NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile Virus NS2B-NS3 protease

BACKGROUND The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor. METHODS In this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution. FINDINGS In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors. CONCLUSION Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.The project was funded by the Australian Research Council (http://www.arc.gov.au), grant DP0877540

Antioxidant effects of antioxidant biofactor on reactive oxygen species in human gingival fibroblasts

The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O2•−) and hydroxyl radical (•OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O2•− and •OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H2O2 (AOB + H2O2 group), containing H2O2 (H2O2 group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H2O2 group > H2O2 group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS

Glutarate and N-acetyl-L-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins

Cell-free protein synthesis provides rapid and economical access to selectively 15N-labelled proteins, greatly facilitating the assignment of 15N-HSQC spectra. While the best yields are usually obtained with buffers containing high concentrations of potassium L-glutamate, preparation of selectively 15N-Glu labelled samples requires non-standard conditions. Among many compounds tested to replace the L-Glu buffer, potassium N-acetyl-L-glutamate and potassium glutarate were found to perform best, delivering high yields for all proteins tested, with preserved selectivity of 15N-Glu labelling. Assessment of amino-transferase activity by combinatorial 15N-labelling revealed that glutarate and N-acetyl-L-glutamate suppress the transfer of the 15N-alpha-amino groups between amino acids less well than the conventional L-Glu buffer. On balance, the glutarate buffer appears most suitable for the preparation of samples containing 15N-L-Glu while the conventional L-Glu buffer is advantageous for all other samples

Ferroelectric properties of Bi3.25Sm0.75V0.02T2.98O12 thin film at elevated temperature

The ferroelectric behavior in terms of electrical polarization and fatigue and dielectric properties at elevated temperature of the ferroelectric Bi3.25Sm0.75V0.02T2.98O12 thin film fabricated by the pulsed laser deposition method were studied. Its switchable polarization increased at elevated temperature, and the coercive field decreased at the same time due to the strong domain depinning process at higher temperature. This film shows almost a polarization-fatigue-free character at room temperature, but the aggregation and diffusion of the thermally activated long-range oxygen vacancies caused strong domain pinning, and thus a poor fatigue resistance was observed at elevated temperature

Spiral magnetic structure in spin-5/2 frustrated trimerized chains in SrMn3P4O14

We study a spin-5/2 antiferromagnetic trimerized chain substance SrMn3P4O14 using neutron powder diffraction experiments. The coplanar spiral magnetic structure appears below T_N1 = 2.2(1) K. Values of several magnetic structure parameters change rapidly at T_N2 = 1.75(5) K, indicating another phase transition, although the magnetic structures above and below T_N2 are the qualitatively same. The spiral magnetic structure can be explained by frustration between nearest-neighbor and next-nearest-neighbor exchange interactions in the trimerized chains.Comment: submitted to Phys. Rev.

Confirmation of a one-dimensional spin-1/2 Heisenberg system with ferromagnetic first-nearest-neighbor and antiferromagnetic second-nearest-neighbor interactions in Rb2{}_{2}Cu2{}_{2}Mo3{}_{3}O12{}_{12}

We have investigated magnetic properties of Rb$_2$Cu$_2$Mo$_3$O$_{12}$ powder. Temperature dependence of magnetic susceptibility and magnetic-field dependence of magnetization have shown that this cuprate is a model compound of a one-dimensional spin-1/2 Heisenberg system with ferromagnetic first-nearest-neighbor (1NN) and antiferromagnetic second-nearest-neighbor (2NN) competing interactions (competing system). Values of the 1NN and 2NN interactions are estimated as $J_1 = -138$ K and $J_2 = 51$ K ($\alpha \equiv J_2 / J_1 = -0.37$). This value of $\alpha$ suggests that the ground state is a spin-singlet incommensurate state. In spite of relatively large $J_1$ and $J_2$, no magnetic phase transition appears down to 2 K, while an antiferromagnetic transition occurs in other model compounds of the competing system with ferromagnetic 1NN interaction. For that reason, Rb$_2$Cu$_2$Mo$_3$O$_{12}$ is an ideal model compound to study properties of the incommensurate ground state that are unconfirmed experimentally.Comment: 6 pages, 4 figure

Multileaf-collimator daily quality assurance of Vero4DRT system: our one-year experience

Purpose: We assessed the daily quality assurance (QA) of multi-leaf collimator (MLC) using the Vero4DRT system. Methods: As part of daily MLC QA, the irradiation field was set to 100 × 150 mm2 with a gantry angle of 0 º. Only the leaf positioning error values only were displayed. We developed an in-house program to easily acquire these values using an open source optical character recognition engine. This test was implemented between 24 August 2015 and 23 August 2016. Results: The maximum leaf positioning error was 0.40 mm in both banks. In addition, the maximum deviation was 0.10 mm in both banks. The average and standard deviation for left and right banks were 0.19 mm ± 0.11 mm and 0.15 mm ± 0.09 mm, respectively. In our one-year measurement, the leaf positioning error was less than 0.50 mm. Therefore, if the leaf position error for daily MLC QA exceeded 0.50 mm, then an external intervention is required.Conclusion: The daily MLC QA of our one-year evaluation of the Vero4DRT system demonstrates an excellent leaf accuracy and reproducibility, thereby giving confidence in the quality of the treatment