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.     

Synthesis and photosensitivity of acryloylmorpholine copolymers with a pendant (meth)acryloyl group

We have developed new photopolymers that have superior waterproof properties and that can easily interact with polyfunctional acrylate compounds, thus serving as hydrophobic photocrosslinking reagents. Acryloylmorpholine monomers whose homopolymers were less moisture absorbing than the usual water‐soluble polymers but were still water soluble to a good degree, were copolymerized with other acryloyl monomers. We then introduced the photosensitive (meth)acryloyl group to side chains of the resulting polymers. Among six copolymers examined, the copolymers composed of acryloyl morpholine, hydroxyethyl acrylate, ethyl, or methyl methacrylate, and methacryloyl isocyanate were found to have nicely balanced hydrophilicity and waterproof properties, in addition to good compatibility with hydrophobic photocrosslinking reagents. The composite polymers thus obtained were confirmed to be promising photopolymers usable even in a highly humid environment. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 684–692, 2003     

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.     

Floating-island TFT leakage caused by process step reduction

The leakage mechanism for a top-gate thin-film transistor (TFT) produced using the fewest process steps in the industry is analyzed in order to achieve a high-contrast liquid crystal display (LCD). Using a T-shaped TFT structure, the OFF and ON channel lengths are defined independently, so that the leakage can be reduced with no ON current decrease     

Amyloid beta-protein deposition in the leptomeninges and cerebral cortex

To further investigate the process of amyloid beta-protein (Abeta) deposition, we determined, using sensitive enzyme immunoassays, the levels of Abeta40 and Abeta42 (Abetas) in the soluble and insoluble fractions of the leptomeninges (containing arachnoid mater and leptomeningeal vessels) and cerebral cortices from elderly control subjects showing various stages of Abeta deposition and from patients affected by Alzheimer's disease (AD). In both locations, insoluble Abeta levels were higher by orders of magnitude than soluble Abeta levels. Soluble Abeta levels in cortices were much lower than those in leptomeninges. In insoluble Abeta in the cortex, Abeta42 was by far the predominant species, and Abeta42 in AD cortices was characterized by the highest degree of modifications in the amino terminus. In contrast, this Abeta42 predominance was not observed in insoluble Abeta in the leptomeninges, which were found to be able to accumulate Abetas to an extent similar to that in the cortex, on a weight basis. The levels of insoluble Abeta in the leptomeninges or cortex generally correlated with the degree of cerebral amyloid angiopathy or the abundance of senile plaque, respectively. However, the presence of plaque-free cortical samples showing significant levels of insoluble Abeta42 suggests that biochemically detectable Abeta accumulation precedes immunocytochemically detectable Abeta deposition in the cortex.     

Photolithographic packaging with selectively occupied repeatedtransfer (PL-Pack with SORT) for scalable film optical linkmultichip-module (S-FOLM)

"Photolithographic packaging (PL-pack) with selectively occupied repeated transfer (SORT)" is proposed for optoelectronic microsystem integration. PL-pack with SORT integrates different types of thin-film device pieces into one substrate with desired configurations using an all-photolithographic process. A process design example is presented for a scalable film optical link multichip-module (S-FOLM). A preliminary estimation reveals that PL-Pack with SORT will achieve III-V epitaxial material saving of <1/100 and module cost reduction of <1/10, compared with flip-chip-bonding-based packaging. The result indicates that the process will save on cost and resources simultaneously. A critical issue is how to simplify the procedure for distributing thin-film device pieces onto a substrate. SORT is found to reduce the distribution step count typically by factor of <1/10-1/10000 compared with the conventional one-by-one method. PL-pack with SORT will be extended to the 3R process (reduce, reuse, recycle), which is generally applied to a variety of device/module fabrications     

Graphite formation on Ni film by chemical vapor deposition

Thin film formation of graphite by chemical vapor deposition using 2-methyl-1,2′-naphthyl ketone as a starting material was carried out on Ni film substrates. On Ni films directly deposited on quartz glass, the graphite films were obtained when the Ni film thickness was above 1 000 Å and above 5 000 Å at 700 °C and 1 000 °C, respectively. Depositions on thinner Ni film substrates comprise amorphous carbon (a-C) or graphite tubes which was owing to the thermal coagulation of the Ni film into droplets. On the other hand, graphite film was obtained on the Ni film with thickness 10 Å when a-C was inserted between the Ni film and the quartz glass. The coagulation of the Ni film is considered to be avoided by inserting a-C layer.