Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

First-dimension separation with the MicroRotoforTM cell prior to SDS-PAGE and LC-MS/MS analysis

Free-flow isoelectric focusing (IEF) is a gel-free method for separating proteins based on their isoelectric point (pl) in a liquid environment and in the presence of carrier ampholytes. this method has been used with the RotoforTM cell at the preparative scale to fractionate proteins from samples containing several hundred milligrams of protein; see the refeences listed in Bio-Rad bulletin 3152. the MicroRotofor cell applies the same method to much sl=maller protein samples without dilution, separating and recoverng milligram quantities of protein in a total volume of about 2 ml.

Comparing tube models for predicting the linear rheology of branched polymer melts

The hierarchical and "bob" (or branch-on-branch) models are tube-based computational models recently developed for predicting the linear rheology of general mixtures of polydisperse branched polymers. These two models are based on a similar tube-theory framework but differ in their numerical implementation and details of relaxation mechanisms. We present a detailed overview of the similarities and differences of these models and examine the effects of these differences on the predictions of the linear viscoelastic properties of a set of representative branched polymer samples in order to give a general picture of the performance of these models. Our analysis confirms that the hierarchical and bob models quantitatively predict the linear rheology of a wide range of branched polymer melts but also indicate that there is still no unique solution to cover all types of branched polymers without case-by-case adjustment of parameters such as the dilution exponent alpha and the factor p(2) which defines the hopping distance of a branch point relative to the tube diameter. An updated version of the hierarchical model, which shows improved computational efficiency and refined relaxation mechanisms, is introduced and used in these analyses.

Synthesis, structure and magnetic properties of mono- and di-nuclear nickel(II) thiocyanate complexes with tridentate N3 donor Schiff bases

The 1:1 condensation of N-methyl-1,3-diaminopropane and N,N-diethyl-1,2-diminoethane with 2-acetylpyridine, respectively at high dilution gives the tridentate mono-condensed Schiff bases N-methyl-N'-(1-pyridin-2-yl-ethylidene)-propane-1,3-diamine (L-1) and N,N-diethyl-N'-(1-pyridin-2-yl-ethylidene)-ethane-1,2-diamine (L-2). The tridentate ligands were allowed to react with methanol solutions of nickel(II) thiocyanate to prepare the complexes [Ni(L-1)(SCN)(2)(OH2) (1) and [{Ni(L-2)(SCN)}(2)] (2). Single crystal X-ray diffraction was used to confirm the structures of the complexes. The nickel(II) in complex 1 is bonded to three nitrogen donor atoms of the ligand L-1 in a mer orientation, together with two thiocyanates bonded through nitrogen and a water molecule, and it is the first Schiff base complex of nickel(II) containing both thiocyanate and coordinated water. The coordinated water initiates a hydrogen bonded 2D network. In complex 2, the nickel ion occupies a slightly distorted octahedral coordination sphere, being bonded to three nitrogen atoms from the ligand L-2, also in a mer orientation, and two thiocyanate anions through nitrogen. In contrast to 1, the sixth coordination site is occupied by a sulfur atom from a thiocyanate anion in an adjacent molecule, thus creating a centrosymmetric dimer. A variable temperature magnetic study of complex 2 indicates the simultaneous presence of zero-field splitting, weak intramolecular ferromagnetic coupling and intermolecular antiferromagnetic interactions between the nickel(II) centers.

Induced random fields in the LiHoxY1-xF4 quantum ising magnet in a transverse magnetic field

The LiHoxY1-xF4 magnetic material in a transverse magnetic field Bxx̂ perpendicular to the Ising spin direction has long been used to study tunable quantum phase transitions in a random disordered system. We show that the Bx-induced magnetization along the x̂ direction, combined with the local random dilution-induced destruction of crystalline symmetries, generates, via the predominant dipolar interactions between Ho3+ ions, random fields along the Ising ẑ direction. This identifies LiHoxY1-xF4 in Bx as a new random field Ising system. The random fields explain the rapid decrease of the critical temperature in the diluted ferromagnetic regime and the smearing of the nonlinear susceptibility at the spin-glass transition with increasing Bx and render the Bx-induced quantum criticality in LiHoxY1-xF4 likely inaccessible.

Rigid ferrocenophane and its metal complexes with transition and alkaline-earth metal ions

The rigid [6]ferrocenophane, L-1, was synthesised by condensation of 1,1'-ferrocene dicarbaldehyde with trans-1,2-diaminocyclohexane in high dilution at r.t. followed by reduction. When other experimental conditions were employed, the [6,6,6]ferrocenephane (L-2) was also obtained. Both compounds were characterised by single crystal X-ray crystallography. The protonation of L-1 and its metal complexation were evaluated by the effect on the electron-transfer process of the ferrocene (fc) unit of L-1 using cyclic voltammetry (CV) and square wave voltammetry (SWV) in anhydrous CH3CN solution and in 0.1 M (Bu4NPF6)-Bu-n as the supporting electrolyte. The electrochemical process of L-1 between 300 and 900 mV is complicated by amine oxidation. On the other hand, an anodic shift from the fc/fc(+) wave of L-1 of 249, 225, 81 and 61 mV was observed by formation of Zn2+, Ni2+, Pd2+ and Cu2+ complexes, respectively. Whereas Mg2+ and Ca2+ only have with L-1 weak interactions and they promote the acid-base equilibrium of L-1. This reveals that L-1 is an interesting molecular redox sensor for detection of Zn2+ and Ni2+, although the kinetics of the Zn2+ complex formation is much faster than that of the Ni2+ one. The X-ray crystal structure of [(PdLCl2)-Cl-1] was determined and showed a square-planar environment with Pd(II) and Fe(II) centres separated by 3.781(1) angstrom. The experimental anodic shifts were elucidated by DFT calculations on the [(MLCl2)-Cl-1] series and they are related to the nature of the HOMO of these complexes and a four-electron, two-orbital interaction.

The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibioticresistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24 h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R2) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24 h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24 h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 102 cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.

Inhibition of Salmonella Typhimurium by tannins in vitro

Given the recent EU ban of antibiotics to promote the growth of farm animals, alternative approaches are needed for animal production systems. Tannins, which are already commercially marketed for animal nutrition, have bacteriostatic and bactericidal properties against pathogenic bacteria. The aim of this study was to investigate the inhibitory effect of various tannins against Salmonella Typhimurium (SL1344nal(r)) to identify potentially effective feed additives. Different sources of condensed and hydrolysable tannins were tested at concentrations between I and 6 mg ml(-1). The tannins tested were either commercial preparations or isolated from such preparations or from plants using Sephadex LH-20 based column chromatography. Some, but not all, of the tannins significantly decreased bacterial growth compared to tannin-free selenite cystine broth following incubation for 24 h at 37 degrees C. Gallotannins were especially effective and tara achieved 1.28 log(10) reductions after 24 hours. Antibacterial activity was also confirmed with inhibition zone diameters in a disc diffusion test. The experiments demonstrated that tannins may have potential as feed additives for reducing Salmonella infections in farm animals.

Long-term acclimation of leaf production, development, longevity and quality following 3 yr exposure to free-air CO2 enrichment during canopy closure in Populus

The effects of elevated CO2 on leaf development in three genotypes of Populus were investigated during canopy closure, following exposure to elevated CO2 over 3 yr using free-air enrichment.• Leaf quality was altered such that nitrogen concentration per unit d. wt (Nmass) declined on average by 22 and 13% for sun and shade leaves, respectively, in elevated CO2. There was little evidence that this was the result of ‘dilution’ following accumulation of nonstructural carbohydrates. Most likely, this was the result of increased leaf thickness. Specific leaf area declined in elevated CO2 on average by 29 and 5% for sun and shade leaves, respectively.• Autumnal senescence was delayed in elevated CO2 with a 10% increase in the number of days at which 50% leaf loss occurred in elevated as compared with ambient CO2.• These data suggest that changes in leaf quality may be predicted following long-term acclimation of fast-growing forest trees to elevated CO2, and that canopy longevity may increase, with important implications for forest productivity.

Evaluation of an improved tracer method to monitor cheese curd syneresis at varying milk fat levels in a cheese vat

Previous studies have reported that cheese curd syneresis kinetics can be monitored by dilution of chemical tracers, such as Blue Dextran, in whey. The objective of this study was to evaluate an improved tracer method to monitor whey volumes expelled over time during syneresis. Two experiments with different ranges of milk fat (0-5% and 2.3-3.5%) were carried out in an 11 L double-O laboratory scale cheese vat. Tracer was added to the curd-whey mixture during the cutting phase of cheese making and samples were taken at 10 min intervals up to 75 min after cutting. The volume of whey expelled was measured gravimetrically and the dilution of tracer in the whey was measured by absorbance at 620 nm. The volumes of whey expelled were significantly reduced at higher milk fat levels. Whey yield was predicted with a SEP ranging from 3.2 to 6.3 g whey/100 mL of milk and a CV ranging from 2.03 to 2.7% at different milk fat levels.

Modern lovers

Modern Lovers was a survey show of contemporary art practices in dialogue with modernism, bringing together established and emerging artists based in London and international artists from Berlin, Jerusalem and Zagreb. The show features video, film, installation, sculpture, music and performance work that addresses the legacy of the avant garde and the survival of its aesthetics within contemporary culture. In 1976, as punk rock was busy smashing the cultural rubble left behind by the second world war and rejecting the consumer society that had emerged from the ruins, one band bravely announced that it wanted no part in this destruction. Jonathan Richman's Modern Lovers sang about how they still loved the old world. Neither parents nor girlfriends could understand, but the decaying inner city with its false promises of progress still held a fascination for Richman, who claimed he wanted to keep his place in this arcane landscape. Punk's assault on culture was the logical conclusion of modernism's linear narrative of art as a force of innovation that must reject preceding artistic movements to establish new ones. Echoing the negations of Dada, it set out to put an end to this narrative, an end to culture. It is partly because of this inherently destructive and totalising side of Modernism that it has come under harsh critique in the post modern era. Nevertheless, we are still caught up in the same dialectic of progress, revolution and destruction. Post modernism has failed to unseat our desire for the revolutionary moment, even as it has been co-opted to the degree of meaninglessness by the discourses of marketing and Capitalism. But, like Jonathan Richman, the artists in the exhibition "Modern Lovers" keep returning to modernism for something else. Instead of taking it at its word when it proffers revolution, they turn to it in search of reform. Still loving the old world and desiring a dialogue with the past, perhaps as an antidote to the eternal present of Capitalism, they are willing to engage with its aesthetics and ideas on equal ground. Leaving behind the ironic deconstructions of post modernism, they find perspectives worth salvaging and juxtapose them with contemporary visual productions. Trading in the grand narratives of modernity for a more personal approach, they don't seek the purity of form that drove the avant garde movements that inspire them but rather revel in adulteration, dilution and contamination of the past by the present". A live performance by sala-manca was sponsored by the British Council and took place May 26th, 19:00. MODERN LOVERS was accompanied by a catalogue (14.80 cm x 14.80 cm) including essays by Avi Pitchon, the sala-manca group and the curators. A discussion panel about the exhibition themes, as well as the catalogue launch,took place at Goldsmiths College's cinema on the 27th of May at 14:00, chaired by Dr. Suhail Malik (Senior Lecturer & Course Leader Postgraduate Fine Art Critical Studies at Goldsmiths College) and with the participation of Tom Morton (curator, Cubitt Gallery, and regular contributor to Frieze magazine), sala-manca (artist group), Dr. Amanda Beech (artist, curator and senior lecturer at the Wimbledon School of Art), Matthew Poole (course director of MA Gallery Studies, dept. of Art History and Theory at the University of Essex).

Contrasting effects of dwarfing alleles and nitrogen availability on mineral concentrations in wheat grain

Background and aim Concentrations of essential minerals in plant foods may have declined in modern high-yielding cultivars grown with large applications of nitrogen fertilizer (N). We investigated the effect of dwarfing alleles and N rate on mineral concentrations in wheat. Methods Gibberellin (GA)-insensitive reduced height (Rht) alleles were compared in near isogenic wheat lines. Two field experiments comprised factorial combinations of wheat variety backgrounds, alleles at the Rht-B1 locus (rht-B1a, Rht-B1b, Rht-B1c), and different N rates. A glasshouse experiment also included Rht-D1b and Rht-B1b+D1b in one background. Results In the field, depending on season, Rht-B1b increased crop biomass, dry matter (DM) harvest index, grain yield, and the economically-optimal N rate (Nopt). Rht-B1b did not increase uptake of Cu, Fe, Mg or Zn so these minerals were diluted in grain. Nitrogen increased DM yield and mineral uptake so grain concentrations were increased (Fe in both seasons; Cu, Mg and Zn in one season). Rht-B1b reduced mineral concentrations at Nopt in the most N responsive season. In the glasshouse experiment, grain yield was reduced, and mineral concentrations increased, with Rht allele addition. Conclusion Effects of Rht alleles on Fe, Zn, Cu and Mg concentrations in wheat grain are mostly due to their effects on DM, rather than of GA-insensitivity on Nopt or mineral uptake. Increased N requirement in semi-dwarf varieties partly offsets this dilution effect.

Effects of urban ventilation patterns on the carbon monoxide concentration in a high-rise mega city

Carbon monoxide (CO) concentration data from 1999–2006, monitored at 5 different pollution stations in a high-rise mega city (Hong Kong), were collected and investigated. The spatio-temporal characteristics of urban CO concentration profiles were obtained. A new approach was put forward to examine the relationship between urban CO concentration and different wind flow patterns. Rather than relying on the meteorological data from a single weather station, usually adopted in previous studies, four weather stations on the boundary of Hong Kong territory were used in the present study so as to identify 16 different wind flow patterns, among which a typical urban heat island circulation (UHIC) can be distinguished. Higher concentrations were observed to be associated with the flow pattern of an inflow from Lau Fau Shan (LFS) station which is located in the northwest of Hong Kong. This suggests that the ability of dilution for north-to-west wind is relatively weak due to the pollutants carried from outside Hong Kong. The effectiveness of wind speed on the alleviation of urban concentration is dependent on the initial concentration of the approaching wind. The increase of wind speed of north-to-west wind from 0 m/s to 6 m/s has little effect on the reduction of urban CO concentration, especially on the non-roadside stations. By contrast, for the southerly marine wind, pollution concentration decreases sharply with an increase in the wind speed. It was also found that urban heat island circulation (UHIC) is conducive of the accumulation of pollutants, especially at night. There exists a positive correlation between CO concentration and UHI intensity. This correlation is much stronger at night compared to during the day. Keywords: urban pollution monitoring, urban ventilation pattern, urban heat island circulation, mega city

The role of SEF14 and SEF17 fimbriae in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces

To gain an understanding of the role of fimbriae and flagella in the adherence of Salmonella enterica serotype Enteritidis to inanimate surfaces, the extent of adherence of viable wild-type strains to a polystyrene microtitration plate was determined by a crystal violet staining assay, Elaboration of surface antigens by adherent bacteria was assayed by fimbriae- and flagella-specific ELISAs, Wild-type Enteritidis strains adhered well at 37 degrees C and 25 degrees C when grown in microtitration wells in Colonisation Factor Antigen broth, but not in other media tested, At 37 degrees C, adherent bacteria elaborated copious quantities of SEF14 fimbrial antigen, whereas at 25 degrees C adherent bacteria elaborated copious quantities of SEF17 fimbrial antigen. Non-fimbriate and non-flagellate knock-out mutant strains were also assessed in the adherence assay. Mutant strains unable to elaborate SEF14 and SEF17 fimbriae adhered poorly at 37 degrees C and 25 degrees C, respectively, but adherence was not abolished. Non-motile mutant strains showed reduced adherence whilst type-1, PEF and LPF fimbriae appeared not to contribute to adherence in this assay. These data indicate that SEF17 and SEF14 fimbriae mediate bacterial cell aggregation on inanimate surfaces under appropriate growth conditions.

Analysis of expression of flagella by Salmonella enterica serotype Typhimurium by monoclonal antibodies recognising both phase specific and common epitopes

Monoclonal antibodies specific for phase 1 ("i" antigen), phase 2 ("1,2" antigen) and common epitopes of the flagellins of Salmonella enterica serotype Typhimurium were raised. Having confirmed their specificity, the monoclonal antibodies were used to develop semi-quantitative ELISAs in order to assess the relative expression of the two phases by strains of Typhimurium. The majority of Typhimurium strains representative of a wide cross-section of definitive types from animal and environmental sources preferentially expressed phase 1 antigen in vitro. DT40 strains were unique in expressing phase 2 preferentially. The ratio of phase 1 to phase 2 expressed by strains tended to be constant for any one strain when strains were grown on a number of conventional laboratory media. However, the ratio of phases was shown to be modulated by incubation at 42 degreesC and buffering media at pH values, notably 4.5, other than neutral. Selenite broth and Rambach media repressed flagellation. Crown Copyright (C) 2001 Published by Elsevier Science B.V. All rights reserved.