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.     

Hot carrier analysis in low-temperature poly-Si thin-film transistors using pico-second time-resolved emission microscope

We have analyzed degradation of N-channel thin-film-transistor (TFT) under dynamic stress using a pico-second time-resolved emission microscope. We have successfully detected emission at pulse fall edge for the first time. Emission intensity increased with the decrease of pulse fall time. As the degradation depended on the pulse fall time, this dependence clearly illustrates that hot electrons are the dominant cause of the degradation under dynamic stress. Based on these dependences, we proposed a model considering electron traps in the poly-Si.     

Development and exploitation of a multipurpose CFD tool for optimisation of microbial reaction and sludge flow.

A numerical analysis technique for optimisation of microbial reaction and sludge flow has been developed in this study. The technique is based on the 3D multiphase Navier-Stokes solver with turbulence models. In order to make numerical analyses of the total processes in wastewater treatment plants possible, four numerical models, the microbial reaction model, a sludge settling model, oxygen mass transfer model from coarse bubbles, and a model from fine bubbles, are added to the solver. All parameters included in those models are calibrated in accordance with experimental results, and good agreements between calculated results and experimental results are found. Finally, this study shows that the numerical technique can be used to optimise wastewater treatment plants with an example of the operational optimisation of an intermittent agitation in anoxic reactors by coarse bubbles. With a proper appreciation of its limit and advantages, the exploitation of the CFD efficiently leads us to the right direction even though it is not quantitatively perfect.     

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.     

Visualization of mitochondria with green fluorescent protein in cultured fibroblasts from patients with mitochondrial diseases

cDNAs for green fluorescent protein (GFP) and for a GFP fusion protein containing the presequence of human ornithine transcarbamylase (pOTC-GFP) were transfected into cultured human fibroblasts. GFP cDNA gave diffuse fluorescence throughout the cytoplasm and the nucleus, whereas pOTC-GFP cDNA gave mitochondria-associated fluorescence. Fluorescent mitochondrial structures could be classified into five patterns: thread-like mitochondria, fine thread-like ones, rod-like ones, granular ones, and granular ones with weak cytosolic fluorescence. pOTC-GFP mutants resulted in a loss of mitochondrial fluorescence and an appearance of weak fluorescence throughout the cytoplasm. pOTC-GFP cDNA was transfected into fibroblasts from patients with various mitochondrial diseases. Higher ratios of fibroblasts with granular mitochondria and those with fine thread-like ones were observed in a patient with Reye's syndrome and a patient with Kearns-Sayre syndrome. Weak cytosolic fluorescence was sometimes observed in fibroblasts from these patients. This method will be useful to analyze mitochondrial structural alterations and disorders of mitochondrial protein import.     

Correlation between serum levels of alanine aminotransferase and ferritin in male blood donors with antibody to hepatitis C virus

Chronic hepatitis C has been demonstrated to be associated with hepatic iron overload, and the hypothesis that the disease activity of hepatitis C is associated with iron cytotoxicity was tested in male volunteer blood donors. Sera with either antibody to hepatitis C virus or hepatitis B surface antigen were selected for determination of ferritin concentration and alanine aminotransferase activity. A correlation between serum ferritin concentration (Y; microgram/l) and alanine aminotransferase activity (X; IU/l) was found in donors with antibody to hepatitis C (log Y = 0.65 x log X + 0.98, r = 0.53, and P < 0.01). The correlation was lower in donors with hepatitis B surface antigen (r = 0.37; P < 0.01). Hepatitis C virus infection probably induces time-dependent iron accumulation associated with the progression of disease activity, while hepatitis B virus infection results in a variety of iron loads with different clinical features. The high disease activity related to hyperferritinemia suggests the presence of iron-induced liver damage in donors with hepatitis C.     

An Analysis of Scheduling Policies In Multiechelon Production Systems

Results of a simulation study of the economics of frequency of rescheduling Material Requirements Planning (MRP) systems are presented for a single-product, two-stage system in which demand is uncertain. The results indicate that for systems with moderate demand uncertainty, frequent rescheduling to maintain customer service may be uneconomical when compared with the alternative of more stable schedules in conjunction with safety stock. This result arises primarily because the cost of “emergency” production setups which occur when rescheduling is frequent exceeds the cost of safety stock required to “protect” stable schedules.     

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.