インドネシアのスラウェシ島固有のマカクにおける種分化の遺伝的基盤

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院京都大学新制・課程博士博士(理学)甲第24185号理博第4876号京都大学大学院理学研究科生物科学専攻(主査)教授 今井 啓雄, 教授 古賀 章彦, 准教授 Huffman Michael Alan学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDGA

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology

Evaluation of Object Surface Edge Profiles Detected with a 2-D Laser Scanning Sensor

Canopy edge profile detection is a critical component of plant recognition in variable-rate spray control systems. The accuracy of a high-speed 270° radial laser sensor was evaluated in detecting the surface edge profiles of six complex-shaped objects. These objects were toy balls with a pink smooth surface, light brown rectangular cardboard boxes, black and red texture surfaced basketballs, white smooth cylinders, and two different sized artificial plants. Evaluations included reconstructed three-dimensional (3-D) images for the object surfaces with the data acquired from the laser sensor at four different detection heights (0.25, 0.50, 0.75, and 1.00 m) above each object, five sensor travel speeds (1.6, 2.4, 3.2, 4.0, and 4.8 km h−1), and 8 to 15 horizontal distances to the sensor ranging from 0 to 3.5 m. Edge profiles of the six objects detected with the laser sensor were compared with images taken with a digital camera. The edge similarity score (ESS) was significantly affected by the horizontal distances of the objects, and the influence became weaker when the objects were placed closer to each other. The detection heights and travel speeds also influenced the ESS slightly. The overall average ESS ranged from 0.38 to 0.95 for all the objects under all the test conditions, thereby providing baseline information for the integration of the laser sensor into future development of greenhouse variable-rate spray systems to improve pesticide, irrigation, and nutrition application efficiencies through watering booms

Functional divergence of the bitter receptor TAS2R38 in Sulawesi macaques

Bitter perception is mediated by G protein‐coupled receptors TAS2Rs and plays an important role in avoiding the ingestion of toxins by inducing innate avoidance behavior in mammals. One of the best‐studied TAS2Rs is TAS2R38, which mediates the perception of the bitterness of synthetic phenylthiocarbamide (PTC). Previous studies of TAS2R38 have suggested that geographical separation enabled the independent divergence of bitter taste perception. The functional divergence of TAS2R38 in allopatric species has not been evaluated. We characterized the function of TAS2R38 in four allopatric species of Sulawesi macaques on Sulawesi Island. We found variation in PTC taste perception both within and across species. In most cases, TAS2R38 was sensitive to PTC, with functional divergence among species. We observed different truncated TAS2R38s that were not responsive to PTC in each species of Macaca nigra and M. nigrescens due to premature stop codons. Some variants of intact TAS2R38 with an amino acid substitution showed low sensitivity to PTC in M. tonkeana . Similarly, this intact TAS2R38 with PTC‐low sensitivity has also been found in humans. We detected a shared haplotype in all four Sulawesi macaques, which may be the ancestral haplotype of Sulawesi macaques. In addition to shared haplotypes among Sulawesi macaques, other TAS2R38 haplotypes were species‐specific. These results implied that the variation in TAS2R38 might be shaped by geographical patterns and local adaptation

EIF3D promoted cervical carcinoma through Warburg effect by interacting with GRP78

The incidence of cervical cancer ranks third among all female tumours globally and second in developing countries. However, the role of eukaryotic translation initiation factor 3 subunit D (EIF3D) in cervical carcinoma is unknown. This study investigated the effects of EIF3D on cell progression of cervical carcinoma and its underlying mechanism in vivo and vitro models. There were increases of EIF3D expression mRNA and protein expression levels in patients with cervical carcinoma. Disease-free survival (DFS) and overall surviva (OS) of EIF3D lower expression in patients with cervical carcinoma was higher than those of EIF3D higher expression. EIF3D mRNA expression levels in cervical carcinoma cell lines (AV3, Hela229, CaSki and Hela cells) were up-regulated, compared with cervical normal cell line (UVECs). EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma. EIF3D interacting with GRP78 to reduce the activity of GRP78 in vitro model of cervical carcinoma. The inhibition of GRP78 reduced the effects of EIF3D on Warburg effect in vitro model of cervical carcinoma. Our work identifies EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma and the inhibition of EIF3D represents a potential therapeutic strategy for the treatment of cervical carcinoma.IMPACT STATEMENT What is already known on this subject? The incidence of cervical cancer ranks third among all female tumours globally and second in developing countries. What do the results of this study add? This study investigated the effects of EIF3D on cell progression of cervical carcinoma and its underlying mechanism in vivo and vitro models. What are the implications of these findings for clinical practice and/or further research? EIF3D promoted cell growth and Warburg effect in vitro model of cervical carcinoma and the inhibition of EIF3D represents a potential therapeutic strategy for the treatment of cervical carcinoma

Role of YAP/TAZ in Cell Lineage Fate Determination and Related Signaling Pathways

The penultimate effectors of the Hippo signaling pathways YAP and TAZ, are transcriptional co-activator proteins that play key roles in many diverse biological processes, ranging from cell proliferation, tumorigenesis, mechanosensing and cell lineage fate determination, to wound healing and regeneration. In this review, we discuss the regulatory mechanisms by which YAP/TAZ control stem/progenitor cell differentiation into the various major lineages that are of interest to tissue engineering and regenerative medicine applications. Of particular interest is the key role of YAP/TAZ in maintaining the delicate balance between quiescence, self-renewal, proliferation and differentiation of endogenous adult stem cells within various tissues/organs during early development, normal homeostasis and regeneration/healing. Finally, we will consider how increasing knowledge of YAP/TAZ signaling might influence the trajectory of future progress in regenerative medicine.ISSN:2296-634

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology

Pore structure and fractal characteristics of coal-bearing Cretaceous Nenjiang shales from Songliao Basin, Northeast China

Shale oil and gas resources mainly exist in the pore and fracture system. Quantitative characterization of pore development characteristics and gas-bearing properties is crucial for shale reservoir evaluation. The pore development of shale reservoir exhibits strong complexity and heterogeneity, and research on pore development characteristics of coal measure shale lags behind that of marine shale reservoir. Hence, it is urgent to investigate the pore heterogeneity characteristics of coal-bearing shale and its influence on gas bearing properties. Therefore, the coal-bearing Cretaceous Nenjiang shales from the Songyuan area of the Songliao Basin were selected as the research object in this study. Through total organic carbon (TOC) analysis, X-ray diffraction experiments, porosity analysis, nitrogen adsorption–desorption experiments, and methane isothermal adsorption experiments, the characteristics of pore structure, heterogeneity, and gas bearing properties of coal-bearing shale were analyzed. The influence of rock and mineral components on pore structure and heterogeneity characteristics, the relationship between pore structure characteristics and fractal characteristics, and the effects of pore structure and heterogeneity on gas bearing properties were also discussed. The results show that: (1) The organic matter abundance of the shale in the Nenjiang Formation does not change significantly (the average TOC content is 2.38%). Ink bottle-shaped pores are mostly developed, and the Nenjiang shale is rich in clay minerals (average content 55.6%), with slit-shaped pores mostly developed. The pore surface of shale exhibits obvious fractal characteristics, with average fractal dimensions D1 and D2 of 2.54 and 2.74, respectively, indicating that the internal structure is more complex than the surface structure. (2) The enrichment of organic carbon increases the specific surface area by affecting the development of micropores and pores, consequently increasing the fractal dimension of pores. Similarly, the development of clay minerals increases the number of mesopores and macropores, thereby increasing the fractal dimension of pores. (3) Small pores develop larger specific surface areas, which increases the complexity and heterogeneity of the pore structure. This promotes remarkable fractal characteristics, expands the adsorption sites, and improves the adsorption capacity. This work will provide a scientific theoretical basis for the comprehensive evaluation of coal-bearing shale reservoirs and research on shale gas reservoir formation theory

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology