Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.     

Radiophotoluminescence phenomenon in copper-doped aluminoborosilicate glass

Radiophotoluminescence phenomena have been widely investigated on various types of materials for dosimetry applications. We report that an aluminoborosilicate glass containing 0.005 mol% copper exhibits intense photoluminescence in the visible region induced by X-ray and γ-ray irradiation. The luminescence is assigned to the 3d⁹4s¹ → 3d¹⁰ transition of Cu⁺. The proportionality of the intensity of the induced photoluminescence to the irradiation dose was confirmed up to 0.5 kGy using ⁶⁰Co γ-ray irradiation. Based on the spectroscopic results, a potential mechanism was proposed for the enhancement of the photoluminescence. The exposure to the ionizing radiation generates electron-hole pairs in the glass, and the electrons are subsequently captured by the Cu²⁺ ions, which are converted to Cu⁺ and emit the luminescence. For the glass containing 0.01 mol% copper, the pronounced enhancement of the photoluminescence was not observed because the reverse reaction, ie, the capture of the holes by the Cu⁺ ions, becomes prominent. The photoluminescence induced by the irradiation was stably observed for the glasses kept at room temperature and even for the glasses heat-treated at 150°C. However, the induced photoluminescence could be eliminated by the heat treatment at a temperature at 500°C, and the glass returned to the initial pre-irradiation state. The Cu-doped aluminoborosilicate glass is a potential candidate for use in dosimetry applications.     

Quantitative risk assessment using a Japanese hydrogen refueling station model

Although hydrogen refueling stations (HRSs) are becoming widespread across Japan and are essential for the operation of fuel cell vehicles, they present potential hazards. A large number of accidents such as explosions or fires have been reported, rendering it necessary to conduct a number of qualitative and quantitative risk assessments for HRSs. Current safety codes and technical standards related to Japanese HRSs have been established based on the results of a qualitative risk assessment and quantitative effectiveness validation of safety measures over ten years ago. In the last decade, there has been much development in the technologies of the components or facilities used in domestic HRSs and much operational experience as well as knowledge to use hydrogen in HRSs safely have been gained through years of commercial operation. The purpose of the present study is to conduct a quantitative risk assessment (QRA) of the latest HRS model representing Japanese HRSs with the most current information and to identify the most significant scenarios that pose the greatest risks to the physical surroundings in the HRS model. The results of the QRA show that the risk contours of 10^?3 and 10^?4 per year were confined within the HRS boundaries, whereas the risk contours of 10^?5 and 10^?6 per year are still present outside the HRS. Comparing the breakdown of the individual risks (IRs) at the risk ranking points, we conclude that the risk of jet fire demonstrates the highest contribution to the risks at all of the risk ranking points and outside the station. To reduce these risks and confine the risk contour of 10^?6 per year within the HRS boundaries, it is necessary to consider risk mitigation measures for jet fires.     

Combustion synthesis of AlN doped with carbon and oxygen

Carbon-and-oxygen-doped AlN specimens were prepared by combustion synthesis using Al, graphite, and AlN. Graphite addition changed the product color from white to blue. By XRD, the lattice constant increased slightly with increasing carbon content. Blue AlN powder was synthesized with a molar ratio of the diluent AlN of 0.2-0.5 with a fixed graphite content of 0.05. At an AlN molar ratio exceeding 0.6, carbon was not successfully incorporated due to the lower reaction temperature. Calcination at 800°C in air removed residual graphite without changing the crystal structure or product color. Oxygen, nitrogen, and carbon analyses revealed that blue AlN powders contained 0.45-0.54 mass% carbon and 1.4-1.6 mass% oxygen, while the undoped AlN contained 0.021 mass% carbon and 0.94 mass% oxygen. The origin of the white-to-blue color change was investigated via reflection measurements. Blue AlN exhibits an absorption peak at 634 nm (1.96 eV). From first-principles electronic structure calculations, the C-doped AlN and carbon-and-oxygen-doped AlN with a 1:1 ratio could be classified as p-type, whereas the O-doped AlN and 1:3 carbon-and-oxygen-doped AlN were n-type. One reason for the absorption peak at 634 nm may be a transition from the conduction band to an upper unoccupied state. These results suggest the possible control of optical and electronic properties of AlN via carbon-and-oxygen doping.     

Graded evolution of anisotropic microstructure during sintering from crystal-oriented powder compact

Anisotropic sintering, including shrinkage and grain growth, was examined for c-axis-oriented (Sr,Ca)₂NaNb₅O₁₅ (SCNN) ceramics, which were prepared by colloidal processing under a magnetic field. In the c-axis-oriented SCNN powder compact, shrinkage and grain growth along the c-axis were higher than those along the a-axis. The anisotropic microstructural development was clearly associated with anisotropic sintering shrinkage. X-ray diffraction, scanning electron microscopy, and energy back scattering diffraction showed that the grain growth of oriented particles by including random grains contribute to the development of the oriented microstructure. Finally, the highly crystal-oriented SCNN ceramics with a densified microstructure were obtained through anisotropic sintering. These results clearly showed the potential to develop a well-defined anisotropic microstructure during sintering by designing and controlling the particle packing structure in a powder compact.     

Hopping and optical absorption of electrons in nano-porous crystal 12CaO·7Al2O3

Recently it has been discovered that a nano-porous main group oxide 12CaO·7Al₂O₃ (C12A7) can be converted from a wide-gap insulator to a good transparent conductor. Using ab initio modelling we explain good conductivity of this material by very small barriers for hopping of localised electrons between neighbouring positive cages. We show that optical absorption of C12A7 in infrared region and at energies higher than 2.7 eV is due to inter-cage and intra-cage electron transitions, respectively. The proposed mechanisms can be useful in further search for conducting transparent media.     

High performance of silicided silicon-sidewall source and drain (S4D) structure

A silicided silicon-sidewall source and drain (S⁴D) structure is proposed for sub-0.1-μm devices. The merit of the S⁴D structure is that the series resistance of the source and drain is significantly reduced since the silicide layer is attached very close to the gate electrode and the silicon sidewall can be doped very highly. Thus, very high drain current drive can be expected, Another advantage of this structure is that the source and drain extensions are produced by the solid-phase diffusion of boron from the highly doped silicon-sidewall. Thus, shallow extensions with very high doping can be realized. A 75-nm gate length pMOSFET fabricated with this structure is shown to exhibit excellent electrical characteristics     

Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs

Zona-free mouse eggs at the pronucleus stage were infected with a replication-defective adenovirus vector containing a nuclear-targeted lacZ gene. Exogenous beta-galactosidase activity was detected in almost all eggs at the two-cell stage. Of 27 mice that developed from infected eggs, three carried the integrated exogenous gene mediated by the adenovirus. Two of the three expressed the lacZ gene, and all three mice transmitted the adenovirus-mediated transgene to F1 progeny Southern blot analysis was consistent with single copy integration. This finding should accelerate the development of new strategies for transgenesis and assist studies on the function of cloned genes in vivo.     

Radiation therapy for stage I-II non-small cell lung cancer in patients aged 75 years and older

Vaccination with peptides that induce a specific immune response is a potential prophylactic or therapeutic strategy against viral infections and tumors. Because of the extensive polymorphism of the HLA loci, synthetic peptide vaccines must consist of a cocktail of peptides that bind specifically to different HLA molecules. Such cocktails should be optimized for the target population as each population has its specific HLA gene frequencies. To achieve maximum population coverage with a minimum number of peptides, information is needed on the ranking of the most frequent HLA phenotypes. We introduce the minimal phenotype panel, which is the smallest combination of HLA antigens selected so that the proportion of individuals in a population that express at least one of the antigens in the panel exceeds a desired minimum value. We developed a method for assembling minimal phenotype panels based on known HLA class I gene frequencies. We give an example based on a set of 2446 well-defined HLA-typed, random, healthy, unrelated, Dutch Caucasoid individuals. In addition, we discuss the possibility of assembling minimal phenotype panels based on two-locus haplotypes, which enables the assembly of phenotype panels from the antigens of both loci.