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.     

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.     

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.     

Optical circulator consisting of a YIG spherical lens, PANDA-fibre polarisers and a fibre-optic polarising beam splitter

A novel optical circulator is reported which employs an yttrium-iron-garnet (YIG) sphere, PANDA-fibre polarisers and a fibre-optic polarising beam splitter. The splitting ratios of the fibre-optic polarising beam splitter were 1.0:99.0 for x-polarisation and 97.5:2.5 for y-polarisation, with an excess loss of 0.5 dB at 1.30 ?m wavelength. The backward isolation of the optical circulator was 30 dB with the total insertion loss of 4.5 dB.     

Linearity stablized fiber-optic thermometer using pseudo-heterodyne phase detection

A temperature sensor using a fiber-optic Fabry-Perot interferometer is described. A pseudo-heterodyne detection scheme is adopted to read the light phase difference in the Fabry-Perot interference output. A higher harmonic components comparison method is used to stabilize the system and to increase detection linearity. This system realizes not only highly sensitive temperature sensing with good linearity and minimal adjusting error, but also application to the sensing of other physical quantities such as vibrations. Additionally, the signal-to-noise ratio and distortion of the detected signal are investigated as functions of fiber end reflectivity. These results will be useful in designing a high performance fiber-optic Fabry-Perot thermometer.     

Clinical and serological study of tsutsugamushi disease in northern Osumi, Kagoshima Prefecture

We report 34 cases of tsutsugamushi disease seen from 1989 to 1993 at Yagi Clinic, northern Osumi, Kagoshima Prefecture. Nineteen patients (55.9%) showed the highest antibody titers against the Kawasaki strain Orientia tsutsugamushi (Ot) and 13 (38.2%) against the Kuroki strain Ot. It is suggested that two antigenic types (Kawasaki and Kuroki) of Ot were distributed in Kagoshima Prefecture, and the Kawasaki type Ot more or less dominates Kuroki type Ot. There was no difference in clinical features between the two groups of patients.     

Right atrial contractile performance in patients with myocardial infarction

To evaluate right atrial (RA) contractile performance in patients with myocardial infarction, we validated a cineangiographic method of RA volume measurement, and investigated RA volume change in 'normal' individuals and patients with a previous myocardial infarction. Sixteen silicone rubber RA casts made from human cadavers were filmed in the postero-anterior and left lateral projections. The cast volumes calculated following Simpson's rule were in good agreement with those measured by water replacement (r = 0.992, P < 0.01). At cardiac catheterization, biplane RA cineangiography was performed in 19 'normal' individuals (N group), in 14 patients with a previous antero-septal infarction (AMI group) and in seven patients with a previous inferior infarction (IMI group). The RA volume-time curve was constructed at 20-40 ms intervals for one cardiac cycle. RA volume at the beginning of the atrial contraction (RAVd), which was defined as the 'preload' of the RA, tended to be larger in both the AMI and IMI groups compared with 'normal' individuals. The RA ejection volume was significantly larger in both the AMI (18.4 +/- 2.1 ml.m-2, P < 0.01) and IMI groups (19.4 +/- 2.8, P < 0.01) than in the N group (14.5 +/- 1.9), even for a comparable level of RAVd (range from 26 to 36 ml.m-2) (18.6 +/- 2.1, P < 0.01, 18.2 +/- 2.0, P < 0.01, 14.7 +/- 1.9, respectively). These results suggest that RA contraction increases in patients with myocardial infarction by increasing both the 'preload' and 'contractility' of the RA.     

A 0.1-μm delta-doped MOSFET fabricated with post-low-energyimplanting selective epitaxy

A simple fabrication technology for delta-doped MOSFETs, named post-low-energy implanting selective epitaxy (PLISE) is presented. The PLISE technology needs no additional photo-lithography mask, deposition step or etching step even for CMOS devices. The only additional step is growing undoped epitaxial channel layers by UHV-CVD after the channel implantation. With this technology, delta-doped NMOSFETs with 0.1-μm gate length were successfully fabricated. By optimizing the epi-layer thickness and the channel doping level, short-channel effects are suppressed enough to achieve 0.1-μm gate length. Moreover, the junction capacitance at zero bias is reduced by 50%