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.     

Novel vitamin E derivative with 4-substituted resorcinol moiety has both antioxidant and tyrosinase inhibitory properties

A novel vitamin E derivative, (6″-hydroxy-2″,5″,7″,8″-tetramethylchroman-2″-yl) methyl 3-(2′,4′-dihydroxyphenyl)propionate (TM4R), which has a chromanoxyl ring and 4-substituted resorcinol moieties, was synthesized; and its inhibitory effects on tyrosinase, antioxidant ability, and lightening effect of ultraviolet B (UVB)-induced hyperpigmentation were estimated. TM4R showed potent inhibitory activity on tyrosinase, which is the rate-limiting enzyme in melanogenesis. The scavenging activities of TM4R on 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals were found to be nearly the same as those of α-tocopherol. Furthermore, an efficient lightening effect was observed following topical application of TM4R to UVB-stimulated hyperpigmented dorsal skin of brownish guinea pigs. These results suggest that TM4R may be a candidate for an efficient whitening agent, possibly by inhibiting tyrosinase activity and biological reactions caused by reactive oxygen species.     

Grafting vinyl monomers onto silk (Bombyx mori) using different initiators: Properties of grafted silk

The graft–copolymerization of silk with methacrylamide (MAA), 2‐hydroxyethyl methacrylate (HEMA), and methyl methacrylate (MMA) was studied using three different free‐radical initiators: an inorganic peroxide [ammonium persulfate (APS)] and two azo compounds [2,2′‐azobisisobutyronitrile (AIBN) and 2,2′‐azobis(2‐methylpropionamidine) dihydrochloride (ADC)]. The rate and yield of grafting followed the order: APS > AIBN > ADC. The performance of AIBN was close to that of APS in terms of weight gain attained. The degree of yellowing of grafted silk varied as a function of the initiator–monomer system used. APS induced the highest degree of yellowing, regardless of which monomer was used, whereas silk grafted with the MAA/AIBN system displayed the lowest level of yellowing. Moisture regain of grafted silk changed as a function of the hydrophilic/hydrophobic character of the grafted polymer, regardless of the kind of initiator used. Accordingly, tensile properties showed a tendency to decrease with increasing weight gain of grafted silk, the extent of which was independent of the kind of monomer and initiator used. The different initiators did not induce any appreciable change in the fine structure of silk, as demonstrated by optical measurements. Uneven surface deposition of homopolymer was detected to variable extent with MMA and HEMA grafting, whereas the surface of MAA‐grafted silk was completely free of any foreign deposit, independently of the initiator used. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 1401–1409, 2001     

Low-Temperature Electrochemical Synthesis of ZrO2 Films on Zirconium Substrates: Deposition of Thick Amorphous Films and in situ Crystallization on Zirconium Anode

The feasibility of synthesizing crystalline ZrO₂ films at low temperatures was evaluated using an electrochemical method. Anodization of zirconium-metal substrates in tetraethyl ammonium hydroxide (TEAOH) solutions under constant applied voltage conditions at ∼25° and ∼100°C was investigated. The chemistry and microstructure of the anodic oxide films deposited on the zirconium-metal substrates under the above conditions were characterized using X-ray diffractometry and scanning electron microscopy. The results indicated that, with sufficiently high applied voltages (in the range of 300 V) at pH ∼9.5, the initial dissolution of the zirconium anode resulted in the local saturation of the electrolyte solution with Zr⁴⁺, forming Zr(OH)⁻₅, which deposited electrophoretically on the anode as a thick, gelatinous film at 25°C. Similar treatments at 100°C resulted in an in situ crystallization of Zr(OH)₄ gel to monoclinic ZrO₂.     

Interfacial Reactions between Sapphire and Silver—Copper—Titanium Thin Film Filler Metal

Wetting and brazing studies of sputtering-deposited, submicrometer thin film filler metal in an Ag—Cu—Ti/Al₂O₃ system were performed. The interfacial reaction layer between the filler metal and Al₂O₃ was investigated. It is possible to make a brazing joint even with a reaction layer of less than 100 nm thickness. Different types of interfacial reaction layers were observed when the Ti content in the filler metal was varied. The Cu—Ti—O system compounds were observed in the samples with high wetting capabilities, but not in the sample with low wetting characteristics. It was found that these compounds are substances that promote effective brazing.     

Homogeneous Tritylation of Cellulose in A Sulfur Dioxide – Diethylamine – Dimethyl Sulfoxide Medium

The present investigation was undertaken to see if a practical method could be developed for homogeneous tritylation of cellulose in a non-aqueous solvent of cellulose. Our new procedure of tritylation of cellulose can easily be carried out under homogeneous conditions by dissolving cellulose in a sulfur dioxide(SO₂)-diethylamine (DEA)-dimethyl sulfoxide(DMSO) solvent system, one or the non-aqueous cellulose solvents, followed by addition of trityl chloride and pyridine. This new method can avoid the time consuming pretreatment for the decrystallization of cellulose which has been necessary in the traditional procedure and can lower the reaction temperature. IR spectra of the products indicated the formation of trityl cellulose. Measurements of dielectric properties of the products confirmed that trityl groups were selectively introduced at the primary hydroxyl groups in cellulose. This conclusion was also confirmed by a H-NMR study in which the tritylated products was first acetylated, detritylated and then trideuterioacetylated and H-NMR spectra were taken at each stage and examined comparatively.     

Enzymic Hydrolysis Of Lignin—Carbohydrate Complexes Isolated From Kraft Pulp

Four carbohydrate samples extracted from kraft pulps are used as model substrates for studying the mechanism by which xylanase enhances subsequent bleaching of kraft pulp. Fourier transform infrared spectroscopy confirms that small amounts of aromatic molecules, probably lignin, remain associated with these carbohydrate samples. When the extracts are hydrolyzed with xylanase or acid, size exclusion chromatography shows a decrease in the molecular mass of their UV-absorbing constituents, as well as their carbohydrate constituents as determined by pulsed amperometric detection. The results are consistent with the hypothesis that xylanase prebleaching hydrolyzes the xylan portion of lignin-carbohydrate complexes to leave smaller lignin-containing macromolecules in pulp fiber, thus facilitating the removal of lignin components by bleaching chemicals.     

Influence of water content in sub-flooring materials using adhesive on chemical compounds emission

Indoor air pollution is caused predominantly by emission of primary pollutants from building materials or finishing materials. Recently, there has been increasing interest in the new secondary pollutants caused by chemical reaction of the primary pollutants. In this study, the pollutants emitted from poly(vinyl chloride) (PVC) flooring material were measured, and 2-ethyl-1-hexanol (2E1H) and volatile organic compounds (VOCs) emitted from composite building materials made of PVC flooring material used as a self-leveling flooring were measured to identify the mechanism of emission of 2E1H. The moisture sorption isotherm was measured with a Magnetic Suspension Balance to examine the moisture content of the self-leveling flooring material and its effect on emission of 2E1H. 2E1H was emitted from the PVC flooring material through hydrolysis or oxidation of di-2-ethylhexyl phthalate (DEHP). The composite building material including the self-leveling flooring material emitted more 2E1H through hydrolysis over time. It was determined that liquid prevails in the self-leveling flooring material when the water content is higher than 4% and vapor prevails when the water content is lower than 4%. The prevailing liquid in the flooring material contributes to the increased emission of 2E1H and the prevailing vapor in the material did not cause increase in emission of 2E1H. It is considered that control of water in the self-leveling flooring material or concrete slab is very important in order to inhibit emission of 2E1H through hydrolysis of DEHP on the floor.     

Production of antimicrobially active silk proteins by use of metal‐containing dyestuffs

The work presented here discusses a new technique for preparing silk fibers and films with persistent antimicrobial activity through use of metallic dyestuffs during the fiber dyeing process. The length of the silk fibers investigated contracted when the fibers were immersed in concentrated neutral salt solutions, such as calcium or potassium nitrate, at elevated temperature levels. The birefringence and molecular orientation of the silk fibroin molecules became less ordered by the action of the neutral salt solutions, resulting in increased dyestuff absorption. Subsequently, contracted silk fibers were dyed with metallic dyestuffs containing Cr or Cu for the purpose of obtaining silk fibers with antimicrobial activity. Silk fibers dyed with metallic dyestuffs showed significant antimicrobial activity against the plant pathogen Cornebacterium and the human pathogen Coli bacillus. Tensile strength of the silk fibers after the salt shrinking and dyeing processes did not show a significant change, whereas the elongation at break was increased slightly. The techniques described here for preparing significantly active antimicrobial silk fibers are effective and economic ways of providing new materials for industrial and biomedical applications. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1181–1188, 2002     

Color control of Japanese soy sauce (shoyu) using membrane technology

The present study systemically decolorized soy sauce using a membrane process to analyze the separation mechanism. An ultrafiltration (UF) membrane (NTU-2120) exhibited only slight decolorization ability. A nanofiltration (NF) membrane with a lower molecular weight cut-off and produced by sulfonated polysulfone (NTR-7400 series) rather than polyvinyl alcohol/polyamide (NTR-7250) had higher decolorization ability. The NF membranes rejected total nitrogen by 17–24%, unsalted soluble solid content by 24–32%, reducing sugar by 25–43%, and amino acids by 10–25%. The NTR-7400 series membrane rejected lactic acid by 6–9%, and pyroglutamic acid by 11–21%; other quality indexes were maintained. In the NF membrane processes, higher rejection of acidic amino acids than neutral and base amino acids was observed. The separation performance was governed by the electrical effect as well as the sieve effect. Soy sauce color could be controlled by blending NF membrane-processed soy sauce with feed soy sauce. Color can be matched to preference in accordance with dishes by suitably blending NF membrane-processed soy sauce with feed soy sauce.