Active Site Crowding of Cytochrome P450 3A4 as a Strategy To Alter Its Selectivity

Substrate‐promiscuous enzymes are a promising starting point for the development of versatile biocatalysts. In this study, human cytochrome P450 3A4, known for its ability to metabolise hundreds of drugs, was engineered to alter its regio‐ and stereoselectivity. Rational mutagenesis was used to introduce steric hindrance in a specific manner in the large active site of P450 3A4 and to favour oxidation at a more sterically accessible position on the substrate. Hydroxylation of a synthetic precursor of (R)‐lisofylline, a compound under investigation for its anti‐inflammatory properties, was chosen as a first proof‐of‐principle application of our protein engineering strategy. In a second example, increasing active site crowding led to an incremental shift in the selectivity of oxidation from an internal double bond to a terminal phenyl group in a derivative of theobromine. The same correlation between crowding and selectivity was found in a final case focused on the hydroxylation of the steroid sex hormone progesterone.     

Cuticular Hydrocarbons as Sex Pheromone of the Bee <Emphasis Type="Italic">Colletes cunicularius</Emphasis> and the Key to its Mimicry by the Sexually Deceptive Orchid, <Emphasis Type="Italic">Ophrys exaltata</Emphasis>

Male Colletes cunicularius bees pollinate the orchid, Ophrys exaltata, after being sexually deceived by the orchid’s odor-mimicry of the female bee’s sex pheromone. We detected biologically active volatiles of C. cunicularius by using gas chromatographic–electroantennographic detection (GC-EAD) with simultaneous flame ionization detection. After identification of the target compounds by coupled gas chromatography–mass spectrometry (GC-MS), we performed behavioral tests using synthetic blends of the active components. We detected 22 EAD active compounds in cuticular extracts of C. cunicularius females. Blends of straight chain, odd-numbered alkanes and (Z)-7-alkenes with 21–29 carbon atoms constituted the major biologically active compounds. Alkenes were the key compounds releasing mating behavior, especially those with (Z)-7 unsaturation. Comparison of patterns of bee volatiles with those of O. exaltata subsp. archipelagi revealed that all EAD-active compounds were also found in extracts of orchid labella. Previous studies of the mating behavior in C. cunicularius showed linalool to be an important attractant for patrolling males. We confirmed this with synthetic linalool but found that it rarely elicited copulatory behavior, in accordance with previous studies. A blend of active cuticular compounds with linalool elicited both attraction and copulation behavior in patrolling males. Thus, linalool appears to function as a long-range attractant, whereas cuticular hydrocarbons are necessary for inducing short-range mating behavior.     

Analytical investigation of the composition of plasma-induced functional groups on carbon nanotube sheets

To increase the applicability of carbon nanotubes (CNTs) oxygen-containing functional groups were generated on their widely inert surface by using glow-discharge plasmas. CNT-sheets (bucky papers) produced from the powder-like raw material were used as substrates allowing for a more defined characterization of one and the same surface by different analytical techniques. The plasma composition was analyzed by optical emission spectroscopy. Since the actual composition of the plasma-induced functional groups has still not been completely resolved, we performed an in-depth characterization of the treated samples by X-ray photoelectron (XPS) and Raman spectroscopy as well as electron spin resonance measurements. To overcome limitations of the XPS-analysis in distinguishing between groups featuring nearby binding energies, alcohol-, keto-/aldehyde-, and carboxyl-groups were tagged by derivatization techniques using fluorine-containing reagents (trifluoroaceticanhydride, trifluoromethylphenylhydrazine, and trifluoroethanol). Differential spectra were calculated to enhance the accuracy of the deconvolution of the XPS-spectra. This enabled us to determine dependencies of the plasma parameters, i.e. treatment time, process pressure, and gas composition (mixtures of Ar, O₂, H₂O, and H₂), on the composition of the generated functional groups as well as an up to 6-fold enhancement in derivatizable groups for switching process gas from Ar/O₂ to Ar/H₂O.     

Method for Accurate Surface Temperature Measurements During Fast Induction Heating

A robust method is proposed for the measurement of surface temperature fields during induction heating. It is based on the original coupling of temperature-indicating lacquers and a high-speed camera system. Image analysis tools have been implemented to automatically extract the temporal evolution of isotherms. This method was applied to the fast induction treatment of a 4340 steel spur gear, allowing the full history of surface isotherms to be accurately documented for a sequential heating, i.e., a medium frequency preheating followed by a high frequency final heating. Three isotherms, i.e., 704, 816, and 927°C, were acquired every 0.3 ms with a spatial resolution of 0.04 mm per pixel. The information provided by the method is described and discussed. Finally, the transformation temperature Ac1 is linked to the temperature on specific locations of the gear tooth.     

Fast Zirconium Oxidation Using Supercritical Water

A novel route for fast zirconium oxidation is reported, using supercritical water, for the case of Zircaloy-4 tubes that are used to clad nuclear fuel pellets in pressurized water reactors. An adherent and dense monoclinic zirconia layer 28 µm thick is obtained after 5 h of supercritical water oxidation at 500 °C. The zirconia crystallites are about 13 ± 2 nm in size. This technique reduces the time needed to oxidize Zircaloy-4 from several weeks to a few hours when standard conditions are being used.     

The Effect of Submicron Second-Phase Particles on the Rate of Grain Refinement in a Copper-Oxygen Alloy During Cold Spray

The effect of non-deformable submicron second-phase particles (d = 200-500 nm) on microstructural refinement during cold spray was examined. Using single particle impact testing, two types of splats were fabricated using two different feedstocks: a Cu-0.21wt.%O powder containing Cu₂O second-phase particles and a single-phase Cu. Microstructural evolution analysis using high-resolution electron backscatter diffraction shows grain refinement occurred at a higher rate in the Cu-0.21wt.%O powder. That was due to dynamic recrystallization initiated by particle-stimulated nucleation (PSN). High-strain-rate deformation of cold spray was found to be the key to activate PSN. The present study suggests cold spray is a possible technique to fabricate ultrafine-grained materials by using feedstock containing second-phase particles.     

A new model for nanofluid conductivity based on the effects of clustering due to Brownian motion

Conductivity values of nanofluids calculated with the model proposed by Nan et al. 1 consistently underestimate the corresponding measured values for 20 sets of experimental data from 12 published studies; thus the conclusion of the recent International Nanofluid Property Benchmark Exercise 2 which stated that it is not necessary to resort to other theories (e.g., Brownian motion, liquid layering, and aggregation) for the interpretation of the INPBE database cannot be generalized. In view of this situation, a new model which takes into account clustering and micro‐convection is proposed and compared with experimental data for five nanofluids (with different particle sizes and a range of particle concentrations) as well as two previously published models. The maximum difference between the predictions of the proposed model and the measured values is 6.7% of the latter. © 2011 Wiley Periodicals, Inc. Heat Trans Asian Res; Published online in Wiley Online Library ( wileyonlinelibrary.com/journal/htj ). DOI 10.1002/htj.20350     

Multi-level analysis of an interaction network between individuals in a mailing-list

It is well known now that most real-world complex networks have some properties which make them very different from random networks. In the case of interactions between authors of messages in a mailing-list, however, a multi-level structure may be responsible for some of these properties. We propose here a rigorous but simple formalism to investigate this question, and we apply it to an archive of the Debian user mailing-list. This leads to the identification of some properties which may indeed be explained this way, and of some properties which need deeper analysis.