Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non- standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocyte/macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-kB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol (R) r was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA. hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.

Insights into redox cycling of sulfur and iron in peatlands using high-resolution diffusive equilibrium thin film (DET) gel probe sampling

High spatial resolution vertical profiles of pore-water chemistry have been obtained for a peatland using diffusive equilibrium in thin films (DET) gel probes. Comparison of DET pore-water data with more traditional depth-specific sampling shows good agreement and the DET profiling method is less invasive and less likely to induce mixing of pore-waters. Chloride mass balances as water tables fell in the early summer indicate that evaporative concentration dominates and there is negligible lateral flow in the peat. Lack of lateral flow allows element budgets for the same site at different times to be compared. The high spatial resolution of sampling also enables gradients to be observed that permit calculations of vertical fluxes. Sulfate concentrations fall at two sites with net rates of 1.5 and 5.0nmol cm− 3 day− 1, likely due to a dominance of bacterial sulfate reduction, while a third site showed a net gain in sulfate due to oxidation of sulfur over the study period at an average rate of 3.4nmol cm− 3 day− 1. Behaviour of iron is closely coupled to that of sulfur; there is net removal of iron at the two sites where sulfate reduction dominates and addition of iron where oxidation dominates. The profiles demonstrate that, in addition to strong vertical redox related chemical changes, there is significant spatial heterogeneity. Whilst overall there is evidence for net reduction of sulfate within the peatland pore-waters, this can be reversed, at least temporarily, during periods of drought when sulfide oxidation with resulting acid production predominates.

Mechatronic design of a high frequency probe for haptic interaction

Current force feedback, haptic interface devices are generally limited to the display of low frequency, high amplitude spatial data. A typical device consists of a low impedance framework of one or more degrees-of-freedom (dof), allowing a user to explore a pre-defined workspace via an end effector such as a handle, thimble, probe or stylus. The movement of the device is then constrained using high gain positional feedback, thus reducing the apparent dof of the device and conveying the illusion of hard contact to the user. Such devices are, however, limited to a narrow bandwidth of frequencies, typically below 30Hz, and are not well suited to the display of surface properties, such as object texture. This paper details a device to augment an existing force feedback haptic display with a vibrotactile display, thus providing a means of conveying low amplitude, high frequency spatial information of object surface properties. 1. Haptics and Haptic Interfaces Haptics is the study of human touch and interaction with the external environment via touch. Information from the human sense of touch can be classified in to two categories, cutaneous and kinesthetic. Cutaneous information is provided via the mechanoreceptive nerve endings in the glabrous skin of the human hand. It is primarily a means of relaying information regarding small-scale details in the form of skin stretch, compression and vibration.

Evaluation of the environmental specificity of fluorescence in situ hybridization (FISH) using fluorescence-activated cell sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil

We explicitly tested for the first time the ‘environmental specificity’ of traditional 16S rRNAtargeted fluorescence in situ hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridised population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted (FACS) -recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz® method for the extraction of bacterial cells from soil.

Neuroprotective effects of phenolic antioxidant tBHQ associate with inhibition of FoxO3a nuclear translocation and activity

The Forkhead transcription factor, FoxO3a induces genomic death responses in neurones following translocation from the cytosol to the nucleus. Nuclear translocation of FoxO3a is triggered by trophic factor withdrawal, oxidative stress and the stimulation of extrasynaptic NMDA receptors. Receptor activation of phosphatidylinositol 3-kinase (PI3K) – Akt signalling pathways retains FoxO3a in the cytoplasm thereby inhibiting the transcriptional activation of death promoting genes. We hypothesised that phenolic antioxidants such as tert-Butylhydroquinone (tBHQ), which is known to stimulate PI3K-Akt signalling, would inhibit FoxO3a translocation and activity. Treatment of cultured cortical neurones with NMDA increased the nuclear localisation of FoxO3a, reduced the phosphorylation of FoxO3a, increased caspase activity and upregulated Fas ligand expression. In contrast the phenolic antioxidant tBHQ caused retention of FoxO3a in the cytosol coincident with enhanced PI3K- dependent phosphorylation of FoxO3a. tBHQ-induced nuclear exclusion of FoxO3a was associated with reduced FoxO-mediated transcriptional activity. Exposure of neurones to tBHQ inhibited NMDA-induced nuclear translocation of FoxO3a prevented NMDA-induced upregulation of FoxO-mediated transcriptional activity, blocked caspase activation and protected neurones from NMDA-induced excitotoxic death. Collectively, these data suggest that phenolic antioxidants such as tBHQ oppose stress-induced activation of FoxO3a and therefore have potential neuroprotective utility in neurodegeneration.

A time-domain probe method for three-dimensional rough surface reconstructions

The task of this paper is to develop a Time-Domain Probe Method for the reconstruction of impenetrable scatterers. The basic idea of the method is to use pulses in the time domain and the time-dependent response of the scatterer to reconstruct its location and shape. The method is based on the basic causality principle of timedependent scattering. The method is independent of the boundary condition and is applicable for limited aperture scattering data. In particular, we discuss the reconstruction of the shape of a rough surface in three dimensions from time-domain measurements of the scattered field. In practise, measurement data is collected where the incident field is given by a pulse. We formulate the time-domain fieeld reconstruction problem equivalently via frequency-domain integral equations or via a retarded boundary integral equation based on results of Bamberger, Ha-Duong, Lubich. In contrast to pure frequency domain methods here we use a time-domain characterization of the unknown shape for its reconstruction. Our paper will describe the Time-Domain Probe Method and relate it to previous frequency-domain approaches on sampling and probe methods by Colton, Kirsch, Ikehata, Potthast, Luke, Sylvester et al. The approach significantly extends recent work of Chandler-Wilde and Lines (2005) and Luke and Potthast (2006) on the timedomain point source method. We provide a complete convergence analysis for the method for the rough surface scattering case and provide numerical simulations and examples.

An iterative contractive framework for probe methods: LASSO

We present a new iterative approach called Line Adaptation for the Singular Sources Objective (LASSO) to object or shape reconstruction based on the singular sources method (or probe method) for the reconstruction of scatterers from the far-field pattern of scattered acoustic or electromagnetic waves. The scheme is based on the construction of an indicator function given by the scattered field for incident point sources in its source point from the given far-field patterns for plane waves. The indicator function is then used to drive the contraction of a surface which surrounds the unknown scatterers. A stopping criterion for those parts of the surfaces that touch the unknown scatterers is formulated. A splitting approach for the contracting surfaces is formulated, such that scatterers consisting of several separate components can be reconstructed. Convergence of the scheme is shown, and its feasibility is demonstrated using a numerical study with several examples.

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2

We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.

Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

High-density oligonucleotide (oligo) arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L.) Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM) probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P) stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ webcite and may be used to facilitate transcriptomic analyses of a wide range of plant and animal species in the absence of custom arrays.

Using coordinated observations in polarized white light and Faraday rotation to probe the spatial position and magnetic field of an interplanetary sheath

Coronal mass ejections (CMEs) can be continuously tracked through a large portion of the inner heliosphere by direct imaging in visible and radio wavebands. White light (WL) signatures of solar wind transients, such as CMEs, result from Thomson scattering of sunlight by free electrons and therefore depend on both viewing geometry and electron density. The Faraday rotation (FR) of radio waves from extragalactic pulsars and quasars, which arises due to the presence of such solar wind features, depends on the line-of-sight magnetic field component B ∥ and the electron density. To understand coordinated WL and FR observations of CMEs, we perform forward magnetohydrodynamic modeling of an Earth-directed shock and synthesize the signatures that would be remotely sensed at a number of widely distributed vantage points in the inner heliosphere. Removal of the background solar wind contribution reveals the shock-associated enhancements in WL and FR. While the efficiency of Thomson scattering depends on scattering angle, WL radiance I decreases with heliocentric distance r roughly according to the expression Ir –3. The sheath region downstream of the Earth-directed shock is well viewed from the L4 and L5 Lagrangian points, demonstrating the benefits of these points in terms of space weather forecasting. The spatial position of the main scattering site r sheath and the mass of plasma at that position M sheath can be inferred from the polarization of the shock-associated enhancement in WL radiance. From the FR measurements, the local B ∥sheath at r sheath can then be estimated. Simultaneous observations in polarized WL and FR can not only be used to detect CMEs, but also to diagnose their plasma and magnetic field properties.

A genome wide association study of mathematical ability reveals an association at chromosome 3q29, a locus associated with autism and learning difficulties: a preliminary study

Mathematical ability is heritable, but few studies have directly investigated its molecular genetic basis. Here we aimed to identify specific genetic contributions to variation in mathematical ability. We carried out a genome wide association scan using pooled DNA in two groups of U.K. samples, based on end of secondary/high school national academic exam achievement: high (n = 419) versus low (n = 183) mathematical ability while controlling for their verbal ability. Significant differences in allele frequencies between these groups were searched for in 906,600 SNPs using the Affymetrix GeneChip Human Mapping version 6.0 array. After meeting a threshold of p<1.5×10-5, 12 SNPs from the pooled association analysis were individually genotyped in 542 of the participants and analyzed to validate the initial associations (lowest p-value 1.14 ×10-6). In this analysis, one of the SNPs (rs789859) showed significant association after Bonferroni correction, and four (rs10873824, rs4144887, rs12130910 rs2809115) were nominally significant (lowest p-value 3.278 × 10-4). Three of the SNPs of interest are located within, or near to, known genes (FAM43A, SFT2D1, C14orf64). The SNP that showed the strongest association, rs789859, is located in a region on chromosome 3q29 that has been previously linked to learning difficulties and autism. rs789859 lies 1.3 kbp downstream of LSG1, and 700 bp upstream of FAM43A, mapping within the potential promoter/regulatory region of the latter. To our knowledge, this is only the second study to investigate the association of genetic variants with mathematical ability, and it highlights a number of interesting markers for future study.

Switchable amplification of vibrational circular dichroism as a probe of local chiral structure

A new method to detect the vibrational circular dichroism (VCD) of a localized part of a chiral molecular system is reported. A local VCD amplifier was implemented, and the distance dependence of the amplification was investigated in a series of peptides. The results indicate a characteristic distance of 2.0±0.3 bonds, which suggests that the amplification is a localized phenomenon. The amplifier can be covalently coupled to a specific part of a molecule, and can be switched ON and OFF electrochemically. By subtracting the VCD spectra obtained when the amplifier is in the ON and OFF states, the VCD of the local environment of the amplifier can be separated from the total VCD spectrum. Switchable local VCD amplification thus makes it possible to “zoom in” on a specific part of a chiral molecule.