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.     

A redundant multivalued logic for a 10-Gb/s CMOS demultiplexer IC

A redundant multivalued logic is proposed for high-speed communication ICs. In this logic, serial binary data are received and converted into parallel redundant multivalued data. Then they are restored into parallel binary data. Because of the multivalued data conversion, this logic makes it possible to achieve higher operating speeds than that of a conventional binary logic. Using this logic, a 1:4 demultiplexer (DEMUX, serial-parallel converter) IC was fabricated using a 0.18-/spl mu/m CMOS process. The IC achieved an operating speed of 10 Gb/s with a supply voltage of only 1.3 V and with power consumption of 38 mW. This logic may achieve CMOS communication ICs with an operating speed several times greater than 10 Gb/s.     

An optically clocked transistor array with dual serial-to-parallel and parallel-to-serial conversion capability for optical label swapping

We propose an optically clocked transistor array optoelectronic integrated circuit (OEIC) for both serial-to-parallel and parallel-to-serial conversion (demux/mux), enabling an interface between high-speed asynchronous burst optical labels and CMOS circuitry for optical label swapping. Dual functionality of the OEIC reduces size, power, and cost of the optical label swapper. The capability for greater than 20-Gb/s conversion operation is demonstrated.     

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.     

Aminoglycosides prevent and dissociate the aggregation of platelets in patients with EDTA-dependent pseudothrombocytopenia

Although EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is of practical importance because failure to recognize this clinical entity may result in misdiagnosis and subsequent mismanagement of the patients, the pathophysiological nature of EDTA-PTCP remains unknown. To develop an effective way to evaluate the platelet counts in patients with EDTA-PTCP, we introduced aminoglycosides-supplemented anticoagulating agents. When kanamycin was pre-supplemented with EDTA for anticoagulating blood samples from EDTA-PTCP patients there was no significant change in the platelet counts and the morphology of blood cells after 150 min of incubation at room temperature. Furthermore, when kanamycin was added to EDTA-anticoagulated blood samples from EDTA-PTCP patients within 30 min after blood withdrawal, rapid dissociation of platelets without apparent morphological changes of blood cells was observed, and complete blood cell counts as well as the histogram patterns were almost the same as those examined immediately after blood sampling. The dissociation of aggregated platelets was also detected when other antibiotics were used, although it was associated with some extent of morphological changes of blood cells. These findings indicate that the supplementation of aminoglycosides either before or after blood sampling is a useful method for the diagnosis EDTA-PTCP and for the evaluation of platelet counts in patients with EDTA-PTCP.     

Microbial sensor for preliminary screening of mutagens utilizing a phage induction test

For the preliminary screening of mutagens, a novel microbial sensor system was developed utilizing a phage induction test. Escherichia coli lysogenic strain GY5027 and nonlysogenic strain GY5026 were used in this study. The number of living cells was determined by measuring the respiration of cells immobilized onto an oxygen electrode. The injection of a mutagen, such as AF-2 and MNNG, caused the phage induction in the lysogenic strain, resulting in the decreased respiration of only the lysogenic strain immobilized onto the oxygen electrode but not of nonlysogenic strain. The rate of current increase correlated well with the concentration of mutagens. The sensor responses to the antibiotics and bactericides were definitely different from those of mutagens. Therefore, utilization of this microbial sensor system makes possible the estimation of a substrate's mutagenicity.     

Acetazolamide challenge and technetium-99m-ECD versus iodine-123-IMP SPECT in chronic occlusive cerebrovascular disease

We compared the acetazolamide challenge test using 99mTc-ECD SPECT and 123I-IMP SPECT images in patients with chronic occlusive cerebrovascular disease. We also evaluated the usefulness of linearization correction for acetazolamide challenge test of 99mTc-ECD SPECT. METHODS: Twenty patients with unilateral chronic occlusive cerebrovascular disease (10 patients had middle cerebral arterial lesion and 10 had internal carotid lesion) were included in the study. Split-dose (a dose fractioning was 1:2), and sequential SPECT technique was used for 99mTc-ECD SPECT studies while only acetazolamide challenge test studies for 123I-IMP SPECT were performed. Permeability surface area product model (PS model) and back-diffusion model (Lassen's correction) were used for linearization correction of acetazolamide challenge with 99mTc-ECD SPECT. RESULTS: Six of 16 patients with reduced vasodilatory capacity in 123I-IMP SPECT were underestimated by 99mTc-ECD SPECT acetazolamide challenge test. Relative ECD uptake normalized by cerebellar uptake compared with IMP uptake showed a nonlinear relationship, indicating relatively less uptake in high flow range. The underestimations of limited vasodilatory capacity observed in 99mTc-ECD SPECT without linearization correction was modified by linearization algorithm. However, the effect of correction based on PS model was superior than that of Lassen's correction. The corrected 99mTc-ECD uptake ratio, based on PS model, and IMP uptake ratio demonstrated a better linear relationship than that of Lassen's correction. CONCLUSION: Technetium-99m ECD SPECT corrected based on the PS model is a better method of linearization for evaluating cerebrovascular reserve using acetazolamide challenge.     

Hair analysis for drugs of abuse. VI. The excretion of methoxyphenamine and methamphetamine into beards of human subjects

The excretion of methoxyphenamine (MOP) and methamphetamine (MA) into beards has been studied. Six healthy male subjects orally took 50 mg of MOP at a single dose and 7 doses for a successive 7 days. Their beard hairs were collected by an electric shaver every morning until MOP disappeared from the beard. After washing with 0.1% SDS, the beard samples were extracted with methanol-5 N HCl (20:1) under ultra-sonication for 1 h and the solution was kept overnight. MOP in the extract was determined by GC/MS using deuterium labelled MOP as an internal standard after trifluoroacetyl-derivatization. The drug concentrations in beard and the reproducibility of analysis were compared with the three procedures, unwashed, 0.1% SDS (wash I) and the additional ethanol (wash II) wash. The drug concentration in beard after SDS wash was 0.5-2.5 ng/mg lower than that in unwashed beard during the first 5-6 days. The drug concentration in beard after ethanol wash was much lower than that in the unwashed beard. The drug excreted into beard was detected 10 approximately 12 days for a single dose and 12-14 days for 7 doses after the last dosage at the cut off level of 1 ng/mg. On the contrary, the drug excreted in urine was not detected after more than 3 days after use. O-Desmethyl MOP, a major metabolite of MOP, was also detected in beard. The procedures were applied to the detection of MA in beard of MA abusers. It was realized that a beard sample was more useful than a urine sample assuming a longer detection.