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.     

Upflow anaerobic sludge blanket (UASB) treatment of supernatant of cow manure by thermal pre-treatment.

Upflow anaerobic sludge blanket (UASB) methane fermentation treatment of cow manure that was subjected to screw pressing, thermal treatment and subsequent solid-liquid separation was studied. Conducting batch scale tests at temperatures between 140 and 180 degrees C, the optimal temperature for sludge settling and the color suppression was found to be between 160-170 degrees C. UASB treatment was carried out with a supernatant obtained from the thermal treatment at the optimal conditions (170 degrees C for 30 minutes) and polymer-dosed solid-liquid separation. In the UASB treatment with a COD(Cr) loading of 11.7 kg/m3/d and water temperature of 32.2 degrees C, the COD(Cr) level dropped from 16,360 mg/L in raw water to 3,940 mg/L in treated water (COD(Cr), removal rate of 75.9%), and the methane production rate per COD(Cr) was 0.187 Nm3/kg. Using wastewater thermal-treated at the optimal conditions, also a methane fermentation treatment with a continuously stirred tank reactor (CSTR) was conducted (COD(Cr) in raw water: 38,000 mg/L, hydraulic retention time (HRT): 20 days, 35 degrees C). At the COD(Cr) loading of 1.9 kg/m3/d, the methane production rate per COD(Cr), was 0.153 Nm3/kg. This result shows that UASB treatment using thermal pre-treatment provides a COD(Cr), loading of four times or more and a methane production rate of 1.3 times higher than the CSTR treatment.     

High-gain hybrid amplifier consisting of cascaded fluoride-based TDFA and silica-based EDFA in 1458-1540 nm wavelength region

A hybrid optical fibre amplifier is described that consists of a fluoride-based thulium-doped fibre amplifier and a silica-based erbium-doped fibre amplifier connected in a cascade. The amplifier has a gain of more than 25 dB and a noise figure of less than 9 dB over a wide wavelength region of 1458-1540 nm.     

High-resolution long-array thermal ink jet printhead fabricated by anisotropic wet etching and deep Si RIE

This paper describes the fabrication and characterization of a thermal ink jet (TIJ) printhead suitable for high speed and high-quality printing. The printhead has been fabricated by dicing the bonded wafer, which consists of a bubble generating heater plate and a Si channel plate. The Si channel plate consists of an ink chamber and an ink inlet formed by KOH etching, and a nozzle formed by inductively couple plasma reactive ion etching (ICP RIE). The nozzle formed by RIE has squeezed structures, which contribute to high-energy efficiency of drop ejector and, therefore, successful ejection of small ink drop. The nozzle also has a dome-like structure called channel pit, which contributes to high jetting frequency and high-energy efficiency. These two wafers are directly bonded using electrostatic bonding of full-cured polyimide to Si. The adhesive-less bonding provided an ideal shaped small nozzle orifice. Use of the same material (Si substrate) in heater plate and channel plate enables the fabrication of high precision long printhead because no displacement and delamination occur, which are caused by the difference in thermal expansion coefficient between the plates. With these technologies, we have fabricated a 1" long printhead with 832 nozzles having 800 dots per inch (dpi) resolution and a 4 pl. ink drop volume.     

Driving voltage reduction in a two-phase CCD by suppression ofpotential pockets in inter-electrode gaps

This study reports an optimum design for a two-phase charge-coupled device (CCD) and limitations on its driving voltage reduction. The two-phase CCD to be used as a horizontal-CCD (H-CCD) in a CCD image sensor requires low-voltage and high-speed operation. Reducing the driving voltage, however, may induce potential pockets in the channel under the inter-electrode gaps which results in a fatal decrease in charge-transfer efficiency. In this case it is necessary to optimize the CCD design to be free of pocket generation. For this requirement, we conducted two-dimensional (2-D) device simulations for the two-phase CCD, whose potential barriers are formed by boron ion-implantation. Our simulations indicated that the edge position of the potential barrier region and the dose of boron-ion implantation would be important parameters for controlling the size of potential pockets. At an optimum edge position and a boron dose, the minimum driving voltage appears to be reducible to 1.1 V. Characteristics of potential pockets and methods of their suppression are also discussed