In the present study we examine the thermodynamics of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titration calorimetry (ITC), X-ray crystallography, and NMR backbone (15)N and methyl side-chain (2)H relaxation measurements. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket.

BackgroundCoxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.Principal FindingIn this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >106 the tissue culture's infectious dose (TCID50) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10−5 was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.ConclusionThese results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.

The vortical flow dynamics in a gas-turbine swirl injector were investigated by means of large eddy simulations. The flow enters the injector through three sets of radial-entry, counter-rotating swirl vanes. The formulation treats the Favre-filtered conservation equations in three dimensions along with a subgrid-scale model, and is solved numerically using a density-based, finite-volume approach with explicit time marching. Several methods, including proper orthogonal decomposition, spectral analysis, and flow visualization, are implemented to explore the flow dynamics in the complex three-dimensional flowfields. Various underlying mechanisms dictating the flow evolution, such as vortex breakdown, the Kelvin–Helmholtz instability, and helical instability, as well as their interactions, are studied for different swirl numbers. The flowfield exhibits well-organized motion in a low swirl-number case, in which the vortex shedding arising from shear instabilities downstream of the guide vanes drives acoustic oscillations of the mixed first tangential and first radial mode. The flowfield, however, becomes much more complicated at high swirl numbers, with each sub-regime dominated by different structures and frequency contents.

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