Extending the biocatalytic scope of regiocomplementary flavin-dependent halogenase enzymes

Flavin-dependent halogenases are potentially valuable biocatalysts for the regioselective halogenation of aromatic compounds. These enzymes, utilising benign inorganic halides, offer potential advantages over traditional non-enzymatic halogenation chemistry that often lacks regiocontrol and requires deleterious reagents. Here we extend the biocatalytic repertoire of the tryptophan halogenases, demonstrating how these enzymes can halogenate a range of alternative aryl substrates. Using structure guided mutagenesis we also show that it is possible to alter the regioselectivity as well as increase the activity of the halogenases with non-native substrates including anthranilic acid; an important intermediate in the synthesis and biosynthesis of pharmaceuticals and other valuable products.     

Simple continuous and discrete models for simulating replica exchange simulations of protein folding

The efficiency of temperature replica exchange (RE) simulations hinge on their ability to enhance conformational sampling at physiological temperatures by taking advantage of more rapid conformational interconversions at higher temperatures. While temperature RE is a parallel simulation technique that is relatively straightforward to implement, kinetics in the RE ensemble is complicated, and there is much to learn about how best to employ RE simulations in computational biophysics. Protein folding rates often slow down above a certain temperature due to entropic bottlenecks. This "anti-Arrhenius" behavior represents a challenge for RE. However, it is far from straightforward to systematically explore the impact of this on RE by brute force molecular simulations, since RE simulations of protein folding are very difficult to converge. To understand some of the basic mechanisms that determine the efficiency of RE, it is useful to study simplified low dimensionality systems that share some of the key characteristics of molecular systems. Results are presented concerning the efficiency of temperature RE on a continuous two-dimensional potential that contains an entropic bottleneck. Optimal efficiency was obtained when the temperatures of the replicas did not exceed the temperature at which the harmonic mean of the folding and unfolding rates is maximized. This confirms a result we previously obtained using a discrete network model of RE. Comparison of the efficiencies obtained using the continuous and discrete models makes it possible to identify non-Markovian effects, which slow down equilibration of the RE ensemble on the more complex continuous potential. In particular, the rate of temperature diffusion and also the efficiency of RE is limited by the time scale of conformational rearrangements within free energy basins.     

Qualitative supercritical fluid extraction coupled to capillary gas chromatography

The usefulness and ease of utilizing supercritical fluid extraction (SFE) directly coupled to capillary gas chromatography (GC) as quantitative or qualitative analytical problem-solving tools will be demonstrated. As an alternative to conventional liquid solvent extractions, SFE presents itself as a means to achieve high extraction efficiencies of different compounds in complex solid matrices in very rapid tims frames. Moreover, SFE has an additional advantage of being able to achieve distinct extraction selectivities as a function of the solubilizing power of the supercritical fluid extracting phase. For on-line SFE/GC, the extraction effluent is directly transferred to the analytical chromatograph. On-line SFE/GC involves the decompression of pressurized extraction effluent directly into a heated, unmodified capillary split injection port of the GC. In this respect, SFE introduction into GC can be used as an alternative means of GC injection, comparable to such modes of injection as pyrolysis and thermal desorption. This paper will show applications of SFE/GC where mass spectrometric detection together with flame ionization detection was used for component identification from environmental, tobacco, and petroleum matrices.     

Iron(II) and zinc(II) monohelical binaphthyl salen complexes

A new chiral binaphthyl salen ligand with rigid polyaromatic sidearms gives monohelical complexes (Fe(II) and Zn(II)) of predetermined handedness.     

Search for neutrino oscillations

A search for ν_μ→ν_e and oscillations has been conducted with the AGS wide-band neutrino beam at the Brookhaven National Laboratory. We find more ν_e ( _e) interactions than were expected on the basis of the number of incident ν_e ( _e) calculated as part of the neutrino beam. The excess is about a factor two over the expectation, the statistical significance being about two and a half standard deviations for ν_e and weaker for _e.     

A high‐order element‐based Galerkin method for the barotropic vorticity equation

A high‐order element‐based Galerkin method is developed to solve the non‐divergent barotropic vorticity equation (BVE). The solution process involves solving a conservative transport equation for the vorticity fields and a Poisson equation for the stream function fields. The discontinuous Galerkin method is employed for solving the transport equation and a spectral element method (continuous Galerkin) is used for the Poisson equation. A third‐order strong stability preserving explicit Runge–Kutta scheme is used for time integration. A series of tests have been performed to validate the model, which include the evolution of an idealized tropical cyclone and interaction of dual vortices in close proximity. The numerical convergence study is performed by solving the BVE on the sphere where the analytic solution is known. The test results are consistent with physical observations, and the model exhibits exponential convergence. Copyright © 2008 John Wiley & Sons, Ltd.     

Biocompatible copper(I) catalysts for in vivo imaging of glycans

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.     

Collinear laser spectroscopy with reverse-extracted bunched beams at TRIUMF

Collinear laser spectroscopy with reverse-extracted cooled bunched beams was performed at TRIUMF with radioactive ion beams. Surface-ionized ^78,78mRb ions were injected into TITAN??s cooler-buncher, and reverse-extracted to the laser beam line. There they were neutralised and excited with a counter-propagating laser beam. The fluorescence signal from the D ₂ line was recorded with a photomultiplier tube. The cooler provided typically 50 ion bunches per second, with each bunch containing approximately 10⁵ ions, temporally distributed with a 1.6 ??s width. A hardware gate was set on the signal, suppressing the background by four orders of magnitude.