Patents and literature

Protein engineering and site-directed mutagenesis is becoming immensely important in both fundamental studies and commercial applications involving proteins and enzymes in biocatalysis. Protein engineering has become a powerful tool to help biochemists and molecular enzymologists elucidate structure-function relationships in enzymic active sites, to understand the intricacies of protein folding and denaturation, and to alter the selectivity of enzymatic catalysis. Commercial applications of engineered enzymes are being developed to increase protein stability, widen or narrow substrate specificity, and to develop novel approaches for use of enzymes in organic synthesis, drug design, and clinical applications. In addition to protein engineering, novel expression systems have been designed to prepare large quantities of genetically engineered proteins. Recent US patents and scientific literature on protein engineering, site-directed mutagenesis, and protein expression systems related to protein engineering are surveyed. Patent abstracts are summarized individually and a list of literature references are given.     

EXAFS and XANES Structure determination of Mn2+ binding in ATP complexes

Summary The binding of Mn²⁺ ions to ATP molecules has been studied by means of the fine-structure analysis of X-ray absorption spectra (EXAFS and XANES) at theK-edge of Mn using the synchrotron, radiation facility PULS in Frascati. The results obtained in both freeze-dried and liquid samples of aqueous solutions at room temperature of the Mn-ATP complex under diffrent values ofpH and Mn:ATP ratio are reported and discussed. The Mn²⁺ ion appears to be octahedrally coordinated, the phosphate oxygen atoms being in the first co-ordination shell. Values of (2.15±0.05) ? and (3.4±0.05) ?, respectively, have been found for Mn-O and Mn-P distances. Theoretical considerations together with experimental results do not support the existence of a direct binding of the Mn to the N(7) atom of the adenine ring. The coordination numbers for the first and second shell are consistent with the formation of a Mn(ATP)₂ complex in solution atpH=9 and Mn: ATP=1:10, while a Mn-ATP 1:1 complex is found at lowerpH and higher Mn: ATP ratios. To speed up publication, the authors of this paper have agreed to not receive the proofs for correction.     

Infrared photoluminescence of erbium-tris(8-hydroxyquinoline) in a distributed feedback cavity

A distributed feedback (DFB) waveguide cavity with erbium-tris(8-hydroxyquinoline) has been fabricated by X-ray interference lithography in a laboratory-scale apparatus. The DFB cavity consists of a large area () one-dimensional grating of polymethylmethacrylate on Si (1 0 0). Structural, morphological and optical properties of the device have been studied. On-grating narrowing of the photoluminescence emission has been observed for the 492 nm grating period in correspondence to the 4f-4f Er band peak (at 1530 nm), indicating the possibility of optical gain for applications in the telecommunications.     

Electrospun poly(ethylene oxide)/chitosan nanofibers with cellulose nanocrystals as support for cell culture of 3T3 fibroblasts

Chitosan/poly(ethylene oxide) (PEO) (5:1) nanofibers with cellulose nanocrystals (CNCs) were produced using an electrospinning technique. The addition of CNCs to the chitosan/PEO solutions allowed the production of uniform fibers (without beads) with a high proportion of chitosan. The fiber diameters were influenced by the concentration of CNCs in the chitosan/PEO solutions. The solutions containing 10% (w/w) of CNCs produced thinner fibers compared to solutions containing 5% (w/w) of CNCs. Thermogravimetric analysis indicated that the nanofibers were thermally stable, despite the CNCs having an effect on the PEO decomposition. Results from the cell assay in cultures of 3T3 fibroblasts indicated that the chitosan/PEO nanofibers (with 10% CNCs) promoted cell attachment with changes in the cytoskeletal organization. The results obtained in this work highlight the favorable effect of CNCs in electrospinning of chitosan/PEO. As expected, the influence of nanofibers on 3T3 fibroblasts F-actin and β-tubulin network revealed alterations in cytoskeleton, leading to changes in cell morphology and spreading.     

Development and characterisation of an XeCl excimer laser-generated soft-X-ray plasma source and its applications

Summary A large-aperture, long-pulse XeCl excimer laser has been used to generate a soft-X-ray plasma source. Several laser optical configurations have been employed to optimise X-ray emission, including positive-branch unstable resonators and injection by seeding the gain region of the laser with a small commercial excimer laser, resulting in power densities in the range 10¹²–10¹⁴ W cm⁻². The characteristics of the plasma source for each different laser configuration and for different target materials are investigated. The most suitable source conditions (spectral energy distribution, time duration, etc.) for specific applications are discussed. Supported by the Royal Society, UK. Supported by INFM, Italy.     

Folding characterization in conservative chaotic fluid flows

We study transport of passive scalar fields in a bidimensional incompressible chaotic fluid flow. For a spatially smooth velocity field with impulsive perturbations, the model is described by a randomized standard mapping. We numerically investigate passive scalar field transport for given initial concentration distributions and their dependence on the nonlinearity and noise amplitude. We show that space and time concentration histograms are determined by the underlying mechanism of stretching and folding. Moreover, to characterize this process we introduce a parameter, the average derivative of a tracer line length, which shows interesting scale properties.     

Three polymorphic forms of the co-crystal 4,4'-bipyridine/pimelic acid and their structural, thermal, and spectroscopic characterization

Three crystal forms of the co-crystal 4,4'-bipy/pimelic acid (bipy: bipyridine), [NH(4)C(5)-C(5)H(4)N][HOOC(CH(2))(5)COOH], have been prepared and their relationship investigated by single-crystal X-ray diffraction, variable-temperature X-ray powder diffraction, differential scanning calorimetry and solid-state NMR spectroscopy. Both X-ray and NMR spectroscopic results indicate that no proton transfer takes place, that is, the three crystal forms are true co-crystals of neutral molecules. Forms I and II both convert into Form III at high temperature, Forms II and III being the thermodynamically stable forms at room and high temperature, respectively.     

Quantification of p-cresol sulphate in human plasma by selected reaction monitoring

Chronic renal failure patients accumulate in the blood molecules that are normally excreted into the urine. p-Cresol Sulphate (pCS), the most representative retained toxin, shows a high level of toxicity. Therefore, its quantification could represent a prediction factor to determine the risk of endothelial dysfunction and cardiovascular complication and response to the haemodialysis treatment. The aim of this study was to evaluate the suitability of the multiple reaction monitoring (MRM) technique in order to improve the sensibility, the selectivity and the timing of pCS detection in a small amount of plasma. Deproteinized plasma of uremic patients was concentrated and dissolved in liquid chromatography (LC) mobile phase solution. pCS was quantified by LC coupled to tandem mass spectrometry (LC-MS/MS) on a triple-quadrupole mass spectrometer. Selective and sensitive detection of pCS was achieved by selecting the specific parent ion and monitoring two specific fragment ions. The MRM assay was carried out using the following transitions: m/z 187?→?80.00 and m/z 187?→?107.00. A good linearity was observed for each calibration curve. The intra-day and inter-day results showed a good precision and repeatability. The percentage recoveries indicate an optimal selectivity of the analytical method. The MRM assay to quantify pCS in a small amount of human plasma is rapid, highly sensitive, selective and with a good repeatability.