Facile identification of photocleavable reactive metabolites and oxidative stress biomarkers in proteins via mass spectrometry

Described herein is a method which combines bond selective fragmentation by photodissociation with online liquid chromatographic separation and mass spectrometric analysis. Photoexcitation of proteins or peptides with 266-nm light does not normally yield abundant fragmentation; however, incorporation of a suitable carbon-sulfur or carbon-halogen bond that is proximal to a chromophore allows access to direct dissociation pathways, resulting in homolytic cleavage of these bonds. Radicals generated through this process can cause further dissociation of the peptide backbone, which is useful for site specifically identifying the point of modification. Two specific applications of this technique for peptide analysis in model systems are presented: (1) identification of reactive metabolites which covalently modify cysteine residues, and (2) characterization of halogenated tyrosine residues which are biomarkers related to oxidative stress. In both cases, these naturally occurring post translational modifications create photocleavable bonds which can be fragmented by 266-nm light. The selectivity offered by photodissociation allows facile identification of the peptides of interest even in complex mixtures, and subsequent selective radical directed backbone fragmentation pinpoints the site of modification. This combination greatly simplifies data analysis and provides more confident assignments.     

Fluorine-directed β-galactosylation: chemical glycosylation development by molecular editing

Validation of the 2-fluoro substituent as an inert steering group to control chemical glycosylation is presented. A molecular editing study has revealed that the exceptional levels of diastereocontrol in glycosylation processes by using 2-fluoro-3,4,6-tri-O-benzyl glucopyranosyl trichloroacetimidate (TCA) scaffolds are a consequence of the 2R,3S,4S stereotriad. This study has also revealed that epimerization at C4, results in a substantial enhancement in β-selectivity (up to β/α 300:1).     

Imaging longitudinal changes in articular cartilage and bone following doxycycline treatment in a rabbit anterior cruciate ligament transection model of osteoarthritis

Objective

The development of osteoarthritis following traumatic anterior cruciate ligament (ACL) injury is well established. However, few reliable indicators of early osteoarthritic changes have been established, which has limited the development of effective therapies. T_1ρ and T₂ mapping techniques have the ability to provide highly accurate and quantitative measurements of articular cartilage degeneration in vivo. Relating these cartilaginous changes to high-resolution bone-densitometric evaluations of the late-stage osteoarthritic bone is crucial in elucidating the mechanisms of development of traumatic osteoarthritis (OA) and potential therapies for early- or late-stage intervention.

Methods

Twelve rabbits were monitored with in vivo magnetic resonance imaging (MRI) scans following ACL transection surgery with a contralateral leg sham operation. Six of the rabbits were treated with oral doxycycline for the duration of the experiment. At 12 weeks, the excised knees from three animals from each group (n=6 overall) were subjected to micro-computed tomography (CT) analysis.

Results

Consistent with previous studies, initial elevations in T_1ρ and T₂ values in ACL-transected animals were observed with relative normalization towards values see in sham-operated legs over the 12-week study. This biphasic pattern could hold diagnostic potential to differentiate osteoarthritic cartilage by tracking the relative proportions of T_1ρ and T₂ values as they rise with inflammation then fall as collagen and proteoglycan loss leads to further dehydration. The addition of doxycycline resulted in inconclusive, yet potentially interesting, cartilaginous changes in several compartments of the rabbit legs. Micro-CT studies demonstrated decreased bone densitometrics in ACL-transected knees. Correlation studies suggest that the cartilaginous changes may be associated with some aspects of bony change and the development of OA.

Conclusion

We conclude that there are definite relationships between cartilaginous changes as seen on MRI and late-stage microstructural bony changes after traumatic ACL injury in rabbits. In addition, doxycycline may show promise in mitigating early-stage cartilage damage that may serve to lessen late-stage osteoarthritic changes. This study demonstrates the ability to track OA progression and therapeutic efficacy with imaging modalities in vivo.     

A theoretical study of the reactivity of Cu2O(111) surfaces: the case of NO dissociation

We compare the electronic properties of Cu(111) and Cu(2)O(111) surfaces in relation to the dissociation of NO using first principles calculations within density functional theory. We note a well-defined three-fold site on both O- and Cu-terminated Cu(2)O surfaces which is verified as the active site for the adsorption and dissociation of NO. The interaction of Cu with O atoms results in the forward shifting of the local density of states and formation of unoccupied states above the Fermi level, compared to the fully occupied d band of pure Cu. These results give valuable insights in the realization of a catalyst without precious metal for the dissociation of NO.     

Priors for Bayesian adaptive spline smoothing

Adaptive smoothing has been proposed for curve-fitting problems where the underlying function is spatially inhomogeneous. Two Bayesian adaptive smoothing models, Bayesian adaptive smoothing splines on a lattice and Bayesian adaptive P-splines, are studied in this paper. Estimation is fully Bayesian and carried out by efficient Gibbs sampling. Choice of prior is critical in any Bayesian non-parametric regression method. We use objective priors on the first level parameters where feasible, specifically independent Jeffreys priors (right Haar priors) on the implied base linear model and error variance, and we derive sufficient conditions on higher level components to ensure that the posterior is proper. Through simulation, we demonstrate that the common practice of approximating improper priors by proper but diffuse priors may lead to invalid inference, and we show how appropriate choices of proper but only weakly informative priors yields satisfactory inference.     

Convergence rate for predictive recursion estimation of finite mixtures

Predictive recursion (PR) is a fast stochastic algorithm for nonparametric estimation of mixing distributions in mixture models. It is known that the PR estimates of both the mixing and mixture densities are consistent under fairly mild conditions, but currently very little is known about the rate of convergence. Here I first investigate asymptotic convergence properties of the PR estimate under model misspecification in the special case of finite mixtures with known support. Tools from stochastic approximation theory are used to prove that the PR estimates converge, to the best Kullback-Leibler approximation, at a nearly root-n rate. When the support is unknown, PR can be used to construct an objective function which, when optimized, yields an estimate of the support. I apply the known-support results to derive a rate of convergence for this modified PR estimate in the unknown support case, which compares favorably to known optimal rates.     

Nuclear data for space radiation

Human space flight requires protecting astronauts from the harmful effects of space radiation. The availability of measured nuclear cross-section data needed for these studies is reviewed in the present paper. The energy range of interest for radiation protection is approximately 100 MeV/n–10 GeV/n. The majority of data are for projectile fragmentation partial and total cross-sections, including both charge changing and isotopic cross-sections. The cross-section data are organized into categories which include charge changing, elemental, isotopic for total, single and double differential with respect to momentum, energy and angle. Gaps in the data relevant to space radiation protection are discussed and recommendations for future experiments are made.     

On the transient response of forced nonlinear oscillators

We consider the transient response of a prototypical nonlinear oscillator modeled by the Duffing equation subjected to near resonant harmonic excitation. Of interest here is the overshoot problem that arises when the system is undergoing free motion and is suddenly subjected to harmonic excitation with a near resonant frequency, which leads to a beating type of transient response during the transition to steady state. In some design applications, it is valuable to know the peak value of this response and the manner in which it depends on system parameters, input parameters, and initial conditions. This nonlinear overshoot problem is addressed by considering the well-known averaged equations that describe the slowly varying amplitude and phase for both transient and steady state responses. For the undamped system, we show how the problem can be reduced to a single parameter χ that combines the frequency detuning, force amplitude, and strength of nonlinearity. We derive an explicit expression for the overshoot in terms of χ, describe how one can estimate corrections for light damping, and verify the results by simulations. For zero damping, the overshoot approximation is given by a root of a quartic equation that depends solely on χ, yielding a simple bound for the overshoot of lightly damped systems.     

Acrolein coupling on reduced TiO2(1 1 0): The effect of surface oxidation and the role of subsurface defects

Reactions of acrolein, water, and oxygen with the vacuum-reduced surface of TiO₂(1 1 0) are reported in a temperature programmed reaction study of the interaction of an aldehydic pollutant with a reducible metal oxide. A total of 25% of the acrolein that binds to the surface is converted to products. Notably, carbon-carbon coupling occurs with 86% selectivity for formation of C₆ products: C₆H₈, identified as 1,3-cyclohexadiene, in a peak at 500 K and benzene immediately thereafter at 530 K. Acrolein is evolved from the surface in three peaks: a peak independent of coverage at 495 K, attributed to decomposition of an intermediate that is partly converted to C₆H₈; a coverage-dependent peak that shifts from 370 K (low coverage) to 260 K (high coverage), which is attributed to adsorption at 5-fold coordinated Ti sites; and a multilayer state at 160 K. Water and acrolein compete for 5-fold coordinated titanium sites when dosed sequentially. The addition of water also opens a new reaction pathway, leading to the hydrogenation of acrolein to form propanal. Water has no effect on the yield of 1,3-cyclohexadiene. Exposure of the surface to oxygen prior to acrolein dosing quenches the evolution of acrolein at 495 K and concurrently eliminates the coupling. From these results, we propose that reduced subsurface defects such as titanium ion interstitials play a role in the reactions observed here. The notion that subsurface defects may contribute to the reactivity of organic molecules over reducible oxide substrates may prove to be general.     

Local derivative post-processing for the discontinuous Galerkin method

Obtaining accurate approximations for derivatives is important for many scientific applications in such areas as fluid mechanics and chemistry as well as in visualization applications. In this paper we discuss techniques for computing accurate approximations of high-order derivatives for discontinuous Galerkin solutions to hyperbolic equations related to these areas. In previous work, improvement in the accuracy of the numerical solution using discontinuous Galerkin methods was obtained through post-processing by convolution with a suitably defined kernel. This post-processing technique was able to improve the order of accuracy of the approximation to the solution of time-dependent symmetric linear hyperbolic partial differential equations from order k+1$k+1$ to order 2k+1$2k+1$ over a uniform mesh; this was extended to include one-sided post-processing as well as post-processing over non-uniform meshes. In this paper, we address the issue of improving the accuracy of approximations to derivatives of the solution by using the method introduced by Thomée [19]. It consists in simply taking the α$\alpha$th-derivative of the convolution of the solution with a sufficiently smooth kernel. The order of convergence of the approximation is then independent   of the order of the derivative, |α|$|\alpha |$. We also discuss an efficient way of computing the approximation which does not involve differentiation but the application of simple finite differencing. Our results show that the above-mentioned approximations to the α$\alpha$th-derivative of the exact solution of linear, multidimensional symmetric hyperbolic systems obtained by the discontinuous Galerkin method with polynomials of degree k$k$ converge with order 2k+1$2k+1$ regardless of the order |α|$|\alpha |$ of the derivative.