A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Effect of Al2O3 and Cr2O3 on loquidus temperatures in the cristobalite and tridymite primary phase fields of the MgO-"FeO"-SiO2 ststem in equilibrium with metallic iron

The effects of alumina and chromite impurities on the liquidus temperatures in the cristobalite/tridymite (SiO2) primary phase fields in the MgO-FeO-SiO, system in equilibrium with metallic iron have been investigated experimentally. Using high temperature equilibration and quenching followed by electron probe X-ray microanalysis (EPMA), liquiclus isotherms have been determined in the temperatures range 1 673 to 1 898 K. The results are presented in the form of pseudo-ternary sections of the MgO-FeO-SiO, system at 2, 3 and 5 wt% Al2O3, 2 wt% Cr2O3, and 2 wt% Cr2O3+2 wt% Al2O3. The study enables the liquidus to be described for a range of SiO2/MgO and MgO/FeO ratios. It was found that liquiclus temperatures in the cristobalite and tridymite primary phase fields, decrease significantly with the addition of Al2O3 and Cr2O3.

Cracking in the welding of cupro-nickel alloys

The problem of variation in weld crack susceptibility caused by small variations in alloy and impurity elements for the 70-30 cupro-nickel alloy has been investigated. Both wrought and cast versions of the alloy have been studied, the main techniques employed being the Varestraint test and weld thermal simulation. In the wrought alloys, cracking has been found to occur mainly in the weld metal, whilst in the cast alloys cracking is extensive in both weld metal and heat affected zone. The previously reported effects of certain impurities (P,S,Si) in increasing cracking have been confirmed, and it has also been shown that Ti and Zr may both have a crack promoting effect at levels commonly found in cupro-nickels, whilst C can interact with several of the other elements investigated to produce a beneficial effect. The testing carried out using the weld thermal simulator has shown that a relationship does exist between hot ductility and weld cracking. In particular, the absence of the peak in ductility in the range 1100°C-900°C on cooling from a temperature near to the solidus is indicative of a highly crack susceptible alloy. Principal practical implications of the investigation concern the relationship of weld metal cracking to alloy composition, especially the level of certain impurities. It would appear that the upper limits permitted by the alloy specifications are unrealistically high. The introduction of lower impurity limits would alleviate the current problems of variability in resistance to cracking during welding.

The copper cladding of steel by the submerged arc welding method

The literature available on submerged arc welding of copper and copper alloys, submerged arc welding with strip electrodes, and related areas has been reviewed in depth. Copper cladding of mild steel substrates by deposition from strip electrodes using the submerged arc welding process has been successful. A wide range of parameters, and several fluxes have been investigated. The range of deposit compositions is 66.4% Cu to 95.7% Cu. The weld beads have been metallographically examined using optical and electron microscopy. Equating weld beads to a thermodynamical equivalent of iron has proven to be an accurate and simplified means of handling quantitative data for multicomponent welds. Empirical equations derived using theoretical considerations characterize the weld bead dimensions as functions of the welding parameters and hence composition. The melting rate for strip electrodes is dependent upon the current-voltage product. Weld nugget size is increased by increased thermal transfer efficiencies resulting from stirring which is current dependent. The presence of Fe2O3 in a flux has been demonstrated to diminish electrode melting rate and drastically increase penetration, making flux choice the prime consideration in cladding operations. A theoretical model for welding with strip electrodes and the submerged arc process is presented.

Cobalt promoted TiO2/GO for the photocatalytic degradation of oxytetracycline and Congo Red

A family of titania derived nanocomposites synthesized via sol-gel and hydrothermal routes exhibit excellent performance for the photocatalytic degradation of two important exemplar water pollutants, oxytetracycline and Congo Red. Low loadings of Co3O4 nanoparticles dispersed over the surfaces of anatase TiO2 confer visible light photoactivity for the aqueous phase decomposition of organics through the resulting heterojunction and reduced band gap. Subsequent modification of these Co3O4/TiO2 composites by trace amounts of graphene oxide nanosheets in the presence of a diamine linker further promotes both oxytetracycline and Congo Red photodegradation under simulated solar and visible irradiation, through a combination of enhanced photoresponse and consequent radical generation. Radical quenching and fluorescence experiments implicate holes and hydroxyl radicals as the respective primary and secondary active species responsible for oxidative photodegradation of pollutants.

Fast pyrolysis and nitrogenolysis of biomass and biogenic residues:production of a sustainable slow release fertiliser

The production of agricultural and horticultural products requires the use of nitrogenous fertiliser that can cause pollution of surface and ground water and has a large carbon footprint as it is mainly produced from fossil fuels. The overall objective of this research project was to investigate fast pyrolysis and in-situ nitrogenolysis of biomass and biogenic residues as an alternative route to produce a sustainable solid slow release fertiliser mitigating the above stated problems. A variety of biomasses and biogenic residues were characterized by proximate analysis, ultimate analysis, thermogravimetric analysis (TGA) and Pyrolysis – Gas chromatography – Mass Spectroscopy (Py–GC–MS) for their potential use as feedstocks using beech wood as a reference material. Beech wood was virtually nitrogen free and therefore suitable as a reference material as added nitrogen can be identified as such while Dried Distillers Grains with Solubles (DDGS) and rape meal had a nitrogen content between 5.5wt.% and 6.1wt.% qualifying them as high nitrogen feedstocks. Fast pyrolysis and in-situ nitrogenolysis experiments were carried out in a continuously fed 1kg/h bubbling fluidized bed reactor at around 500°C quenching the pyrolysis vapours with isoparaffin. In-situ nitrogenolysis experiments were performed by adding ammonia gas to the fast pyrolysis reactor at nominal nitrogen addition rates between 5wt.%C and 20wt.%C based on the dry feedstock’s carbon content basis. Mass balances were established for the processing experiments. The fast pyrolysis and in-situ nitrogenolysis products were characterized by proximate analysis, ultimate analysis and GC– MS. High liquid yields and good mass balance closures of over 92% were obtained. The most suitable nitrogen addition rate for the in-situ nitrogenolysis experiments was determined to be 12wt.%C on dry feedstock carbon content basis. However, only a few nitrogen compounds that were formed during in-situ nitrogenolysis could be identified by GC–MS. A batch reactor process was developed to thermally solidify the fast pyrolysis and in-situ nitrogenolysis liquids of beech wood and Barley DDGS producing a brittle solid product. This was obtained at 150°C with an addition of 2.5wt% char (as catalyst) after a processing time of 1h. The batch reactor was also used for modifying and solidifying fast pyrolysis liquids derived from beech wood by adding urea or ammonium phosphate as post processing nitrogenolysis. The results showed that this type of combined approach was not suitable to produce a slow release fertiliser, because the solid product contained up to 65wt.% of highly water soluble nitrogen compounds that would be released instantly by rain. To complement the processing experiments a comparative study via Py–GC–MS with inert and reactive gas was performed with cellulose, hemicellulose, lignin and beech wood. This revealed that the presence of ammonia gas during analytical pyrolysis did not appear to have any direct impact on the decomposition products of the tested materials. The chromatograms obtained showed almost no differences between inert and ammonia gas experiments indicating that the reaction between ammonia and pyrolysis vapours does not occur instantly. A comparative study via Fourier Transformed Infrared Spectroscopy of solidified fast pyrolysis and in-situ nitrogenolysis products showed that there were some alterations in the spectra obtained. A shift in frequencies indicating C=O stretches typically related to the presence of carboxylic acids to C=O stretches related to amides was observed and no double or triple bonded nitrogen was detected. This indicates that organic acids reacted with ammonia and that no potentially harmful or non-biodegradable triple bonded nitrogen compounds were formed. The impact of solid slow release fertiliser (SRF) derived from pyrolysis and in-situ nitrogenolysis products from beech wood and Barley DDGS on microbial life in soils and plant growth was tested in cooperation with Rothamsted Research. The microbial incubation tests indicated that microbes can thrive on the SRFs produced, although some microbial species seem to have a reduced activity at very high concentrations of beech wood and Barley DDGS derived SRF. The plant tests (pot trials) showed that the application of SRF derived from beech wood and barley DDGS had no negative impact on germination or plant growth of rye grass. The fertilizing effect was proven by the dry matter yields in three harvests after 47 days, 89 days and 131 days. The findings of this research indicate that in general a slow release fertiliser can be produced from biomass and biogenic residues by in-situ nitrogenolysis. Nevertheless the findings also show that additional research is necessary to identify which compounds are formed during this process.

The combined impact of urban heat island, thermal bridge effect of buildings and future climate change on the potential overwintering of Phlebotomus species in a Central European metropolis

Leishmaniasis is one of the most important emerging vector-borne diseases in Western Eurasia. Although winter minimum temperatures limit the present geographical distribution of the vector Phlebotomus species, the heat island effect of the cities and the anthropogenic heat emission together may provide the appropriate environment for the overwintering of sand flies. We studied the climate tempering effect of thermal bridges and the heat island effect in Budapest, Hungary. Thermal imaging was used to measure the heat surplus of heat bridges. The winter heat island effect of the city was evaluated by numerical analysis of the measurements of the Aqua sensor of satellite Terra. We found that the surface temperature of thermal bridges can be at least 3-7 °C higher than the surrounding environment. The heat emission of thermal bridges and the urban heat island effect together can cause at least 10 °C higher minimum ambient temperature in winter nights than the minimum temperature of the peri-urban areas. This milder micro-climate of the built environment can enable the potential overwintering of some important European Phlebotomus species. The anthropogenic heat emission of big cities may explain the observed isolated northward populations of Phlebotomus ariasi in Paris and Phlebotomus neglectus in the agglomeration of Budapest.

A Comparati ve Study of Mechanical and Tribological Properties of AISI-304 and AISI-316 Submitted to Glow Discharge Nitriding

Mechanical and tribological properties of AISI 304 and AISI 316 stainless steels submitted to glow discharge ion nitriding are reported. The atmosphere was 20:80 - N2:H2 with substrate temperatures ranging from 300 to 500 °C. Treatment at 300 °C produced expanded austenite (γN) in both steels. Increasing the temperature, the phases γ′-Fe4N and ε- Fe2+xN were present and the latter is the major phase for AISI 304. At 500 °C, the CrN phase was also identified in both steels. Hardnesses of about 13-14 GPa at near surface regions were obtained in both steels. Moreover, AISI 316 nitrided at 500 °C has the deepest hard layer. Tribological tests showed that wear can be reduced by up to a factor of six after the nitriding processes, even for a working temperature of 300 °C. The profiles during and after nanoscratch tests did not reveal significant differences after nitriding processes in both steels.

A Comparative Study of Mechanical and Tribological Properties of AISI-304 and AISI-316 Submitted to Glow Discharge Nitriding

Mechanical and tribological properties of AISI 304 and AISI 316 stainless steels submitted to glow discharge ion nitriding are reported. The atmosphere was 20:80 - N2:H2 with substrate temperatures ranging from 300 to 500 °C. Treatment at 300 °C produced expanded austenite (γN) in both steels. Increasing the temperature, the phases γ′-Fe4N and ε- Fe2+xN were present and the latter is the major phase for AISI 304. At 500 °C, the CrN phase was also identified in both steels. Hardnesses of about 13-14 GPa at near surface regions were obtained in both steels. Moreover, AISI 316 nitrided at 500 °C has the deepest hard layer. Tribological tests showed that wear can be reduced by up to a factor of six after the nitriding processes, even for a working temperature of 300 °C. The profiles during and after nanoscratch tests did not reveal significant differences after nitriding processes in both steels.

A Comparati ve Study of Mechanical and Tribological Properties of AISI-304 and AISI-316 Submitted to Glow Discharge Nitriding

Mechanical and tribological properties of AISI 304 and AISI 316 stainless steels submitted to glow discharge ion nitriding are reported. The atmosphere was 20:80 - N2:H2 with substrate temperatures ranging from 300 to 500 °C. Treatment at 300 °C produced expanded austenite (γN) in both steels. Increasing the temperature, the phases γ′-Fe4N and ε- Fe2+xN were present and the latter is the major phase for AISI 304. At 500 °C, the CrN phase was also identified in both steels. Hardnesses of about 13-14 GPa at near surface regions were obtained in both steels. Moreover, AISI 316 nitrided at 500 °C has the deepest hard layer. Tribological tests showed that wear can be reduced by up to a factor of six after the nitriding processes, even for a working temperature of 300 °C. The profiles during and after nanoscratch tests did not reveal significant differences after nitriding processes in both steels.

A Comparative Study of Mechanical and Tribological Properties of AISI-304 and AISI-316 Submitted to Glow Discharge Nitriding

Mechanical and tribological properties of AISI 304 and AISI 316 stainless steels submitted to glow discharge ion nitriding are reported. The atmosphere was 20:80 - N2:H2 with substrate temperatures ranging from 300 to 500 °C. Treatment at 300 °C produced expanded austenite (γN) in both steels. Increasing the temperature, the phases γ′-Fe4N and ε- Fe2+xN were present and the latter is the major phase for AISI 304. At 500 °C, the CrN phase was also identified in both steels. Hardnesses of about 13-14 GPa at near surface regions were obtained in both steels. Moreover, AISI 316 nitrided at 500 °C has the deepest hard layer. Tribological tests showed that wear can be reduced by up to a factor of six after the nitriding processes, even for a working temperature of 300 °C. The profiles during and after nanoscratch tests did not reveal significant differences after nitriding processes in both steels.

Applications of advanced and dual energy computed tomography in proton therapy

This thesis focuses on advanced reconstruction methods and Dual Energy (DE) Computed Tomography (CT) applications for proton therapy, aiming at improving patient positioning and investigating approaches to deal with metal artifacts. To tackle the first goal, an algorithm for post-processing input DE images has been developed. The outputs are tumor- and bone-canceled images, which help in recognising structures in patient body. We proved that positioning error is substantially reduced using contrast enhanced images, thus suggesting the potential of such application. If positioning plays a key role in the delivery, even more important is the quality of planning CT. For that, modern CT scanners offer possibility to tackle challenging cases, like treatment of tumors close to metal implants. Possible approaches for dealing with artifacts introduced by such rods have been investigated experimentally at Paul Scherrer Institut (Switzerland), simulating several treatment plans on an anthropomorphic phantom. In particular, we examined the cases in which none, manual or Iterative Metal Artifact Reduction (iMAR) algorithm were used to correct the artifacts, using both Filtered Back Projection and Sinogram Affirmed Iterative Reconstruction as image reconstruction techniques. Moreover, direct stopping power calculation from DE images with iMAR has also been considered as alternative approach. Delivered dose measured with Gafchromic EBT3 films was compared with the one calculated in Treatment Planning System. Residual positioning errors, daily machine dependent uncertainties and film quenching have been taken into account in the analyses. Although plans with multiple fields seemed more robust than single field, results showed in general better agreement between prescribed and delivered dose when using iMAR, especially if combined with DE approach. Thus, we proved the potential of these advanced algorithms in improving dosimetry for plans in presence of metal implants.

Pipkiiα Is Widely Expressed In Hematopoietic-derived Cells And May Play A Role In The Expression Of Alpha- And Gamma-globins In K562 Cells.

Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.

Desorption/ionization Efficiencies Of Triacylglycerols And Phospholipids Via Easi-ms.

Knowledge of the major effects governing desorption/ionization efficiency is required for the development and application of ambient mass spectrometry. Although all triacylglycerols (TAG) have the same favorable protonation and cationization sites, their desorption/ionization efficiencies can vary dramatically during easy ambient sonic-spray ionization because of structural differences in the carbon chain. To quantify this somewhat surprising and drastic effect, we have performed a systematic investigation of desorption/ionization efficiencies as a function of unsaturation and length for TAG as well as for diacylglycerols, monoacylglycerols and several phospholipids (PL). Affinities for Na(+) as a function of unsaturation level have also been assayed via comprehensive metadynamics calculations to understand the influence of this phenomenon on the ionization efficiency. The results suggest that dipole-dipole interactions within a carbon chain tuned by unsaturation sites govern ionization efficiency of TAG and PL.

Prognostic Value Of Dna And Mrna E6/e7 Of Human Papillomavirus In The Evolution Of Cervical Intraepithelial Neoplasia Grade 2.

This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.