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.     

Relationship between Expression of Onco-Related miRNAs and the Endoscopic Appearance of Colorectal Tumors

Accumulating data indicates that certain microRNAs (miRNAs or miRs) are differently expressed in samples of tumors and paired non-tumorous samples taken from the same patients with colorectal tumors. We examined the expression of onco-related miRNAs in 131 sporadic exophytic adenomas or early cancers and in 52 sporadic flat elevated adenomas or early cancers to clarify the relationship between the expression of the miRNAs and the endoscopic morphological appearance of the colorectal tumors. The expression levels of miR-143, -145, and -34a were significantly reduced in most of the exophytic tumors compared with those in the flat elevated ones. In type 2 cancers, the miRNA expression profile was very similar to that of the exophytic tumors. The expression levels of miR-7 and -21 were significantly up-regulated in some flat elevated adenomas compared with those in exophytic adenomas. In contrast, in most of the miR-143 and -145 down-regulated cases of the adenoma-carcinoma sequence and in some of the de novo types of carcinoma, the up-regulation of oncogenic miR-7 and/or -21 contributed to the triggering mechanism leading to the carcinogenetic process. These findings indicated that the expression of onco-related miRNA was associated with the morphological appearance of colorectal tumors.     

A comparative study on thermomechanical and rheological characteristics of graphite/polypropylene nanocomposites: Highlighting the role of mixing

The thermomechanical and rheological properties of various graphite/long‐chain branched polypropylene (PP) nanocomposites prepared by different mixing methods have been addressed in the current work. To improve the degree of dispersion of graphite throughout the PP domains, solid‐state milling was carried out and followed by low‐temperature melt processing in the range of melting point and the crystallization temperature of PP. As a result, the well‐dispersed graphite/PP nanocomposites revealed a higher degree of plastic deformation and energy to break in the tensile test; moreover, interestingly, nanocomposites based on branched PP showed a lower zero shear viscosity and lower isothermal crystallization rate. It was speculated that the unexpected decrease in zero shear viscosity arose from the disentanglement of the long‐chain branches in particular, when an exfoliated morphology existed within the PP matrix. J. VINYL ADDIT. TECHNOL., 21:12–17, 2015. © 2014 Society of Plastics Engineers     

Structural Characterization of Neutral Glycosphingolipids from 3T3‐L1 Adipocytes

In recent years, obesity has been considered a pathological stage of early lifestyle‐related diseases, and adipose tissue and adipocyte research has been active. Glycosphingolipids are involved in the pathogenesis of type 2 diabetes induced by insulin resistance, but the details of the glycosphingolipid molecular species composition of adipocytes have yet to be elucidated. We used 3T3‐L1 adipocytes and the 1,2‐dichloroethane‐wash method to remove triacylglycerols, which are abundant in adipocytes, and analyzed the structures of glycosphingolipids, particularly neutral glycosphingolipids, using liquid chromatography–mass spectrometry.     

Polyacrylonitrile/Nylon 6 Blends: Preparation and Characterization

Summary: Polyacrylonitrile (PAN) particles with micro‐size ranges (0.15–2 μm) were prepared by emulsion and dispersion polymerizationa and in supercritical carbon dioxide media. The PAN particles were blended with Nylon 6 (PA6) at 220 °C by using a miniature mixer; it was found that melt‐mixing was possible for PAN‐rich compositions as high as 70 wt.‐%. Blends were characterized by scanning electron microscopy, IR, viscosity measurements, differential scanning calorimetry, and dynamic mechanical thermal analysis (DMTA). The size and shape of original PAN particles were retained in PAN/PA6 blends. The useful range to blend PAN particles size was less than 1 μm in terms of shape retention of the PAN particles in blends. Blends with 40 wt.‐% PAN content were found to be melt‐processable. The elastic modulus was higher for PAN/PA6 blends than pure PA6.

SEM photograph of PAN‐SC/PA6 blend with a 40/60 weight ratio.     

Actuation and blocking force of stacked nanocarbon polymer actuators

We have developed stacked nanocarbon polymer actuators that are composed of several nanocarbon polymer actuator films using nonwoven fabric as insulation layers. The nonwoven fabric prepared through electrospinning methods has extremely-low-density structures, which do not significantly prevent the motions of each nanocarbon actuator layer. Therefore, stacking several thin nanocarbon polymer actuators using nonwoven fabric as insulation layers is expected to increase generated force without decreasing the displacement of a one-layer actuator. We have prepared stacked actuators with one, two, three, four, and seven layers using this method. The displacement and blocking force of these actuators are measured and compared with those of one-layer actuators of different thicknesses. Displacement is weakly dependent on the thickness of the actuator films of the stacked actuators. On the contrary, it decreases considerably as the thickness of the actuator film of the one-layer actuator increases. In both cases, blocking force is proportional to the thickness of actuator films. We have developed a stacked actuator model based on a trilayer actuator model and confirmed the experimental results using the model.     

Liquid‐phase sintering of highly Na+ ion conducting Na3Zr2Si2PO12 ceramics using Na3BO3 additive

Na₃Zr₂Si₂PO₁₂ (NASICON) is a promising material as a solid electrolyte for all‐solid‐state sodium batteries. Nevertheless, one challenge for the application of NASICON in batteries is their high sintering temperature above 1200°C, which can lead to volatilization of light elements and undesirable side reactions with electrode materials at such high temperatures. In this study, liquid‐phase sintering of NASICON with a Na₃BO₃ (NBO) additive was performed for the first time to lower the NASICON sintering temperature. A dense NASICON‐based ceramic was successfully obtained by sintering at 900°C with 4.8 wt% NBO. This liquid‐phase sintered NASICON ceramic exhibited high total conductivity of ~1 × 10^?3 S cm^?1 at room temperature and low conduction activation energy of 28 kJ mol^?1. Since the room‐temperature conductivity is identical to that of conventional high‐temperature‐sintered NASICON, NBO was demonstrated as a good liquid‐phase sintering additive for NASICON solid electrolyte. In the NASICON with 4.8 wt% NBO ceramic, most of the NASICON grains directly bonded with each other and some submicron sodium borates segregated in particulate form without full penetration to NASICON grain boundaries. This characteristic composite microstructure contributed to the high conductivity of the liquid‐phase sintered NASICON.     

Polyhydrides of Sc,Zr and Hf and Their Proposed Formation.

Hydrogenation at 500 psi of (PNP)Sc(CH₃)₂ results in formation of a trinuclear polyhydride complex [(PNP)Sc]₃(μ₂-H)₄(μ₃-H)₂ ( 1 ) in 55 % yield. The solid-state structure shows a non-symmetric trinuclear species resulting from one pincer phosphine arm being demetallated, and where two hydrides bridge all three Sc centers, whereas the other four bridge two. Hydrogenation of (PNP)Zr(CH₃)₃ at 200 psi results instead in formation of a dinclear polyhydride species [(PNP)Zr(H)]₂(μ₂-H)₄ ( 2 ). Conducting the hydrogenation at atmospheric pressure, resulted instead in formation of the bridging methylidene complex [(PNP)Zr(CH₃)]₂(μ₂-H)₂(μ₂-CH₂) ( 3 ), which cleanly converted to 2 , upon hydrogenation at higher pressure. Both 2 and 3 were also structurally characterized. Hydrogenation of (PNP)Hf(CH₃)₃ at 200 psi resulted in incomplete hydrogenation with some formation of dinuclear mono- and dimethyl-polyhydride complexes [(PNP)Hf(CH₃)]₂(μ₂-H)₄ ( 4 ) and [(PNP)Hf(CH₃)][(PNP)Hf(H)](μ₂-H)₄ ( 5 ), which were identified by solid-state X-ray structural studies. Based on these results, we propose a pathway for the complete hydrogenation of (PNP)Zr(CH₃)₃ to 2 .     

Growth of β-Ga2O3 nanocolumns crossing perpendicularly each other on MgO (1 0 0) surface

β-Ga₂O₃ nanocolumns straightened and crossed perpendicularly each other were deposited on MgO (1 0 0) substrate by vapor phase transport method. Growth of the nanocolumns was examined at steps of 1000, 1050, and 1200 °C in elevation of source-boat temperature. We have drawn out the substrate from deposition-tube at each source-boat temperatures of 1000, 1050, and 1200 °C. Scanning electron microscopy of the sample with source-boat temperature of 1200 °C demonstrated that the straightened and elongated nanocolumns are crossing perpendicularly each other. Typical lengths of the nanocolumns were in the range of several hundreds nanometers below 1050 °C, and those of 1200 °C were in the range of ten to fifteen hundreds nanometers. Diameters of the nanocolumns stayed in the range of few hundreds nanometers, notwithstanding variation of the source temperature. X-ray diffraction and transmission electron microscopy with energy dispersive X-ray spectroscopy revealed that the nanocolumns are monoclinic β-Ga₂O₃ crystal, and the (4 0 0) plane of β-Ga₂O₃ nanocolumns is parallel to the (1 0 0) plane of MgO substrate.