A Proton ENDOR Study of Azurin

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cβ atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein

PhyloPat: an updated version of the phylogenetic pattern database contains gene neighborhood

Phylogenetic patterns show the presence or absence of certain genes in a set of full genomes derived from different species. They can also be used to determine sets of genes that occur only in certain evolutionary branches. Previously, we presented a database named PhyloPat which allows the complete Ensembl gene database to be queried using phylogenetic patterns. Here, we describe an updated version of PhyloPat which can be queried by an improved web server. We used a single linkage clustering algorithm to create 241 697 phylogenetic lineages, using all the orthologies provided by Ensembl v49. PhyloPat offers the possibility of querying with binary phylogenetic patterns or regular expressions, or through a phylogenetic tree of the 39 included species. Users can also input a list of Ensembl, EMBL, EntrezGene or HGNC IDs to check which phylogenetic lineage any gene belongs to. A link to the FatiGO web interface has been incorporated in the HTML output. For each gene, the surrounding genes on the chromosome, color coded according to their phylogenetic lineage can be viewed, as well as FASTA files of the peptide sequences of each lineage. Furthermore, lists of omnipresent, polypresent, oligopresent and anticorrelating genes have been included. PhyloPat is freely available at http://www.cmbi.ru.nl/phylopat

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes

Contains fulltext : 124321.pdf (publisher's version ) (Open Access)BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome

Temperature Determination by EPR at 275 GHz and the Detection of Temperature Jumps in Aqueous Samples

Second-moment analysis along two dimensions of continuous-wave EPR spectra of nitroxides enables EPR thermometry in a broad temperature range. Simulations show that the temperature can be derived in both the slow-motion and the fast-motion regime, which is experimentally verified at 275 GHz for H₂O/glycerol (50/50% by volume) and pure water. We demonstrate that this tool allows the calibration of temperature jumps induced by infrared laser irradiation of a submicroliter sample in the single-mode cavity of a 275 GHz spectrometer, which prepares for kinetic studies of processes involving paramagnetic species

A novel TiO2 composite for photocatalytic wastewater treatment

A novel TiO2 composite consisting of Anatase interacting with a Ti3+-containing Rutile phase was synthesized by heating a mixture of TiO2 (Hombikat) and Ti2O3 in air at different temperatures ranging from 300 °C up to 900 °C. The preparation of the samples was analyzed by Thermal Gravimetric Analysis (TGA), and the resulting composites characterized by X-ray powder diffraction (XRD), Raman and UV–Vis spectroscopy, X-ray Photoelectron Spectroscopy (XPS), Electron Paramagnetic Resonance (EPR) spectroscopy, and Scanning Electron Microscopy. Characterization data show a phase transformation from Ti2O3 to Ti3+-containing Rutile at temperatures of around 600 °C. Moreover, Hombikat is gradually converted from amorphous to crystalline Anatase. The Ti3+-content and the degree of Anatase crystallinity are respectively inversely and directly proportional to an increasing preparation temperature. The composite which was synthesized at 600 °C showed the highest photocatalytic rate in the decolorization of Methyl Orange (MO). The rate constant was significantly larger than obtained for Evonik P25 after identical thermal treatment (600 °C). Photodeposition of Pt further not only enhanced the photocatalytic activity of the optimized composite, but surprisingly also the stability. The methyl orange degradation results are discussed on the basis of hole and electron transfer phenomena between Anatase and Rutile phases, the latter containing (surface) oxygen vacancies (Ti3+). The presence of surface oxygen vacancies and/or Pt nanoparticles is proposed to be of benefit to the rate determining oxygen reduction reactio

Single-Crystal EPR Study at 95 GHz of the Type 2 Copper Site of the Inhibitor-Bound Quercetin 2,3-Dioxygenase

An electron-spin-echo-detected, electron-paramagnetic-resonance study has been performed on the type 2 copper site of quercetin 2,3-dioxygenase from Aspergillus japonicus. In the protein, copper is coordinated by three histidine nitrogens and two sulfurs from the inhibitor diethyldithiocarbamate. A single crystal of the protein was studied at 95 GHz and the complete g-tensor determined. The electron-paramagnetic-resonance data are compatible with two orientations of the principal g-axes in the copper center, one of which is preferred on the basis of an analysis of the copper coordination and the d-orbitals that are involved in the unpaired-electron orbital. For this orientation, the principal z-axis of the g-tensor makes an angle of 19° with the Cu-N(His112) bond and the N of His112 may be considered the axial ligand. The singly occupied molecular orbital contains a linear combination of copper d(xy) and d(yz)-orbitals, which are antibonding with atomic orbitals of histidine nitrogens and diethyldithiocarbamate sulfurs. The orientation of the g-tensor for the quercetin 2,3-dioxygenase is compared with that for type 1 copper sites