Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.     

Diamond-like carbon thin film with controlled zeta potential for medical material application

With the purpose of applying diamond-like carbon (DLC) thin films as a biocompatible material, we experimented with introducing functional groups such as amino and carboxyl groups to the surface of DLC thin films by plasma surface treatment. From the results, it was found that the contact angle values of the DLC thin films surface were decreased with increasing in the OCO bonded network on the surface. Measurement of the zeta potential when the amounts of the functional groups were varied showed that this successfully varied the zeta potential over the wide range of − 48 mV to + 12 mV. It was found that when carboxyl groups are introduced by O₂ plasma modification of the DLC thin films surface, the zeta potential was lower than that of untreated DLC thin films sample. It was also found that amino groups can be introduced to the DLC thin films surface by NH₃ plasma treatment, the zeta potential was higher than that of untreated DLC thin films sample. This means that zeta potential of the DLC thin films can also be controlled by controlling these two functional groups. Therefore, we have succeeded in developing a multifunctional DLC thin films that does not use polymers and is suitable as a biocompatible material.     

Quantitative evaluation of die casting surface defect severity by analyzing surface height

It is necessary in factories to assess the severity of the surface defects of castings, as a slight surface defect will be taken as qualified when it brings no bad effect or it can be removed by the subsequent processing. In practical production, professional technicians visually inspect the surface defect severity according to their individual experience. Therefore, it is difficult for them to maintain the same standard and accuracy in the subjective, tedious and labor-intensive work. Recently, image processing techniques based on optical images have been applied to achieve better accuracy and high efficiency. Unfortunately, optical images cannot directly quantify surface depth, which works as a crucial factor in the practical assessment of surface defect severity. The surface roughness evaluation algorithm, which takes into account of both area and depth information of the assessed surface, was applied to directly characterize surface defect severity based on surface asperity rather than optical image. The results using standard casting pieces show that surface defect severity has no apparent dependence on surface roughness. However, the subsequent results show that the root-mean-squared-deviation (RMSD) of surface gradient of flow line defects positively correlates with the increase of defect severity. The other types of defect do not present such tendency. Thus, practical workpieces with flow line defects on the surface were used to verify the universality of this tendency. The results show that surface roughness of an unqualified workpiece is larger than that of a qualified workpiece after surface slope adjustment, but presents no obvious coincidence before the adjustment. In contrast, the RMSD of an unqualified workpiece, no matter before or after the adjustment, is larger than that of a qualified one.     

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.     

Microstructure characterization of Cu-rich nanoprecipitates in a Fe–2.5 Cu–1.5 Mn–4.0 Ni–1.0 Al multicomponent ferritic alloy

The evolution of precipitates in a Fe–2.5 Cu–1.5 Mn–4.0 Ni–1.0 Al multicomponent ferritic alloy during annealing at 500 °C was systematically investigated by aberration-corrected scanning transmission electron microscopy. The atomic-scale structure and chemistry characterization reveal that primary precipitates with enriched Cu, Ni, Mn and Al originate from continuous growth of B2 ordered domains in the as-quenched alloy. The formation of a Cu-rich body-centered cubic (bcc) phase takes place by the decomposition of the B2 ordered primary phase, which forms a Cu-rich bcc core and ordered B2-Ni(Al,Mn) shell. The B2 shells serve as a buffer layer to moderate the coherent strain and to prohibit the inter-diffusion between the Cu-rich precipitates and bcc-Fe matrix, giving rise to a low coarsening rate of the precipitates. The Cu-rich precipitates experience a structural transformation from bcc to 9R at a critical size of ～6 nm during long time annealing, corresponding to obvious coarsening of the precipitates and dramatic loss in hardness of the alloy.     

Synergistic alloying effect on microstructural evolution and mechanical properties of Cu precipitation-strengthened ferritic alloys

We report the influence of alloying elements (Ni, Al and Mn) on the microstructural evolution of Cu-rich nanoprecipitates and the mechanical properties of Fe–Cu-based ferritic alloys. It was found that individual additions of Ni and Al do not give rise to an obvious strengthening effect, compared with the binary Fe–Cu parent alloy, although Ni segregates at the precipitate/matrix interface and Al partitions into Cu-rich precipitates. In contrast, the co-addition of Ni and Al results in the formation of core–shell nanoprecipitates with a Cu-rich core and a B2 Ni–Al shell, leading to a dramatic improvement in strength. The coarsening rate of the core–shell precipitates is about two orders of magnitude lower than that of monolithic Cu-rich precipitates in the binary and ternary Fe–Cu alloys. Reinforcement of the B2 Ni–Al shells by Mn partitioning further improves the strength of the precipitation-strengthened alloys by forming ultrastable and high number density core–shell nanoprecipitates.     

Effect of chemical composition on bead formation in TIG arc welding. Effect of chemical composition on weldability in fabrication in high alloyed steel (2nd Report)

The flux cored arc welding process has grown in use in recent times as a function of its characteristics of high rate of deposition, associated with suitable mechanical properties in the welded joint. However, many aspects still remain obscure in regard to greater utilization of this process. As a result, the intention of this work is the analysis and optimization of the voltage, wire feed speed and contact tip part distance welding parameters, in a process using tubular wire with shielding gas, on the penetration and convex index of the weld. By means of statistical techniques, a mathematical model was developed with subsequent optimization of the responses. Based on the results obtained, the strong influence of the wire feed speed on the process was seen, followed by the voltage and, with less intensity, the contact tip to workpiece distance (CTWD). Statistical analyses indicated that the best condition for the parameters analysed was obtained with voltage values between 32 and 34 V, associated with a wire feed speed of 12 m/min and a CTWD of 20 mm. On the other hand, tensions at around 36 V caused surface defects that prejudiced the quality of the weld. Later tests showed an acceptable forecast of the results of the mathematical models when compared to the actual results.     

Effect of chemical composition on anode area formation in TIG arc welding. Effect of chemical composition on weldability in fabrication in high alloyed steel (1st Report)

Summary

This paper describes a fundamental investigation of the weldability of A5052 wrought alloy and AC7A castings in electron beam welding and gas tungsten arc welding. Microscopic observations, hardness, tensile, and impact tests were used. In electron beam welding, the tensile and impact properties of the wrought alloy weld metal and heat affected zone are satisfactory. Micro-solidification cracks are found in the weld metal, whereas micro-liquation cracks are found in the AC7A HAZ. The tensile properties of the welds produced by GTAW (or TIG) are virtually equivalent or slightly inferior to those of the electron beam welded joints.     

Effect of REM addition of wire on CO2 gas shielded arc phenomenon

Carbon dioxide (CO₂) gas shielded arc welding is the main arc welding method, but it generates a large amount of spatter during welding. The root cause of spatter lies in the fact that the droplet undergoes repeated irregular shaking. To solve this problem, spatter generation modes were clarified and the effects of polarity and rare earth metal (REM) addition of the wire on CO₂ gas shielded arc welding were investigated. As a result, when welding is performed with an electrode negative (DCEN) polarity using REM added wire, it was found that a conical arc plasma is formed, and the droplet which is transferred from the wire tip to the molten pool is fine and continuous, in what is termed ‘spray transfer’. Thus, spatter generation was reduced to 10% of amount of the conventional CO₂ gas shielded arc welding (from 0.058 to 0.005g/s).     

Photocatalytic H2 evolution under visible light irradiation on Zn1-x Cu x S solid solution

The H₂ evolution reaction from an aqueous Na₂SO₃ solution proceeded with 3.7% quantum yield under visible light irradiation (λ > 420 nm) on a Zn_0.957Cu_0.043S solid solution photocatalyst without co‐catalysts such as Pt. This revised version was published online in July 2006 with corrections to the Cover Date.