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.     

Effects of post annealing on removal of defect states in silicon oxynitride films grown by oxidation of silicon substrates nitrided in inductively coupled nitrogen plasma

Ultra thin (5 nm) silicon oxynitride (SiON) films were fabricated at a low temperature using nitrogen plasma generated by an inductively coupled plasma system. Effects of post-metalization annealing (PMA) of Al/SiON/Si MOS structure on the electrical properties of the SiON films were studied and correlations between the charge trapping states and the leakage current were established. Positive charge trapping by interface states generated by plasma damage was characterized by the hysteresis in high-frequency capacitance-voltage (C-V) characteristics. Hysteresis was observed to be completely removed by PMA while interface state density at the Si mid band gap reduced from 2.2×10¹³ to 3.7×10¹¹/eV/cm² and the oxide fixed charge density changed from 3.3×10¹² to −4×10¹¹/cm². The leakage current also decreased significantly, by more than two orders of magnitude, with PMA. The analysis of the leakage current using trap assisted tunneling (TAT) mechanism indicated that with PMA, the trap energy level in the SiON film becomes shallower from 1.3 to 0.7 eV. The positive trapped charges were observed to be annihilated by PMA and the trapping sites became neutral trap centers in the SiON film. This could lead to the reduction in the leakage current component given rise to by TAT.     

High performance liquid chromatographic separation of diacylglycerol acetates to quantitate disaturated species of lung phosphatidylcholine

A high-performance liquid chromatographic (HPLC) separation of diacylglycerol acetates to quantitatite disaturated species of lung phosphatidylcholine (PC) was studied. The diacylglycerol acetates were applied on a reversed phase column, eluted by an isocratic solvent, acetonitrile/isopropanol/water (35:15:1, v/v/v) at a flow rate of 1 ml/min, and detected by differential refractometry (RI). This isocratic HPLC method was useful to separate disaturated species from the others of lung PC. The quantitative analysis of the molecular species separated by HPLC was studied by RI detection. Chroamtograms obtained by RI detection and radioactivity determination of diacylglycerol [³H]acetates prepared by [³H]acetic anhydride were almost identical. The RI detector responsed in the same degree for different, authentic standards of diacylglycerol acetates. The detection limit with RI detection was about 30 nmoles. Molecular species of PCs from human lung and carcinoma tissues were analyzed by this HPLC method. The contents of disaturated species were very similar to those reported previously. These results indicate that RI detection is very useful in the nmole range for the quantitative analysis among the molecular species containing disaturated species.     

Transmittance and cathodoluminescence of AlN ceramics sintered with Ca3Al2O6 as sintering additive

Aluminum nitride ceramics were prepared by sintering with 0–4.8 mass% of Ca₃Al₂O₆ (C₃A) as a sintering additive. The transmittance in the range of 260–550 nm increased with increasing amount of C₃A. The cathodoluminescence intensity attributed to oxygen-induced defects decreased with increasing amount of C₃A. From the results, the increase of the transmittance in the range of 260–550 nm was considered to be related to the decrease of the oxygen-induced defect density.     

Ulcerative colitis with skip lesions at the mouth of the appendix: a clinical study

OBJECTIVE: We examined the clinical characteristics of ulcerative colitis patients who demonstrated endoscopically discontinuous lesions at the mouth of the appendix. METHODS: Of patients with initial or recurrent active ulcerative colitis who underwent total colonoscopy during the past 3 yr at Osaka City General Hospital, we selected those who had skip lesions in the mouth of the appendix before treatment, and examined their gender, age, disease type, sites of lesions, inflammatory reaction, severity of disease, effects of treatment, and posttreatment course. RESULTS: Discontinuous lesions at the mouth of the appendix were found in 10 patients, who had the following common clinical features: the major lesion was usually present in the lower part of the large bowel including the rectum, many of the patients had suffered an initial attack only, all patients had mild disease, and many of the patients responded quite satisfactorily to treatment with salicylazosulfapyridine. CONCLUSION: Numerous patients with ulcerative colitis with discontinuous lesions at the mouth of the appendix were observed and their clinical characteristics were examined. Determination of the clinical significance of skip lesions in the appendix will contribute to elucidation of the pathogenesis of ulcerative colitis.     

Formation of toxic aldehydes in cod liver oil after ultraviolet irradiation

Formaldehyde, acrolein, malonaldehyde (MA), acetaldehyde and propanal produced from cod liver oil upon ultraviolet irradiation (λ_max=300 nm) were derivatized into nitrogenor sulfur-containing compounds and then analyzed by capillary gas chromatography with a nitrogen-phosphorus detector or a flame photometric detector. Acrolein and MA were formed at levels of 10.9±3.06 and 190.2±38.4 nmol/mg of fish oil, respectively. Maximum levels of formaldehyde, acetaldehyde and propanal formed were 7.0±0.90, 49.1±4.5 and 35.8±4.0 nmol/mg of oil, respectively. Formation of propanal in large quantities corresponded to the high content of ɛ-3 fatty acids in cod liver oil.     

A new type of arc plasma reactor with 12-phase alternating current discharge for synthesis of carbon nanotubes

A new type of arc plasma reactor with 12-phase alternating current (AC) discharge for synthesis of carbon nanotubes (CNTs) is proposed. A couple of six discharge electrodes by which have mutually electrical connection between them to enlarge the high-temperature regions in the reactor are arranged to three-dimensional locations. A new method of CNTs fabrication by this reactor, which accomplishes to enlarge the suitable growth region in high purity and at high yield, was developed.     

Generation of adaptive gait patterns for quadruped robot with CPG network including motor dynamic model

This paper describes the generation of adaptive gait patterns using new Central Pattern Generators (CPGs) including motor dynamic models for a quadruped robot under various environments. The CPGs act as the flexible oscillators of the joints and adjust joint angles to required values. The CPGs are interconnected with each other and sets of their coupling parameters are adjusted by a genetic algorithm so that the quadruped robot can realize stable and adequate gait patterns. Generation of gait patterns results in the formation of the CPG networks suitable for the formation of not only a straight walking pattern but also of rotating gait patterns. Experimental results demonstrate that the proposed CPG networks are effective for the automatic adjustment of the adaptive gait patterns for the tested quadruped robot under various environments. Furthermore, the target tracking control based on image processing is achieved by combining the general gait patterns. © 2006 Wiley Periodicals, Inc. Electr Eng Jpn, 155(1): 35–43, 2006; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/eej.20225