Chenhui Ding

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

ABSTRACTGenome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

Piezo-assisted nuclear transfer affects cloning efficiency and may cause apoptosis.

Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the cloning technique which has not yet been well investigated. There are two main procedures for transferring nuclei into enucleated oocytes: fusion and piezoelectric microinjection, the latter being used mostly in mice. We have, therefore, decided to compare the quality and the developmental ability, both in vivo and in vitro, of embryos reconstructed with electrofusion or piezoelectric injection. In addition, the effect of piezo setups of differing electric strengths was investigated. Along with the record of the rate of development, we compared the nuclear integrity in the blastomeres during the first cleavages as well as the morphological and cellular quality of the blastocysts. Our results show that the piezo-assisted micromanipulation can induce DNA damage in the reconstructed embryos, apoptosis, and reduced cell numbers in blastocysts as well as a lower rate of development to term. Even if piezo-driven injection facilitates a faster and more efficient rate of reconstruction, it should be used with precaution and with as low parameters as possible.

Improvement in wear resistance of plasma sprayed yttria stabilized zirconia coating using nanostructured powder

Abstract Plasma sprayed yttria stabilized zirconia coatings were prepared using nanostructured and conventional powders with optimized process parameters for the highest deposition efficiency, the smallest porosity and the highest microhardness. The tribological properties of these coatings against 100C6 steel were then tested with a ball-on-disc arrangement. Results showed that although the friction coefficients of the coatings sprayed using the nanostructured powder were slightly different from those of the coatings sprayed using the conventional powder, the former coatings were more wear resistant than the latter coatings. The wear mechanisms of all the coatings were explained in terms of adhesion-induced spallation and micro-fracturing of lamellae. The improvement in wear resistance of the coatings sprayed using the nanostructured powder could be mainly ascribed to the decrease of micrometer-sized defects such as pores and interlamellar and intralamellar cracks in the coatings.

Mechanical and tribological properties of plasma-sprayed Cr3C2-NiCr, WC-Co, and Cr2O3 coatings

Mechanical properties such as Young’s moduli and fracture toughness of plasma-sprayed Cr3C2-NiCr, WC-Co and Cr2O3 coatings were measured. The tribological properties of the three kinds of coatings were investigated with a block-on-ring self-mated arrangement under water-lubricated sliding. Furthermore, the influences of the mechanical properties on the tribological properties of the coatings were also examined. It was found that the Young’s moduli, bend strengths and fracture toughness of the coatings were lower than the corresponding bulk materials, which may be attributed to the existence of pores and microcracks in the coatings. Among the three kinds of coatings, the magnitude of wear coefficients, in decreasing order, is Cr3C2-NiCr, WC-Co and Cr2O3, and the wear coefficient of Cr2O3 coating was less than 1 × 10−6mm3N−1m−1. The wear mechanisms of the coatings were explained in terms of microcracking and fracturing, and water deteriorated wear performance of the coatings. The higher the fracture toughness and the lower the porosity and length of microcracking of the coating, the more the wear-resistance of the coating.

Successful generation of cloned mice using nuclear transfer from induced pluripotent stem cells

Successful generation of cloned mice using nuclear transfer from induced pluripotent stem cells

Tribological properties of silicon carbide under water-lubricated sliding

Abstract The friction and wear performances of three kinds of self-mated silicon carbide (SiC), reaction sintered SiC, pressureless sintered SiC and toughened SiC, were investigated with a block-on-ring arrangement under water-lubricated sliding. Furthermore, the morphologies, phases and element valences of the worn surface were observed, examined and analysed employing scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectrometer and Fourier transform infrared spectroscopy respectively. In addition, the effects of microstructure and mechanical properties of the ceramics on their tribological properties were also discussed. It was found that the three kinds of SiC obviously showed different tribological properties. Higher friction coefficient and wear coefficient occurred for reaction sintered SiC and pressureless sintered SiC. The wear mechanisms of them were explained in term of pull-outs of grains and grain fracture. However, excellent tribological property, friction coefficient of 0.01, which suggested the characterization of hydrodynamic lubrication and wear coefficient of 4.2 × 10 −7 mm 3 N −1 m −1 , was obtained for toughened SiC. This resulted from that tribochemical oxidation took place and a ultraflat surface was formed on the worn track as a result that the toughened SiC possessed higher fracture toughness.

Correction of β-thalassemia mutant by base editor in human embryos

Abstractβ-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G) mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Wear mechanism of plasma-sprayed TiO2 coating against stainless steel

Abstract Wear of plasma-sprayed TiO 2 coating against stainless steel was tested with a block-on-ring arrangement at different loads and sliding speeds. It was found that wear mass loss of TiO 2 coating increased with increase in loads but decreased with increase in sliding speeds. The mechanism was explained in terms of grinding wear, adhesive wear, micromechanical delamination wear, plastic deformation wear, melting wear and formation of Fe 2 O 3 Fe 3 O 4 thin film.

Decreased cofilin1 expression is important for compaction during early mouse embryo development

Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarization by which cellular asymmetry is first established. During this process many molecules and organelles undergo polarized distribution, but the cytoskeletal basis for these distribution specifications remains to be explored. The present study focused on cofilin1, an actin-binding protein that depolymerizes actin filaments. We showed that cofilin1 expression decreased at the compaction stage, and that down-regulation of cofilin1 expression by siRNA microinjection accelerated compaction. Continuous observation using time-lapse video miscroscopy confirmed these findings. That is, the embryonic cells microinjected with anti-cofilin1 antibody exhibit earlier adherence properties compared to uninjected cells. Pronuclear microinjection of a site-directed mutated cofilin1 plasmid, in which cofilin1 is sustained in its active form produced embryos with blastomeres that did not adhere, suggesting that inactivation of cofilin1 is critical for cell flattening and adherence. Fluorescein-phalloidin staining indicated that decreased cofilin1 expression promoted the formation of the apical pole, which is a marker for polarity. Scanning electron microscopy results demonstrated the appearance of microvilli on the outer face of blastomeres in cofilin1 knockdown embryos. Our results suggest that cofilin1 plays an important role in cortical cytoplasmic organization during embryo compaction.

Wear mechanism of plasma-sprayed Cr3C2NiCr against TiO2 coating

Abstract The friction and wear performance of plasma-sprayed Cr 3 C 2 NiCr against TiO 2 coating was investigated with a block-on-ring arrangement at different sliding speeds and loads. Furthermore, the morphologies, elements, phases and element valences of the worn surface and debris were observed, examined and analysed employing scanning electron microscopy, energy disperse spectrum, X-ray diffraction and X-ray photoelectron spectrometer, respectively. It was found that the friction coefficient decreased with increase in loads and sliding speeds. However, the exponent of load against wear rate of the C 3 C 2 NiCr coating appeared as two values on changing from the lower to higher load. The wear mechanism of the coating was explained in terms of fatigue-induced detachment of transferred TiO 2 layer at lower load, and plastic deformation, shear fracture and melting wear at higher load.