Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs

Prevalence of allergic rhinitis based on the SACRA questionnaire among Japanese nursing professionals with asthma

Although adult asthma is attributable to occupational factors and asthma and rhinitis are related, relatively few studies have investigated the prevalence of occupational rhinitis based on occupation, and knowledge of occupational rhinitis in Japan is currently limited. The objective of this cross-sectional study was to estimate the prevalence of allergic rhinitis among Japanese nursing professionals with asthma. A postal survey was conducted from October to December 2013 using translated versions of the European Community Respiratory Health Survey for the prevalence of asthma and State of the Impact of Allergic Rhinitis on Asthma Control questionnaire for the prevalence of rhinitis. Of 4,634 Japanese nursing professionals, 497 subjects had asthma, and 270 of these 497 subjects had allergic rhinitis (54.3% ; 95% confidence interval [CI], 49.7-58.7). Latex allergy was significantly associated with allergic rhinitis (odds ratio, 1.77 ; 95% CI, 1.21-2.60). There was no relationship between employment period and prevalent allergic rhinitis. The results of this study provide fundamental information regarding occupational health among Japanese nursing professionals, including the prevalence of allergic rhinitis among Japanese nursing professionals with asthma and latex allergy as a potential risk factor for prevalent allergic rhinitis

Two-dimensional direct numerical simulation of spray flames – Part 2: Effects of ambient pressure and lift, and validity of flamelet model

The effect of ambient pressure on spray flames is investigated by means of two-dimensional direct numerical simulation (DNS), and the validity of an extended flamelet/progress-variable approach (EFPV) is examined under the high-pressure condition. The DNS is performed not only for a simple jet spray flame with a pilot burner but also for a lifted recirculation spray flame without any pilot burner at ambient pressures of 0.1 and 0.5 MPa. n-decane (C[10]H[22]) is used as liquid spray fuel, and the evaporating droplets’ motions are tracked by the Lagrangian method. The results show that the behaviors of jet and lifted recirculation spray flames are strongly affected by ambient pressure. The effects of the change of the ambient pressure on these spray flame behaviors can be well captured by EFPV and EFPV coupled with G-equation model (EFPV-G), respectively

数値解析による液滴蒸発および燃焼振動に関する研究

京都大学0048新制・課程博士博士(工学)甲第19685号工博第4140号新制||工||1639(附属図書館)32721京都大学大学院工学研究科機械理工学専攻(主査)教授 小森 悟, 教授 中部 主敬, 教授 稲室 隆二学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA

Evaporation and combustion of multicomponent fuel droplets

The effects of difference in fuel components on the droplet evaporation and combustion are numerically investigated. Jet-A is used as liquid fuel, and one (n-decane)-, two (n-decane and 1, 2, 4-trimethyl-benzene)- and three (n-dodecane, iso-octane and toluene)-component fuels are used as the surrogate fuels of Jet-A. The results show that the evaporation of the three-component surrogate fuel becomes faster and slower than those of the one- and two-component surrogate fuels in the initial and subsequent evaporating periods, respectively. The differences in the gas temperature evolution among these three different surrogate fuels are remarkable right after the ignition, but become small with time

Effects of ambient pressure, gas temperature and combustion reaction on droplet evaporation

The effects of ambient pressure, initial gas temperature and combustion reaction on the evaporation of a single fuel droplet and multiple fuel droplets are investigated by means of three-dimensional numerical simulation. The ambient pressure, initial gas temperature and droplets’ mass loading ratio, ML, are varied in the ranges of 0.1–2.0 MPa, 1000–2000 K and 0.027–0.36, respectively, under the condition with or without combustion reaction. The results show that both for the conditions with and without combustion reaction, droplet lifetime increases with increasing the ambient pressure at low initial gas temperature of 1000 K, but decreases at high initial gas temperatures of 1500 K and 2000 K, although the droplet lifetime becomes shorter due to combustion reaction. The increase of ML and the inhomogeneity of droplet distribution due to turbulence generally make the droplet lifetime longer, since the high droplets’ mass loading ratio at local locations causes the decrease of gas temperature and the increase of the evaporated fuel mass fraction towards the vapor surface mass fraction

Rice ERECT LEAF 1 acts in an alternative brassinosteroid signaling pathway independent of the receptor kinase OsBRI1

<p>ERECT LEAF 1 (ELF1) was previously identified as a component of brassinosteroid signaling in rice. A double mutant obtained by crossing <i>elf1-1</i> (a null mutant of <i>ELF1</i>) with <i>d61-1</i> (a leaky mutant of <i>OsBRI1</i>) showed a more severe phenotype than did the <i>elf1-1</i> single mutant, resembling that of a severe brassinosteroid-deficient mutant. Microarray analysis showed that the gene expression profile of <i>elf1-1</i> was distinct from that of <i>d61-12</i> (a leaky mutant of <i>OsBRI1</i> with a phenotype similar to that of <i>elf1-1</i>), and fewer than half of genes differentially expressed between the wild-type and <i>elf1-1</i> showed similar differences in <i>d61-12</i> relative to the wild-type. These results indicate that less than half of ELF1-regulated genes in rice seedlings are affected by OsBRI1, and suggest that ELF1 acts in a rice brassinosteroid signaling pathway different from that initiated by OsBRI1. Gene expression analysis showed that some stress response-related genes were induced in <i>elf1-1</i> but not in <i>d61-12</i>, and 8 of 9 genes oppositely regulated in <i>elf1-1</i> and <i>d61-12</i> were significantly up- or down-regulated in both <i>elf1-1</i> and jasmonic acid-treated wild-type. These results imply that ELF1 suppresses stress-induced signalling, and that jasmonic acid signaling is stimulated in <i>elf1-1</i>; therefore, ELF1 may be involved in the brassinosteroid-mediated suppression of jasmonic acid response in rice.</p