担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

Founder animals carrying high proportions of somatic mutation induced by CRISPR–Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR–Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype–phenotype correlations in CRISPR–Cas9-based loss-of-function screening of numerous target genes in various organisms.

DOI： 10.1111/gtc.12775

Scopus

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記述言語：英語   掲載種別：研究論文（学術雑誌）

Developmental exposure to bisphenol A (BPA) is associated with liver dysfunction and diseases in adulthood. The aims of this study were to assess the effects of prenatal BPA exposure on the hepatic transcriptome and proteome in female and male offspring and to understand adverse outcome pathways (AOPs) to observed phenotypic effects. Pregnant Wistar rats were exposed to 50 or 5000 μg BPA/kg bw/day, or 17β-estradiol (E2, 50 μg/kg bw/day) from embryonic day 3 to 18. The liver transcriptome and proteome profiles were analyzed in the newborn (postnatal day 1; PND1) and weaning (PND21) rat offspring. Based on the differentially expressed genes/proteins derived from transcriptome and proteome profiles, we performed pathway, transcription factor, and disease enrichment analyses. A principal component analysis of transcriptome data demonstrated that prenatal BPA exposure caused masculinization of the hepatic transcriptome in females. Both of transcriptomic and proteomic data showed that prenatal BPA exposure led to the disruption of cell cycle, lipid homeostasis, and hormone balance in offspring. Most of the effects at the transcript level were extended from newborn to weaning in males, but were moderated until weaning in females. The alterations at the transcript and protein levels were accordant with the observation of increases in body weight and anogenital distance and changes in hepatosomatic index in the offspring. Collectively, we constructed AOPs with evidence of sex- and age-specific actions of prenatal BPA exposure in the offspring.

DOI： 10.1016/j.scitotenv.2020.137568

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）

Motivation: Disease states are distinguished from each other in terms of differing clinical phenotypes, but characteristic molecular features are often common to various diseases. Similarities between diseases can be explained by characteristic gene expression patterns. However, most disease-disease relationships remain uncharacterized. Results: In this study, we proposed a novel approach for network-based characterization of disease-disease relationships in terms of drugs and therapeutic targets. We performed large-scale analyses of omics data and molecular interaction networks for 79 diseases, including adrenoleukodystrophy, leukaemia, Alzheimer's disease, asthma, atopic dermatitis, breast cancer, cystic fibrosis and inflammatory bowel disease. We quantified disease-disease similarities based on proximities of abnormally expressed genes in various molecular networks, and showed that similarities between diseases could be explained by characteristic molecular network topologies. Furthermore, we developed a kernel matrix regression algorithm to predict the commonalities of drugs and therapeutic targets among diseases. Our comprehensive prediction strategy indicated many new associations among phenotypically diverse diseases.

DOI： 10.1093/BIOINFORMATICS/BTAA439

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記述言語：英語   掲載種別：研究論文（学術雑誌）

<p>In this study, we assessed embryonic developmental toxicity and teratogenicity of lithium (Li) on medaka (<i>Oryzias latipes</i>) and predict the molecular mechanisms of their effects using a nanosecond pulsed electric field (nsPEF) technique and bioinformatics analysis. The microscopic observation revealed that the 1 mg/L LiCl treatment causes the most severe deformation effects, such as thrombus, heart hypertrophy, deformation of eyes, and growth retardation to embryos. The RNA-seq analysis identified 2,483 up- and down-regulated genes, such as histogenesis and organ growth related genes, in 2 day post-fertilization embryos after treatment with nsPEF and 1 mg/L LiCl. In addition, bioinformatic analyses showed that LiCl affects several aspects of gene ontology, such as molecular functions and cellular components, and some pathways, such as spliceosome, cell cycle, selenocompound metabolism, TGF-β signaling, and RNA degradation. The upregulation of <i>GSK3B</i> (signal transduction and cell growth), <i>BAX </i>(apoptosis), and <i>MAP3K8</i> (cell death, arrest of cell cycle, and inflammation) genes were also observed in embryos exposed to LiCl. Our results suggest that the incorporation of Li compounds into medaka eggs using nsPEF shows adverse effects to the development and teratogenicity, and that these toxic effects may be affected by the alterations of certain gene expression in medaka embryos.</p>

DOI： 10.2131/fts.6.31

CiNii Article

CiNii Research

その他リンク： https://www.jstage.jst.go.jp/article/fts/6/2/6_31/_pdf

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

Environmental contaminant exposure can pose significant risks to human health. Therefore, evaluating the impact of this exposure is of great importance; however, it is often difficult because both the molecular mechanism of disease and the mode of action of the contaminants are complex. We used network biology techniques to quantitatively assess the impact of environmental contaminants on the human interactome and diseases with a particular focus on seven major contaminant categories: persistent organic pollutants (POPs), dioxins, polycyclic aromatic hydrocarbons (PAHs), pesticides, perfluorochemicals (PFCs), metals, and pharmaceutical and personal care products (PPCPs). We integrated publicly available data on toxicogenomics, the diseasome, protein–protein interactions (PPIs), and gene essentiality and found that a few contaminants were targeted to many genes, and a few genes were targeted by many contaminants. The contaminant targets were hub proteins in the human PPI network, whereas the target proteins in most categories did not contain abundant essential proteins. Generally, contaminant targets and disease-associated proteins were closely associated with the PPI network, and the closeness of the associations depended on the disease type and chemical category. Network biology techniques were used to identify environmental contaminants with broad effects on the human interactome and contaminant-sensitive biomarkers. Moreover, this method enabled us to quantify the relationship between environmental contaminants and human diseases, which was supported by epidemiological and experimental evidence. These methods and findings have facilitated the elucidation of the complex relationship between environmental exposure and adverse health outcomes.

DOI： 10.1016/j.ecoenv.2018.05.065

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) have been detected in tissues of both wild animals and humans. Several previous studies have suggested adverse effects of OH-PCBs on the endocrine and nervous systems in mammals. However, there have been no studies on transcriptome analysis of the effects of OH-PCBs, and thus, the whole picture and mechanisms underlying the adverse effects induced by OH-PCBs are still poorly understood. We therefore investigated the mRNA expression profile in the liver of adult male Wistar rats treated with 4-hydroxy-2,3,30,40,5-pentachlorobiphenyl (4-OH-CB107) to explore the genes responsive to OH-PCBs and to understand the potential effects of the chemical. Next-generation RNA sequencing analysis revealed changes in the expression of genes involved in the circadian rhythm and fatty acid metabolism, such as nuclear receptor subfamily 1, group D, member 1, aryl hydrocarbon receptor nuclear translocator-like protein 1, cryptochrome circadian clock 1, and enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase, in 4-OH-CB107-treated rats. In addition, biochemical analysis of the plasma revealed a dose-dependent increase in the leucine aminopeptidase, indicating the onset of liver damage. These results suggest that OH-PCB exposure May induce liver injury as well as disrupt the circadian rhythm and peroxisome proliferator-activated receptor-related fatty acid metabolism.

DOI： 10.1093/toxsci/kfy123

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記述言語：英語   掲載種別：研究論文（学術雑誌）

Body-size relationships between predators and their prey are important in ecological studies because they reflect the structure and function of food webs. Inspired by studies on the impact of global warming on food webs, the effects of temperature on body-size relationships have been widely investigated; however, the impact of environmental factors on body-size relationships has not been fully evaluated because climate warming affects various ocean environments. Thus, here, we comprehensively investigated the effects of ocean environments and predator-prey body-size relationships by integrating a large-scale dataset of predator-prey body-size relationships in marine food webs with global oceanographic data. We showed that various oceanographic parameters influence prey size selection. In particular, oxygen concentration, primary production and salinity, in addition to temperature, significantly alter body-size relationships. Furthermore, we demonstrated that variability (seasonality) of ocean environments significantly affects body-size relationships. The effects of ocean environments on body-size relationships were generally remarkable for small body sizes, but were also significant for large body sizes and were relatively weak for intermediate body sizes, in the cases of temperature seasonality, oxygen concentration and salinity variability. These findings break down the complex effects of ocean environments on body-size relationships, advancing our understanding of how ocean environments influence the structure and functioning of food webs.

DOI： 10.1098/rsos.180707

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記述言語：英語   掲載種別：研究論文（学術雑誌）

Herein, we propose using a nanosecond pulsed electric field (nsPEF) technique to assess teratogenicity and embryonic developmental toxicity of estradiol-17β (E 2 ) and predict the molecular mechanisms of teratogenicity and embryonic developmental defects caused by E 2 on medaka (Oryzias latipes). The 5 hour post-fertilization embryos were exposed to co-treatment with 10 μm E 2 and nsPEF for 2 hours and then continuously cultured under non-E 2 and nsPEF conditions until hatching. Results documented that the time to hatching of embryos was significantly delayed in comparison to the control group and that typical abnormal embryo development, such as the delay of blood vessel formation, was observed. For DNA microarray analysis, 6 day post-fertilization embryos that had been continuously cultured under the non-E 2 and nsPEF condition after 2 hour co-treatments were used. DNA microarray analysis identified 542 upregulated genes and one downregulated gene in the 6 day post-fertilization embryos. Furthermore, bioinformatic analyses using differentially expressed genes revealed that E 2 exposure affected various gene ontology terms, such as response to hormone stimulus. The network analysis also documented that the estrogen receptor α in the mitogen-activated protein kinase signaling pathway may be involved in regulating several transcription factors, such as FOX, AKT1 and epidermal growth factor receptor. These results suggest that our nsPEF technique is a powerful tool for assessing teratogenicity and embryonic developmental toxicity of E 2 and predict their molecular mechanisms in medaka embryos.

DOI： 10.1002/jat.3579

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記述言語：英語   掲載種別：研究論文（学術雑誌）

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRβ, is indispensable for amphibian metamorphosis. However, specific roles for the TRβ isoform in metamorphosis are poorly understood. To address this issue, we generated trβ-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of trβ in metamorphosis. trβ-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trβ itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of trβ. Surprisingly, trβ-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRβ is not required but contributes to the timing of resorptive events of metamorphosis.

DOI： 10.1242/bio.030338

Scopus

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記述言語：英語   掲載種別：研究論文（学術雑誌）

Dioxins cause various toxic effects through the aryl hydrocarbon receptor (AHR) in vertebrates, with dramatic species and strain differences in susceptibility. Although inbred mouse strains C3H/HeJ-lpr/lpr (C3H/lpr) and MRL/MpJ-lpr/lpr (MRL/lpr) are known as dioxin-sensitive and dioxin-resistant mice, respectively, the molecular mechanism underlying this difference remains unclear. The difference in the hepatic proteome of the two mouse strains treated with vehicle or 2,3,7,8-tetrabromodibenzo-p-dioxin (TBDD) was investigated by a proteomic approach of two-dimensional electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF). To confirm the strain-difference in response to TBDD treatment, cytochrome P450 (CYP) 1A1 and 1A2 protein levels were measured in both strains. A dose of 10 µg/kg body weight of TBDD induced hepatic CYP1A1 and CYP1A2 expression in both strains, but the expression levels of both CYP1A proteins were higher in C3H/lpr mice than in MRL/lpr mice, supporting that C3H/lpr mice are more sensitive to dioxins than MRL/lpr mice. Proteins that were more induced or suppressed by TBDD treatment in C3H/lpr mice were successfully identified by 2-DE and MALDI-TOF/TOF, including proteins responsible for AHR activation through production of endogenous ligands such as aspartate aminotransferase, indolethylamine N-methyltransferase, and aldehyde dehydrogenases, as well as proteins reducing oxidative stress, such as superoxide dismutase and peroxiredoxins. Taken together, our results provide insights into the molecular mechanism underlying the high dioxin susceptibility of the C3H/lpr strain, in which AHR activation by TBDD is more prompted by the production of endogenous ligands, but the adaptation to oxidative stress is also acquired.

DOI： 10.1007/s00204-016-1834-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）

The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5′-flanking regions of AHR1 and AHR2 genes. Both of the 5′-flanking regions in these AHR genes contained three potential xenobiotic-responsive elements (XREs). To assess whether the 5′-flanking region is transactivated by rsAHR1 and rsAHR2 proteins, we measured the transactivation potency of the luciferase reporter plasmids containing the 5′-flanking regions by AHR1 and AHR2 proteins that were transiently co-expressed in COS-7. Only reporter plasmid (pGL4-rsAHR2-3XREs) that contained three putative XRE sites in the 5′-flanking region of AHR2 gene showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins. TCDD-EC50 values for the rsAHR2-derived XRE transactivation were 1.3 and 1.4 nM for AHR1 and AHR2, respectively. These results suggest that the putative XREs of AHR2 gene have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene, is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.

DOI： 10.1007/s00204-016-1732-9

Scopus

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開催期間： 2022年06月30日 - 2022年07月02日   記述言語：日本語   開催地：札幌コンベンションセンター   国名：日本国  

疾患や有害事象といった生命現象は、たんぱく質などの生体分子の相互作用の結果として起こる。このため、これらの現象を理解するためには、生体分子がどのように相互作用しているのかを明らかにする必要がある。生体分子の相互作用からなる複雑なネットワークを理解するための学問は、ネットワーク生物学と呼ばれる。我々はこれまで、ネットワーク生物学を用いて化学物質がヒトに与える影響を明らかにしてきた。

まず我々は、環境中の化学物質がヒトの健康に与える影響をネットワーク生物学の観点から明らかにした。環境化学物質による健康への影響を評価・予測するためには、化学物質と疾患の関連性を評価することが不可欠である。しかし、疾患の発症メカニズムと化学物質の作用機序はともに複雑であり、疾患と化学物質の関連性を評価することは難しい。そこで、我々はネットワーク生物学の解析手法を用いて、化学物質がヒトの疾患に与える影響を定量的に評価した。この結果、ヒトのインタラクトームに重大な影響を与える化学物質群や化学物質感受性バイオマーカー遺伝子を同定することに成功した (Iida et al., Ecotoxicol. Environ. Saf., 2018) 。

さらに我々は、ネットワーク生物学とAI技術を統合することで、ヒトのインタラクトーム上で似ている疾患の情報を取り入れ、疾患の治療効果がある化学物質を効率よく探索する機械学習の手法を開発した (Iida et al., Bioinformatics, 2020)。本手法を用いることで、生物実験が行われていない化学物質の疾患に対する有効性を予測した。本手法は、有害事象を有する化学物質の探索にも応用できる。そこで現在、ヒトのインタラクトーム上で似ている化学物質の情報を取り入れ、有害事象を有する化学物質の探索を試みている。これらの方法および知見は、化学物質と生体の複雑な関係の解明に貢献すると期待できる。

その他リンク： https://doi.org/10.14869/toxpt.49.1.0_S13-2

開催期間： 2022年06月14日 - 2022年06月16日   記述言語：日本語   開催地：富山国際会議場   国名：日本国  

開催期間： 2021年09月27日 - 2021年09月29日   記述言語：日本語   開催地：On line   国名：日本国  

開催期間： 2020年09月01日 - 2020年09月03日   記述言語：日本語   開催地：On line   国名：日本国  

開催期間： 2020年06月13日 - 2020年06月16日   記述言語：英語   開催地：On line   国名：カナダ  

開催期間： 2021年09月12日 - 2021年09月13日   記述言語：日本語   開催地：On line   国名：日本国  

開催期間： 2018年11月04日 - 2018年11月08日   記述言語：英語   開催地：Sacramento   国名：アメリカ合衆国  

開催期間： 2018年09月11日 - 2018年09月12日   記述言語：英語   開催地：岐阜大学サテライトキャンパス   国名：日本国  

人間活動に起因する化学的な環境汚染から、環境汚染物質による健康障害が懸念されている。このような健康障害を予測し安全性を判定するには、汚染物質と疾患の関連性を評価することが不可欠である。しかし、疾患の発症メカニズムと化学物質の作用機序はともに複雑であり、疾患と化学物質の関連性を評価することは難しい。ネットワーク生物学１、２）はこのような複雑な関連性の理解に役立つ。そこで、我々はネットワーク生物学の解析手法を用いて、環境化学物質がヒトのインターラクトームや疾患に与える影響を定量的に評価した３）。

本研究では、環境中に放出されている様々な化学物質のうち、環境への影響が強く懸念されている７つの化学物質カテゴリー（残留性有機汚染物質（POPs）、ダイオキシン類、多環芳香族炭化水素化合物（PAHs）、殺虫剤、フッ素化合物、金属、製薬およびパーソナルケア製品（PPCPs））に着目した。

まず、化学物質と標的遺伝子の関係を調査するために、データベースに基づきヒト標的遺伝子－化学物質ネットワークを作成した。このネットワークを分析し、TCDDなどの一部の汚染物質は多様な遺伝子を標的としており、エストロゲンレセプターなどのいくつか遺伝子は多くの環境化学物質の標的となっていること見出した。

次に、汚染物質がヒトインターラクトームに与える影響を評価するため、ヒトのタンパク質－タンパク質相互作用（PPI）ネットワークを作成し、化学物質の標的遺伝子と相互作用する遺伝子の数（次数）を調査した。この結果、化学物質の標的遺伝子の平均次数はPPIに存在する全ての遺伝子の平均次数に比べ有意に高かった。これはPPIに対する汚染物質の影響が大きいことを意味する。また、生存に必須な遺伝子は一般に次数が高い１）ことから、化学物質の標的遺伝子は生存に必須な遺伝子であると予想した。これを検証するため、各化学物質カテゴリーの標的遺伝子に占める生存必須遺伝子の割合を調べた。この結果、PAHsと金属の標的遺伝子にのみ、生存必須遺伝子が有意にエンリッチしていた。これは、PAHsと金属の標的遺伝子は必須遺伝子である傾向にあることを示す。一方で、その他の化学物質は非必須遺伝子を標的にしていた。

最後に、化学物質と疾患の関係を定量的に評価するため、ヒトPPIネットワーク上で、ヒトの疾患原因遺伝子と化学物質標的遺伝子の距離を調べた。この距離は分子レベルの反応過程数を示し、距離が短いほどふたつの遺伝子の関連性が強いことを意味する２）。結果として、疾患原因遺伝子と化学物質標的遺伝子の距離はランダムに選んだ遺伝子間の平均距離よりも短いことを見出した。これは、その化合物が疾患原因遺伝子（またはこれに関連する遺伝子）と有意に関連することを示す。さらに、特定の疾患に注目し、化合物との関連性を定量的に評価した。疾患原因遺伝子をがん、消化器系疾患、内分泌系疾患等の１４グループに分け、グループごとに化学物質標的遺伝子とそれら疾病関連遺伝子の距離を求めた。この結果、全ての化学物質カテゴリーでがんや消化器系疾患との有意な関連性が認められた。また、POPsやダイオキシン類、殺虫剤、金属、PPCPs標的遺伝子は内分泌系疾患原因遺伝子との距離がランダムに比べ有意に短かった。これは化学物質特異的な疾患があることを意味する。

本研究では、ネットワーク生物学の技術を応用して、ヒトのインタラクトームに重大な影響を与える化学物質や汚染物質感受性バイオマーカ遺伝子を同定した。さらに、環境汚染物質とヒト疾患との関係を定量化した。得られた知見は疫学的および実験的証拠によって裏付けられた。これらの方法および知見は、環境汚染物質と健康障害の複雑な関係の解明に貢献すると期待できる。