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.     

Improvement in the DOA estimation of broadband signals usingpartially IIR beamformers

An ideal broadband beamformer requires an increased number of taps for each filter, which in turn increases the complexity of the design. It is shown that for angles near the broadside of the antenna the beamforming is more accurate compared to the angles near the endfire, and it is advantageous to use recursive filters combined with a computed number of zeros for angles near the endfire. The proposed method is used for estimating the arrival angle of the incoming signals and displays uniform accuracy for all angles     

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.     

Eosinophil chemotaxis induced by several biologically active substances and the effects of apafant on it in vitro

Chemotaxis of guinea pig eosinophils induced by various stimuli in use of a modified Boyden chamber technique in vitro and the effect of a platelet-activating factor (PAF) antagonist, apafant (CAS 105219-56-5, WEB 2086 BS), on it were examined. The eosinophils were obtained by bronchoalveolar lavage from the animals treated by i.v. injection with Sephadex G-200 and purified by Percoll density gradient centrifugation. PAF significantly and potently induced the chemotaxis at a broad range of 10(-17) to 10(-7) mol/l, where no concentration-dependency was observed. Leukotriene B4 also induced the chemotaxis in a concentration-dependent manner at 10(-14) to 10(-12) mol/l and the enhanced migration was not declined until 10(-7) mol/l. Interleukin-5 (IL-5), IL-8 and regulated on activation normal T expressed and secreted (RANTES) only modestly enhanced the chemotaxis in some concentrations at 10(-13) to 10(-7) mol/l with or without significance and with no concentration-dependency while formyl-methionyl-leucyl-phenylalanine (FMLP), a known chemoattractant, increased the migration at 10(-7) to 10(-5) mol/l. Apafant at 10(-8) to 10(-6) mol/l strongly and concentration-dependently inhibited 10(-8) mol/l PAF-induced chemotaxis. However, the drug showed nominal or no influences on their chemotaxis stimulated by the other agonists, at the concentrations of which the enhanced migration was observed. From these results, it is concluded that IL-5, IL-8 and RANTES, different from PAF and LTB4, are not potent stimuli for the eosinophil chemotaxis and that apafant is a selective antagonist of PAF, which is expected to be therapeutically effective for PAF-associated diseases including bronchial asthma.     

Percutaneous ethanol injection therapy by ethanol mixed with CO2 microbubble for hepatocellular carcinoma

One of the shortcomings of percutaneous ethanol injection therapy (PEIT) for hepatocellular carcinoma (HCC) is that many sessions are necessary to accomplish the treatment. This may be caused by which the ultrasonography (US) image does not reflect correctly to the kinetics of injected ethanol into HCC nodule. It is considered that number of treatment sessions are able to be reduced if we just enough injected labelled ethanol under US into HCC nodule. Therefore, we tried PEIT by ethanol mixed with CO2 microbubble (CO2 ethanol). The injected CO2 ethanol was aquired as hyperechoic image without strong acoustic shadow to the end of injection. Consequently we could reduce the number of treatment sessions to almost 1 for lesions < or = 3 cm in diameter and markedly reduce total dose of injected ethanol. The detectable rate of CO2 ethanol leaked out HCC nodule was high. No serious complication occurred. There have been only 1 lesion of local recurrence and no case of intrahepatic and peritoneal dissemination for 11.5 months on average of observation after PEIT by CO2 ethanol (CO2PEIT). These findings suggest that CO2PEIT is useful method for reducing the number of treatment sessions and total dose of injected ethanol, moreover preventing complication by ethanol leakage.     

Effects of pramipexole on contraversive rotation and functional motor impairments in 1-methyl-4-phenyl1,2,3, 6-tetrahydropyridine-induced chronic hemiparkinsonian monkeys

Rotational and functional motor behavioral changes were studied in five MPTP lesioned chronic hemiparkinsonian Macaca nemestrina monkeys after i.m. pramipexole, a predominant D2 subfamily agonist. Pramipexole induced contraversive rotations in a dose-dependent manner with an optimal dose of 56 microg/kg for approximately 2 to 4 hr after injection. Three different rating scales were used to determine drug-induced functional improvement. They included a monkey parkinsonism rating scale, volitional responses to fruit presentations, and number of hand movements that appeared volitional. A dose of 56 microg/kg of pramipexole produced functional improvements on hand disability, and on a parkinsonian rating scale for monkeys in a dose-dependent manner from 32 to 100 microg/kg. These doses produced an increase in significant hand movements in the affected (contralateral) as well as in the normal (ipsilateral) hand to the side of the brain lesion compared with 5% dextrose in water vehicle control. With a dose of 100 microg/kg, the therapeutic effects of pramipexole on hand movements were less than with 56 microg/kg, due to side effects such as scratching.     

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.