Improving Protein Crystal Quality by the Without-Oil Microbatch Method: Crystallization and Preliminary X-ray Diffraction Analysis of Glutathione Synthetase from Pseudoalteromonas haloplanktis

Glutathione synthetases catalyze the ATP-dependent synthesis of glutathione from l-γ-glutamyl- l-cysteine and glycine. Although these enzymes have been sequenced and characterized from a variety of biological sources, their exact catalytic mechanism is not fully understood and nothing is known about their adaptation at extremophilic environments. Glutathione synthetase from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhGshB) has been expressed, purified and successfully crystallized. An overall improvement of the crystal quality has been obtained by adapting the crystal growth conditions found with vapor diffusion experiments to the without-oil microbatch method. The best crystals of PhGshB diffract to 2.34 Å resolution and belong to space group P2₁2₁2₁, with unit-cell parameters a = 83.28 Å, b = 119.88 Å, c = 159.82 Å. Refinement of the model, obtained using phases derived from the structure of the same enzyme from Escherichia coli by molecular replacement, is in progress. The structural determination will provide the first structural characterization of a psychrophilic glutathione synthetase reported to date.     

Longitudinal study on the sources of Listeria monocytogenes contamination in cold-smoked salmon and its processing environment in Italy

The aim of the present study was to investigate the sources of Listeria monocytogenes contamination in a cold smoked salmon processing environment over a period of six years (2003-2008). A total of 170 samples of raw material, semi-processed, final product and processing surfaces at different production stages were tested for the presence of L. monocytogenes. The L. monocytogenes isolates were characterized by multiplex PCR for the analysis of virulence factors and for serogrouping. The routes of contamination over the six year period were traced by PFGE. L. monocytogenes was isolated from 24% of the raw salmon samples, 14% of the semi-processed products and 12% of the final products. Among the environmental samples, 16% were positive for L. monocytogenes. Serotyping yielded three serovars: 1/2a, 1/2b, 4b, with the majority belonging to serovars 1/2a (46%) and 1/2b (39%). PFGE yielded 14 profiles: two of them were repeatedly isolated in 2005-2006 and in 2007-2008 mainly from the processing environment and final products but also from raw materials. The results of this longitudinal study highlighted that contamination of smoked salmon occurs mainly during processing rather than originating from raw materials, even if raw fish can be a contamination source of the working environment. Molecular subtyping is critical for the identification of the contamination routes of L. monocytogenes and its niches into the production plant when control strategies must be implemented with the aim to reduce its prevalence during manufacturing.     

Antimicrobial applications of electroactive PVK-SWNT nanocomposites

The antibacterial properties of a nanocomposite containing an electroactive polymer, polyvinyl-N-carbazole (PVK) (97 wt %), and single-walled carbon nanotubes (SWNT) (3 wt %) was investigated as suspensions in water and as thin film coatings. The toxic effects of four different PVK-SWNT (97:3 wt %) nanocomposite concentrations (1, 0.5, 0.05, and 0.01 mg/mL) containing 0.03, 0.015, 0.0015, and 0.0003 mg/mL of SWNT, respectively, were determined for planktonic cells and biofilms of Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). The results showed that the nanocomposite PVK-SWNT had antibacterial activity on planktonic cells and biofilms at all concentration levels. Higher bacterial inactivation (94% for E. coli and 90% for B. subtilis) were achieved in planktonic cells at a PVK-SWNT concentration of 1 mg/mL. Atomic force microscopy (AFM) imaging showed significant reduction of biofilm growth on PVK-SWNT coated surfaces. This study established for the first time that the improved dispersion of SWNTs in aqueous solutions in the presence of PVK enhances the antimicrobial effects of SWNTs at very low concentrations. Furthermore, PVK-SWNT can be used as an effective thin film coating material to resist biofilm formation.     

Prebiotic potential of a refined product containing pectic oligosaccharides

Apple pomace samples were subjected to Simultaneous Saccharification and Fermentation (SSF) to yield a medium containing mainly lactic acid and oligosaccharides. After lactic acid removal by ion exchange, the SSF medium was refined by a two-step (diafiltration-concentration) membrane treatment. Limited losses of glucooligosaccharides, galactooligosaccharides, xylooligosaccharides and arabinooligosaccharides were noticed in permeates, whereas the concentrate from the second stage showed low concentrations of lactic acid, arabinose and NaCl, which were extensively removed in the refining process. About 60% other the non-saccharide compounds present in the solution fed to membranes were removed in permeates, whereas oligogalacturonides were quantitatively recovered in the final concentrate. The refined product was assayed for its prebiotic potential by means of in vitro fermentability assays performed with individual microbial strains or human fecal inocula. In this latter case, the dynamics of microbial populations was assessed by Fluorescent In Situ Hybridization (FISH). The experimental results proved the prebiotic effects caused by the refined product obtained in this work, which included the promotion of beneficial bacterial growth and the increase of short chain fatty acids (SCFA) concentrations.     

Revealing the proliferation of hydrogen scavengers in a single-chamber microbial electrolysis cell using electron balances

The bioelectrochemical generation of hydrogen in microbial electrolysis cells (MECs) is a promising technology with many bottlenecks to be solved. Among them, the proliferation of hydrogen scavengers drastically reduces the cell efficiency leading to unrealistic coulombic efficiencies (CE) and cathodic gas recoveries (r_CAT). This work provides a novel theoretical approach to understand, through electron equivalent balances, the fate of hydrogen in these systems. It was validated with a long term operated single-chamber membrane-less MEC. In the short term, H₂-recycling (i.e. hydrogen being derived to the anode) resulted in r_CAT of only 4% and in CE up to 463%. The 80.5% of the current intensity came from H₂-recycling and only the 19.5% from substrate oxidation. In the long term, methane was produced from hydrogen, thus decreasing r_CAT to 0 (r_CAT = 94.5% when considering methane production). CE was 74.5% suggesting that H₂-recycling only took place when methanogenic activity was marginal.     

Dispersion and surface functionalization of oxide nanoparticles for transparent photocatalytic and UV-protecting coatings and sunscreens

Abstract

This review describes recent efforts on the synthesis, dispersion and surface functionalization of the three dominating oxide nanoparticles used for photocatalytic, UV-blocking and sunscreen applications: titania, zinc oxide, and ceria. The gas phase and liquid phase synthesis is described briefly and examples are given of how weakly aggregated photocatalytic or UV-absorbing oxide nanoparticles with different composition, morphology and size can be generated. The principles of deagglomeration are reviewed and the specific challenges for nanoparticles highlighted. The stabilization of oxide nanoparticles in both aqueous and non-aqueous media requires a good understanding of the magnitude of the interparticle forces and the surface chemistry of the materials. Quantitative estimates of the Hamaker constants in various media and measurements of the isoelectric points for the different oxide nanoparticles are presented together with an overview of different additives used to prepare stable dispersions. The structural and chemical requirements and the various routes to produce transparent photocatalytic and nanoparticle-based UV-protecting coatings, and UV-blocking sunscreens are described and discussed.     

Ammonium Ion Adsorption and Settleability Improvement Achieved in a Synthetic Zeolite-Amended Activated Sludge

Municipal wastewater treatment plants typically exhibit two classic problems:high ammonium concen-tration in water after conventional biological treatment and, in some cases, poor activated sludge sedi...     

Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits

A method based on solid-phase microextraction (SPME) and capillary electrophoresis/mass spectrometry (CE/ MS) is described for determining simultaneously five acidic pesticides (o-phenylphenol, ioxynil, haloxyfop, acifluorfen, picloram) in fruits. The CE device is coupled to an electrospray interface by a commercial sheath-flow adapter. Emphasis is placed on fulfillment of the speed and sensitivity requirements. The best separation is achieved using 32 mM ammonium formate/acid formic buffer at pH 3.1, with a working voltage of 25 kV. The MS detection of the five pesticides was performed in negative ionization mode. Full-scan spectra with base peaks corresponding to [M-H]- were obtained except for acifluorfen, which gives [M-H-CO2]- as most abundant ion. Compared with the conventional EC-UV, the limits of detection were lower for acifluorfen, haloxyfop, ioxynil, and picloram, by a factor of 20, 20, 50, and 2, respectively. Extraction involved fruit sample homogenization with an acetone-water solution (5:1), filtration, and acetone evaporation prior to fiber extraction. SPME conditions such as time, pH, ion strength, stationary phase of the fiber, sample matrix, and desorption solvents were examined. The recovery of the analytes ranged from 7 to 94%, and the relative standard deviation was between 3 and, 13%. The method was found to be linear between 0.02 and 500 mg kg(-1) with correlation coefficients ranging from 0.992 to 0.997. The limits of quantification were from 0.02 to 5 mg kg(-1). The optimized method was successfully applied to the analysis of acid pesticides in fruit samples.     

Comparison of dissolution profiles for albendazole tablets using USP apparatus 2 and 4

The in vitro dissolution of albendazole from three different commercially available products (200 mg tablets) was studied using U.S. Pharmacopeia (USP) Apparatus 2 and USP Apparatus 4 in order to compare the release performance of the drug in two essentially different dissolution systems. For both cases, 0.1 N HCl was used as dissolution medium. Only the reference product and one of the generic products studied met the 80% USP 24 specification for albendazole dissolved at 30 min, using USP Apparatus 2. Although the reference product reached 80% of albendazole dissolved at 30 min when Apparatus 4 was used, the generic products' dissolution performance was markedly reduced in this system. Though dissolution rate was slower using Apparatus 4, the total quantity of albendazole dissolved from the reference product, represented by area under the dissolution profile, was practically the same regardless of the system used. Dissolution kinetics of albendazole was adequately described by Weibull's function for all the products. The dissolution time (t_d) derived from data fitting to this function showed significant differences among the products studied. Data analysis based on analysis of variance (ANOVA) showed nonequivalence among the dissolution profiles of generic products compared with the reference product either with the dissolution vessel system or the flow-through cell, as well as nonequivalence among the dissolution profiles using both apparatuses with the same product. Though differences in the dissolution profiles for generic products against the reference product in both systems were found, USP Apparatus 4 showed higher discriminative capacity in differentiating the release characteristics of the products tested.     

Iodine-131 metaiodobenzylguanidine scintigraphy for localization of lesions in children with neuroblastoma: comparison with computed tomography and ultrasonography

Iodine-131 metaiodobenzylguanidine (MIBG) scintigraphy, computed tomography (CT) and ultrasonography (US) were used to localize tumour lesions in 28 children with histologically proven neuroblastoma. Overall, a total of 73 lesions were detected on imaging studies. MIBG scintigraphy, CT and US localized 63 (86%), 49 (67%) and 36 (49%) of these lesions, respectively. The findings of the three imaging techniques were concordant in respect of only 31 (42%) of the lesions. The best agreement among MIBG scintigraphy, CT and US was observed for abdominal lesions (the techniques were concordant for 22 of 23 lesions, i.e. 96%). MIBG scintigraphy detected nine out of ten (90%) liver metastases, but agreement with CT and US was observed in only six instances (60%). The imaging findings were concordant in respect of only two (33%) out of six lymph node metastases; the MIBG scan was normal in the other four cases. Imaging agreement was observed for a lesion located in the pelvis. MIBG and CT findings were concordant in four lesions located in the chest, but US was not performed. MIBG scintigraphy depicted the majority (96%) of the skeletal lesions (23/24); CT showed five of these, but, again, US was not performed. The imaging findings were not concordant as regards the remaining five lesions located in different anatomical sites. The results indicated that MIBG imaging is more sensitive that CT and US in localizing the majority of neuroblastoma lesions. Since the metastatic spread of neuroblastoma is unpredictable, we recommend MIBG scintigraphy as the initial imaging modality for staging of these patients.(ABSTRACT TRUNCATED AT 250 WORDS)