A high-throughput digital imaging screen for the discovery and directed evolution of oxygenases.

BACKGROUND: Oxygenases catalyze the hydroxylation of a wide variety of organic substrates. An ability to alter oxygenase substrate specificities and improve their activities and stabilities using recombinant DNA techniques would expand their use in processes such as chemical synthesis and bioremediation. Discovery and directed evolution of oxygenases require efficient screens that are sensitive to the activities of interest and can be applied to large numbers of crude enzyme samples. RESULTS: Horseradish peroxidase (HRP) couples the phenolic products of hydroxylation of aromatic substrates to generate colored and/or fluorescent compounds that are easily detected spectroscopically in high-throughput screening. Coexpression of the coupling enzyme with a functional mono- or dioxygenase creates a pathway for the conversion of aromatic substrates into fluorescent compounds in vivo. We used this approach for detecting the products of the toluene-dioxygenase-catalyzed hydroxylation of chlorobenzene and to screen large mutant libraries of Pseudomonas putida cytochrome P450cam by fluorescence digital imaging. Colors generated by the HRP coupling reaction are sensitive to the site of oxygenase-catalyzed hydroxylation, allowing the screen to be used to identify catalysts with new or altered regiospecificities. CONCLUSIONS: The coupled oxygenase-peroxidase reaction system is well suited for screening oxygenase libraries to identify mutants with desired features, including higher activity or stability and altered reaction specificity. This approach should also be useful for screening expressed DNA libraries and combinatorial chemical libraries for hydroxylation catalysts and for optimizing oxygenase reaction conditions.     

Localization of weakly disordered flat band states

Certain tight binding lattices host macroscopically degenerate flat spectral bands. Their origin is rooted in local symmetries of the lattice, with destructive interference leading to the existence of compact localized eigenstates. We study the robustness of this localization to disorder in different classes of flat band lattices in one and two dimensions. Depending on the flat band class, the flat band states can either be robust, preserving their strong localization for weak disorder W, or they are destroyed and acquire large localization lengths ξ that diverge with a variety of unconventional exponents ν, ξ ~ 1 /W ^ν .     

Eisenhart lift in pseudo–Euclidean space and higher rank killing tensors

It is emphasized that the Eisenhart lift applied to integrable systems in pseudo–Euclidean space may result in Ricci–flat metrics of ultrahyperbolic signature which admit higher rank Killing tensors.     

Non-Hydroxamate Zinc-Binding Groups as Warheads for Histone Deacetylases

Histone deacetylases (HDACs) remove acetyl groups from acetylated lysine residues and have a large variety of substrates and interaction partners. Therefore, it is not surprising that HDACs are involved in many diseases. Most inhibitors of zinc-dependent HDACs (HDACis) including approved drugs contain a hydroxamate as a zinc-binding group (ZBG), which is by far the biggest contributor to affinity, while chemical variation of the residual molecule is exploited to create more or less selectivity against HDAC isozymes or other metalloproteins. Hydroxamates have a propensity for nonspecificity and have recently come under considerable suspicion because of potential mutagenicity. Therefore, there are significant concerns when applying hydroxamate-containing compounds as therapeutics in chronic diseases beyond oncology due to unwanted toxic side effects. In the last years, several alternative ZBGs have been developed, which can replace the critical hydroxamate group in HDACis, while preserving high potency. Moreover, these compounds can be developed into highly selective inhibitors. This review aims at providing an overview of the progress in the field of non-hydroxamic HDACis in the time period from 2015 to present. Formally, ZBGs are clustered according to their binding mode and structural similarity to provide qualitative assessments and predictions based on available structural information.     

Computational study of singlet and triplet sulfonylnitrenes insertion into 1,3‐butadienes: 1,2‐ or 1,4‐cycloaddition?

The formation of N‐trifluoromethylsulfonyl‐2‐vinylaziridine and N‐trifluoromethylsulfonyl‐3‐pyrroline by the reaction of the singlet and triplet trifluoromethanesulfonylnitrenes with s‐cis‐ and s‐trans‐1,3‐butadienes was studied theoretically at the B3LYP/6‐311++G(d,p) and M06‐2X/6‐311++G(d,p) levels of theory. The singlet trifluoromethanesulfonylnitrene adds to s‐cis‐ and s‐trans‐1,3‐butadiene exothermally in one step to give the product of 1,2‐cycloaddition, N‐trifluoromethylsulfonyl‐2‐vinylaziridine, the energy decreasing by 88.5 and 86.2 kcal/mol at the B3LYP level and by 105.2 and 103.0 kcal/mol at the M06‐2X level, respectively. The formed 2‐vinylaziridine can undergo rotation about the C(2)–C_sp2 bond with the barrier not exceeding 3.5 kcal/mol and to rearrange into N‐trifluoromethylsulfonyl‐3‐pyrroline. The triplet trifluoromethanesulfonylnitrene reacts with s‐cis‐ and s‐trans‐1,3‐butadiene in two steps. The first exothermic step is the formation of the triplet diradical adducts. The second step is the spin inversion with the energy raising by 5.8 and 17.8 kcal/mol at the B3LYP level and by 11.0 and 20.8 kcal/mol at the M06‐2X level for the adducts to s‐cis‐ and s‐trans‐1,3‐butadiene, respectively. Recombination of the radical centers occurs selectively to give N‐trifluoromethylsulfonyl‐2‐vinylaziridine that is exothermally rearranged into N‐trifluoromethylsulfonyl‐3‐pyrroline with the energy barrier of 40 kcal/mol at the B3LYP level and of 50 kcal/mol at the M06‐2X level. Copyright © 2014 John Wiley & Sons, Ltd.     

Ultra‐Precision Surface Machining with Reactive Plasma Jets

Atmospheric Plasma Jet Machining (PJM) is a technology for non‐mechanical ultra‐precision surface shape generation, shape error correction and smoothing based on atmospheric plasma jets. PJM is favorably applied to generate optical surfaces like aspheres, acylinders, or free‐forms but also to improve the surface shape accuracy in a very fast and cost‐efficient way. For that purpose a mainly fluorine containing plasma jet is brought into contact with a surface to locally remove material by a chemical reaction forming volatile products. Hence, the technology is limited to materials like silicon, fused silica and similar, or silicon carbide. Furthermore, the etch profile results from a convolution of the radical and the temperature distribution at the surface. Since the temperature distribution is also influenced by the plasma jet this leads to a non‐linear dependence of the removal function of the plasma tool on its velocity. Using the dwell‐time algorithm for deterministic surface machining by superposition of the local removal function of the plasma tool an advanced process simulation is necessary. In a first local approximation the velocity dependence of the removal function, which has to be determined previously, must be incorporated. Second order thermal effects due to inhomogeneous heating caused by the part geometry and the tool path can be managed by a sophisticated calculation of the surface temperature evolution during machining based on the Finite Element Method (FEM). With the help of this procedure the accuracy and convergence of the machining process can be significantly improved. In the article several examples of surface processing using plasma jet machining are presented. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The microscopic visualisation of the sonocrystallisation of ice using a novel ultrasonic cold stage

This work reports dynamic video images of the influence of ultrasonic cavitation on the sonocrystallisation of ice at a microscopic level. This has been achieved through the construction of a unique ultrasonic system for an optical microscope. The system consists of (1). an ultrasonic cold stage, (2). a temperature control system, and (3). a microscope and imaging setup. This allows the temperature of a sample to be systematically controlled while it is subjected to simultaneous excitation with alternating pressures in the ultrasonic frequency range. Both the amplitude of excitation and the frequency can be varied. Experiments on ice crystals in pure water and sucrose solutions were conducted. Three distinct phenomena were observed. Firstly, there is a tendency for cavitation bubbles to form at the grain boundaries between ice crystals. Secondly, there is a progressive melting of ice by cavitation bubbles which appear to eat their way into the ice phase. Thirdly, the dendritic ice structures may fragment under the influence of ultrasound, thus increasing the number of nuclei which may subsequently grow (secondary nucleation). These observations form the basis of a significantly enhanced understanding and exploitation of the sonocrystallisation of ice.     

Chiral mirror and optical resonator designs for circularly polarized light: suppression of cross-polarized reflectances and transmittances

A left-handed chiral sculptured thin film (STF) that reflects strongly at the wavelength of the circular Bragg resonance tends to partially convert the handedness of incident LCP (left-circularly-polarized) light to RCP (right-circularly-polarized). We show that the cross-polarized component of the reflected RCP beam can be eliminated by interference with an additional RCP beam that is reflected at the interface of an isotropic cover and an AR (antireflecting) layer. For best results the refractive index and thickness of the AR layer need to accommodate a phase change on reflection that occurs at the chiral film. Effective suppression of the reflectances R_RR, R_RL, R_LR and the transmittances T_RL, T_LR can be achieved by sandwiching the chiral reflector between such amplitude and phase-matched AR coatings. Co-polarized chiral reflectors of this type may form efficient handed optical resonators. For LCP light the optical properties of such a handed resonator are formally the same as the properties of the isotropic passive or active Fabry–Perot resonators, but the handed resonator is transparent to RCP light.     

Compressible Navier–Stokes Equations on Thin Domains

We consider the barotropic Navier–Stokes system describing the motion of a compressible viscous fluid confined to a straight layer \({\Omega_\varepsilon = \omega\times (0, \varepsilon)}\) , where ω is a particular 2-D domain (a periodic cell, bounded domain or the whole 2-D space). We show that the weak solutions in the 3D domain converge to a (strong) solutions of the 2-D Navier–Stokes system on ω as \({\varepsilon \to 0}\) on the maximal life time of the strong solution.