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.     

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.     

Molecular cloning of human Frizzled-6

The Frizzled genes encode receptors for WNTs, secreted glycoproteins implicated in development as well as in carcinogenesis. In this paper, we report molecular cloning of Hfz6, the human homologue of Mfz6. Nucleotide sequence analysis showed that the Hfz6 gene encodes the 706 amino-acid protein with seven transmembrane domains, a cystein-rich domain in the N-terminal extracellular region, two N-linked glycosylation sites, and two cystein residues in the second and third extracellular loops. Hfz6 mRNA 4.4-kb in size was detected in various normal adult and fetal tissues, and a larger amount of Hfz6 mRNA was detected in both fetal lung and fetal kidney. The Hfz6 gene has been mapped to human chromosome 8q22.3-q23.1. In conclusion, we have cloned Hfz6, which encodes a seven-transmembrane receptor with the cystein-rich domain in the N-terminal extracellular region, but without the Ser/Thr-X-Val motif in the C-terminus.     

Preparation and antibacterial activity of novel fluoroalkyl end-capped oligomers/silica nanocomposites-encapsulated low molecular biocides

Fluoroalkyl end-capped N-(1,1-dimethyl-3-oxobutyl)acrylamide oligomer [R_F-(DOBAA) _n -R_F] reacted with tetraethoxysilane (TEOS) and silica nanoparticles in the presence of low-molecular weight biocides such as hibitane, hinokitiol, and hinokioil under alkaline conditions to afford R_F-(DOBAA) _n -R_F/silica nanocomposites-encapsulated these biocides in excellent to moderate isolated yields. Fluoroalkyl end-capped N,N-dimethylacrylamide oligomer [R_F-(DMAA) _n -R_F] and acrylic acid oligomer [R_F-(ACA) _n -R_F]/silica nanocomposites-encapsulated hibitane were obtained under similar conditions. Dynamic light scattering measurements showed that the size of these fluorinated nanocomposites-encapsulated biocides thus obtained is nanometer size-controlled. Additionally, these fluorinated nanocomposites were shown to have a good dispersibility and stability in methanol and water. Of particular interest, these fluorinated nanocomposites-encapsulated biocides were found to have a good antibacterial activity against Staphylococcus aureus, and these nanocomposites were applied to the surface modification of traditional organic polymers such as poly(methyl methacrylate).     

Magnetic resonance imaging study on the results of surgery for cervical compression myelopathy

The morphologic changes and signal intensity of the spinal cord on preoperative magnetic resonance images were correlated with postoperative outcomes in 74 patients undergoing decompressive cervical surgery for compressive myelopathy. The transverse area of the spinal cord on T1-weighted images at the level of maximum compression was closely correlated with the severity of myelopathy, duration of disease, and recovery rate as determined by the Japanese Orthopaedic Association score. In patients with ossification of the posterior longitudinal ligament or cervical spondylotic myelopathy, the increased intramedullary T2-weighted magnetic resonance imaging signal at the site of maximal cord compression and duration of disease significantly influenced the rate of recovery. A multiple regression equation was then developed with these three variables to predict surgical outcomes.     

Enhancement by ganglioside GT1b of annexin I phosphorylation in bovine mammary gland in the presence of phosphatidylserine and Ca2+

Ganglioside GT1b and, to a lesser extent, GD3, enhanced phosphorylation of a 36 kDa protein (the substrate of protein kinase C) in the particulate fraction from bovine mammary gland. Sialic acids, asialogangliosides, and GM3 were without effect, and GD1a conversely inhibited phosphorylation of the 36 kDa protein. The enhanced phosphorylation by GT1b required the simultaneous presence of phosphatidylserine (PS) and Ca²⁺. The 36 kDa protein reacted with anti-annexin I in Western blot analysis. Addition of purified annexin I to the reaction mixture containing the particulate fraction increased the extent of phosphorylated 36 kDa protein, and the phosphorylation was further enhanced by GT1b. The enhanced phosphorylation of annexin I by GT1b was also dependent on PS and Ca²⁺. When annexin I was phosphorylated by purified protein kinase C, GT1b inhibited the annexin I phosphorylation. Addition of epidermal growth factor or insulin to the particulate fraction had little effect on the enhancement. These results suggest that an enzyme or enzymes other than protein kinase C, epidermal growth factor receptor kinase, or insulin receptor kinase is responsible for the GT1b- and GD3-enhanced phosphorylation of annexin I in the presence of PS and Ca²⁺.     

Electron transport in InGaO3(ZnO)m (m=integer) studied using single-crystalline thin films and transparent MISFETs

We have investigated the characteristics of transparent metal-insulator-semiconductor field-effect transistors (MISFETs) fabricated using InGaO₃(ZnO)_m (m=integer) single-crystalline thin films as n-channel layers and amorphous alumina as gate insulator films. The MISFETs exhibit good characteristics such as insensitivity to visible light illumination, off-current as low as ∼1 nA with a positive threshold voltage of ∼3 V and on/off current ratio of 10⁵. The field-effect mobility increased from ∼1 to ∼10 cm² (V s)⁻¹ as the m-value increased. Room temperature Hall mobility also increased. However, unexpectedly these values were lower than the field-effect mobility. It is explained by existence of shallow localized state in the homologous compounds.     

Evaluation of intraductal ultrasonography in the diagnosis of pancreatic cancer

An intraductal ultrasound (IDUS) probe which is inserted via the papilla into the main pancreatic duct (MPD) was evaluated in the diagnosis of 20 patients with pancreatic cancer. The examination was successfully performed with the probe in 17 of the patients (85%). Due to its high frequency (30 MHz) the probe only allowed visualization of the ductal wall and the immediate periductal vicinity (up to about 10 mm). In 15 of the 20 patients the tumors were surgically resected and IDUS scanning was also performed in vitro on the resection specimens, the results being compared to those of histopathological examination. Of these 15 patients, 13 were found to have ductal adenocarcinomas and all but one had been unequivocally diagnosed as having such by ultrasonography (US), computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasonography (EUS). IDUS showed an echorich area (corresponding to cancerous canaliculi on histopathological examination), surrounded by an echopoor area (abundant stroma). This pattern was classified as type I. In two patients with intraductal papillary carcinomas in whom a conclusive diagnosis was not established on US, CT, EUS or ERCP, IDUS showed tumorous tissue with an inhomogeneous echopattern outside (type II) or within (type III) the duct. These results show that IDUS offers valuable diagnostic information complementary to ERCP especially in cases of intraductal papillary tumors. Whether the high resolution imaging of the duct and the paraductal tissue can be used to differentiate between the different forms of pancreatic lesions (inflammation, neoplasms) has to be investigated further.