One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.     

Precision die-casting of optical MU/SC conversion sleeve

The performance of MU/SC conversion sleeve produced by bulk metallic glass (Zr₅₅Al₁₀Ni₅Cu₃₀) was examined. A precision die-casting method was applied to improve size accuracy. The size accuracy of the conversion sleeve produced by the precision die-casting method was ±1 μm, and optical insertion loss (Li) was less than 0.3 dB for a standard value. The wear resistance of metallic glass is improved by surface oxidation treatment in air at 673 K. The MU/SC conversion sleeve produced from bulk metallic glass has superior characteristics for optical parts.     

Preparation of carbon-based transparent and conductive thin films by pyrolysis of silylated graphite oxides

Thin films of silylated graphite oxide were obtained from a chloroform/cyclohexane dispersion of n-hexadecylamine-intercalated silylated graphite oxide by a casting method at a low temperature. Carbon-based thin films were obtained from the pyrolysis of the resulting films under a reduced pressure at 500 °C or higher temperatures. The resulting samples were well adhered to the substrate because of the presence of silicon containing species as a “glue”. The resistivity decreased with an increase in the film thickness or a decrease in the transparency. Based on the data obtained for the samples prepared from graphite with different particle sizes and graphite oxide with different oxygen contents, the conduction of the electrons within each carbon sheet seemed important for large film thickness and conduction through the boundary seemed important when the film thickness was small. A low sheet resistance of 3.7 kΩ/sq for 80% of transmittance was achieved, when graphite oxide with a lower oxygen content was prepared from graphite with smaller particle sizes and the precursor film was heated at 500 °C. At 900 °C, it further decreased to a value of 700 Ω/sq.     

The effect of ethane on the performance of commercial polyphenylene oxide and Cardo-type polyimide hollow fiber membranes in CO<Subscript>2</Subscript>/CH<Subscript>4</Subscript> separation applications

Impurities such as hydrogen sulfide, water vapor and heavy hydrocarbons in natural gas have considerable effects on the membrane performance. Small amounts of condensable and polymer soluble components in the feed gas cause swelling or plasticization of glassy membranes, leading to a reduction in membrane selectivity. In the present research the influence of ethane was investigated on the permeance and selectivity of two commercially available hollow fiber membranes, namely Cardo-type polyimide and PPO hollow fibers for CO₂/CH₄ separations. It was concluded that the gas mixture permeation rate was increased in the presence of C₂H₆. However, the CO₂/CH₄ separation factors remained almost the same in the presence and absence of the C₂H₆.     

Ring-opening polymerization of various oxirane derivatives using organotin phosphate condensate; Selective synthesis of the polyether containing oxirane ring in the side chain

Ring-opening polymerization (ROP) of various oxirane derivatives of the type, 2,2-R¹,R²-CCH₂O [R¹ = H (1), CH₃ (2); R² = CH₃ (a), CH₂Cl (b), CH₂OCH₃ (c)], using organotin phosphate (Bu₂SnO-Bu₃PO₄) condensate has been explored. The ROP of monosubstituted oxiranes (1a-c) afforded ring-opened polymers in high yields (1a, 1c = 99% and 1b = 69%); the resultant polymers from monomers 1a and 1b possessed high molecular weights (M_n = 9.49 × 10⁴, 10.60 × 10⁴, respectively). In contrast, both polymer yields and molecular weights for resultant polymers in the polymerization of disubstituted oxiranes (2a-c) were considerably lower than those in the polymerization of monosubstituted monomers (1a-c). ROP of glycidyl 2-methylglycidyl ether (3) possessing two oxirane groups with different reactivity was thus conducted by organotin catalyst; the high molecular weight polyether (M_n = 9.17 × 10⁴) containing oxirane ring in the side chain has successfully been obtained in moderate yield.     

Synthesis of Polypeptide-Polyether Conjugates from an Activated Urethane Derivative of γ-Benzyl-L-glutamate as a Monomer

N-(4-Nitrophenoxycarbonyl)-γ-benzyl-L-glutamate (1) was heated at 60 °C in N,N-dimethylacetoamide (DMAc) in the presence of p-(tert-butyl)phenylmethylamine (2) to afford poly(γ-benzyl-L-glutamate) in high yield. ¹H NMR analysis of the obtained polymer revealed that the p-(tert-butyl)phenylmethyl group was successfully introduced into the polymer chain end. Under the same conditions, polycondensations of 1 in the presence of poly(ethylene glycol)s having amino terminals were performed. Both of the components were consumed quantitatively to afford the corresponding block copolymers having poly(γ-benzyl-L-glutamate) and poly(ethylene glycol) segments. The molecular weights of the copolymers were controlled by the feed ratio [1]₀/[amino group in the macroinitiator]₀. Electronic Supplementary Material  The online version of this article at contains supplementary material, which is available to authorized users.     

Magnetic properties and phase diagram of Ni50Mn50−xGax ferromagnetic shape memory alloys

We present the magnetic properties and the magnetic phase diagram of Ni₅₀Mn_50?xGa_x ferromagnetic shape memory alloys across a wide concentration range. Martensitic transformation, intermediate transformation, B2–L2₁ order–disorder transformation, Néel and Curie temperatures are determined for the prepared samples. The martensitic transformation temperature decreases with increasing Ga concentration and bends two times when crossing the Curie temperature and the intermediate-phase transformation temperature. Spontaneous magnetization and its composition dependence were also investigated. Composition dependence of the transformation temperatures and the spontaneous magnetization in the martensite phase of Ni₅₀Mn_50?xGa_x are compared with those of Ni₅₀Mn_50?xIn_x and Ni₅₀Mn_50?xSn_x, revealing a similarity in the NiMn-based alloy systems.     

Re-evaluation of Radiation-Energy Transfer to an Extraction Solvent in a Minor-Actinide-Separation Process Based on Consideration of Radiation Permeability

ABSTRACT

Absorbed-dose estimation is essential for evaluation of the radiation tolerance of minor-actinide-separation processes. We propose a dose-evaluation method based on radiation permeability, with comparisons of heterogeneous structures seen in the solvent-extraction process, such as emulsions forming in the mixture of the organic and aqueous phases. A demonstration of radiation-energy-transfer simulation is performed with a focus on the minor-actinide-recovery process from high-level liquid waste with the aid of the Monte Carlo radiation-transport code PHITS. The simulation results indicate that the dose absorbed by the extraction solvent from alpha radiation depends upon the emulsion structure, and that from beta and gamma radiation depends upon the mixer-settler-apparatus size. Non-negligible contributions of well-permeable gamma rays were indicated in terms of the plant operation of the minor-actinide-separation process.     

Application of isoscattering points to the analysis of globular solute structures

There are specific points at which the X-ray (neutron) scattering intensity of solutes is independent of the electron density (scattering density) contrast between solutes and solvent. These points are referred to as “isoscattering points”. They have been observed in the X-ray small-angle scattering data of globular particles such as micelles and bacteriophages. The condition for the appearance of an isoscattering point is briefly reported. The nature of the isoscattering point is described in view of the application to analysis of solute structures.     

Studies on Epoxy Resin Film Coating by the Flame Spraying Method

The authors have shown that special formulations are required to give complete covering of steel surface (with no swelling, break away, cracks, etc) with flame sprayed film coating of epoxy resin. For this purpose, there should be minimum stress in the film, and it was concluded that treatments effective for this purpose involve adding adequate filler to reduce the thermal expansion coefficient, controlling the degree of cure, holding small closed pores in the film and semi-curing immediately after spray to keep the required tensile strength.