Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri

Background

Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host.

Results

In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.

Conclusions

Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.     

Experimental evaluation and constitutive modeling of non-proportional deformation for asymmetric steels

Recently non-proportional deformation has received increased attention from researchers working in the area of experimental and computational modeling of metal deformation. However, most of them are numerical in nature with limited experimental data available, making it further difficult to model non-proportional deformation. In the present work, two-stage uniaxial tests, along with uniaxial cyclic and biaxial tests for different stress ratios, have been performed to evaluate deformation behavior of ultra-low carbon high strength automotive steel. Behaviors like cross-effect and hardening stagnation, which are attributed to the evolution of complex dislocation structures, were observed in this steel. It was also noticed that this steel exhibits tension-compression asymmetry. As for constitutive modeling, a modified asymmetric yield function is proposed to be used with a combined isotropic-kinematic hardening model. Also methods to account for the hardening stagnation during reverse loading and the cross-effect during two-stage deformation are proposed. The resulting constitutive model showed reasonably good agreement with experimental results.     

X-ray topography, photopyroelectric and two-photon absorption studies on solution grown benzimidazole single crystal

Benzimidazole (BMZ) is an organic nonlinear optical material, and it is possible to grow it in the laboratory by a slow evaporation solution growth technique using methanol as the solvent. Its nonlinear optical harmonic generation efficiency is 4.5 times higher than that of standard potassium dihydrogen phosphate (KDP) single crystal. It crystallizes with orthorhombic structure, which is noncentrosymmetric in nature, with lattice parameters a=13.58 Å, b=6.85 Å, c=6.97 Å. The functional groups of the grown specimen are confirmed by heteronuclear chemical shift correlation (HETCOR) analysis and it is found that other elements are not incorporated in the compound. The crystalline perfection is examined by high resolution X-ray diffraction (HRXRD), and its topographic image was recorded for the as-grown single crystal indicates the presence of some grain boundaries in the grown specimen. Thermal parameters such as thermal diffusivity (α), thermal conductivity (k), thermal effusivity (e), and heat capacity (c _p ) have been determined by a photopyroelectric thermal wave method. In addition, piezoelectricity, birefringence, and third-order nonlinear optical properties have been examined. The two-photon absorption coefficient of the title compound is determined by the open-aperture Z-scan technique.     

Energy transfer in CaSiO3 phosphors doped with Ce3+ and Tb3+

Calcium metasilicate phosphors activated by Ce³⁺ and Tb³⁺ have been studied for their emission characteristics. In two series of phosphors, one activator was kept at its optimum value while the other was varied. In another two series, one activator was kept below its optimum value and the other was varied. Concentration quenching effects start when each activator gives its maximum emission. There is clear evidence of an energy transfer from Ce³⁺ to Tb³⁺ because the⁵ D ₃ lines appear on addition of Ce³⁺ while they were conspicuously absent when Tb³⁺ alone was present. Their absence in singly activated phosphors could not have been due to cross-relaxation. Obviously X-ray excitation does not lead to⁵ D ₃ transitions which are achieved only by energy transfer. Further, considering the features of the emission spectra and the concentrations of activators used, the transfer could only be of the dipole-dipole type.     

KTB iron mineralogy at iridium deficient layers revealed by Mössbauer spectroscopy

Mössbauer spectroscopic studies of two sedimentary layers from Petriccio section in Gubbio formation are presented. It is found that these layers have almost the same iron phases nanophase hematite and phyllosilicate and with the same compositions as those in the Cretaceous-Tertiary Boundary (KTB) in Petriccio. The two layers also show enhancement in the siderophile elements such as Cr, Ni, Fe, Ca etc. to that found in the KTB layer. The two layers are well separated from the KTB layer, 128 and 149 cm below this layer, and show no enhancement of iridium. From the data it is suggested that the two layers also represent some kind of impact by extraterrestrial (ET) bolides.     

Uhf to Ka band upconverter

A balanced upconverter circuit has been developed in planar transmission line configuration using NEC GaAs beam lead Schottky barrier diodes. RF power output of +1.6 dBm SSB is obtained at Ka-band when up converter is driven by +17 dBm IF and +20 dBm LO signal.     

Electron diffraction data as a constraint in the determination of force field of thallous halide dimers

Electron diffraction data have been used as a constraint in the determination of force field for Tl₂F₂ having planar rhombic structure. The L-F approximation method, recently given by us, has also been applied to evaluate force constants for thallous halide dimers,e.g. T1₂F₂ and T1₂Cl₂. The results have been compared with the available experimental data in order to check the validity of the present work. It is concluded that non-bond experimental mean amplitudeU ₁..._Tl for Tl₂F₂ is capable of fixing the force field and L-F approximation gives reasonably good force fields for the two thallous halide dimers now under study.