodicals, Inc

Compensatory Upregulation of Myelin Protein Zero-Like 2 Expression in Spermatogenic Cells in Cell Adhesion Molecule-1-Deficient Mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells

Tandem metalloenzymes gate plant cell entry by pathogenic fungi

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase–peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection. Fungal phytopathogens secrete tandem metalloenzymes that catalyze cuticle oxidation and drive plant cell entryThis study was supported by the “Agence Nationale de la Recherche” and by the Natural Sciences and Engineering Research Council of Canada through the ANR-NSERC project “FUNTASTIC” (ANR-17-CE07-0047, STPGP 493781-16). We are grateful to MANE & Fils and the “Association Nationale Recherche Technologie” (ANRT) for funding the Ph.D. fellowship of D.R. (grant no. 2017/1169). Work in Japan was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research—KAKENHI, grant numbers 15H05780 and 20H02989 to Y.K., and 20K15529 to S.K.Peer Reviewed"Article signat per 18 autors/es: Bastien Bissaro and Sayo Kodama and Takumi Nishiuchi and Anna Maria Díaz-Rovira and Hayat Hage and David Ribeaucourt and Mireille Haon and Sacha Grisel and A. Jalila Simaan and Fred Beisson and Stephanie M. Forget and Harry Brumer and Marie-Noëlle Rosso and Victor Guallar and Richard O’Connell and Mickaël Lafond and Yasuyuki Kubo and Jean-Guy Berrin "Postprint (published version

Visualization of Neural Activity in Insect Brains Using a Conserved Immediate Early Gene, Hr38

Many insects exhibit stereotypic instinctive behavior [1-3], but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates [4, 5]. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. Here, we identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, we mapped comprehensive activity patterns of the silkmoth brain in response to female sex pheromones. We found that Hr38 can also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, we constructed a neural activity map of the fly brain that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies. © 2013 Elsevier Ltd. All rights reserved

Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma. Nevertheless, mechanisms for a phenotypic change have remained to be addressed. Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics. In metastatic melanoma, the cells are composed of Met-low and Met-high populations. Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation. Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs. Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Met-high cells facilitates lung metastasis by Met-low cells. Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations. Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high. Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells. These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression

Different growth and metastatic phenotypes associated with a cell-intrinsic change of Met in metastatic melanoma

A dynamic phenotypic change contributes to the metastatic progression and drug resistance in malignant melanoma. Nevertheless, mechanisms for a phenotypic change have remained to be addressed. Here, we show that Met receptor expression changes in a cell-autonomous manner and can distinguish phenotypical differences in growth, as well as in metastatic and drug-resistant characteristics. In metastatic melanoma, the cells are composed of Met-low and Met-high populations. Met-low populations have stem-like gene expression profiles, are resistant to chemotherapeutic agents, and have shown abundant angiogenesis and rapid tumor growth in subcutaneous inoculation. Met-high populations have a differentiated phenotype, are relatively resistant to B-RAF inhibitor, and are highly metastatic to the lungs. Met plays a definitive role in lung metastasis because the lung metastasis of Met-high cells requires Met, and treatment of mice with the Met-containing exosomes from Methigh cells facilitates lung metastasis by Met-low cells. Clonal cell fate analysis showed the hierarchical phenotypical changes from Met-low to Met-high populations. Met-low cells either showed self-renewal or changed into Met-high cells, whereas Met-high cells remained Met-high. Clonal transition from Met-low to Met-high cells accompanied changes in the gene expression profile, in tumor growth, and in metastasis that were similar to those in Met-high cells. These findings indicate that malignant melanoma has the ability to undergo phenotypic change by a cell-intrinsic/autonomous mechanism that can be characterized by Met expression