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.

Guided growth of neurons and glia using microfabricated patterns of parylene-C on a SiO2 background

This paper describes a simple technique for the patterning of glia and neurons. The integration of neuronal patterning to Multi-Electrode Arrays (MEAs), planar patch clamp and silicon based ‘lab on a chip’ technologies necessitates the development of a microfabrication-compatible method, which will be reliable and easy to implement. In this study a highly consistent, straightforward and cost effective cell patterning scheme has been developed. It is based on two common ingredients: the polymer parylene-C and horse serum. Parylene-C is deposited and photo-lithographically patterned on silicon oxide (SiO2) surfaces. Subsequently, the patterns are activated via immersion in horse serum. Compared to non-activated controls, cells on the treated samples exhibited a significantly higher conformity to underlying parylene stripes. The immersion time of the patterns was reduced from 24 to 3 h without compromising the technique. X-ray photoelectron spectroscopy (XPS) analysis of parylene and SiO2 surfaces before and after immersion in horse serum and gel based eluant analysis suggests that the quantity and conformation of proteins on the parylene and SiO2 substrates might be responsible for inducing glial and neuronal patterning.

Seal Formation in Silicon Planar Patch-Clamp Microstructures

This paper presents a microfabricated planar patch-clamp electrode design and looks at the impact of several physical characteristics on seal formation. The device consists of a patch aperture, 1.5-2.5 mum in diameter and 7-12 mum in depth, with a reverse-side deep-etched 80-mum well. The patch aperture was coated with either thermal oxide or plasma-enhanced chemical vapor deposited (PECVD) SiO2. Some of the thermal oxide devices were converted into protruding nozzle structures, and some were boron-doped. Seal formation was tested with cultured N2a neuroblastoma cells. The PECVD oxide devices produced an average seal resistance of 34 MOmega(n = 24), and the thermal oxide devices produced an average seal resistance of 96 MOmega(n = 59). Seal resistance was found to positively correlate with patch aperture depth. Whole-cell recordings were obtained from 14% of cells tested with the thermal oxide devices, including a single recording where a gigaohm seal was obtained.

Arrays of chiral nanotubes and a layered coordination polymer containing gallium-sulfide supertetrahedral clusters

We report here a unique chiral hybrid gallium sulfide, [NC2H8]2[Ga10S16(N2C12H12)(NC2H7)2] 1, consisting of helical chains of organically-functionalised supertetrahedral clusters which form quadruple-stranded helical nanotubes of ca. 3 nm diameter. This material therefore consists of discrete metal-organic nanotubes which, to the best of our knowledge, are extremely rare. Whilst solvothermal reactions involving 1,2-di(4-pyridyl)ethylene (DPE) resulted in the formation of such single-walled chiral nanotubes, the use of longer 4,4’-trimethylenedipyridine (TMP) ligands resulted in the synthesis of a two-dimensional hybrid gallium sulfide, [C5H6N]3[Ga10S16(OH)(N2C13H14)] 2 in which, for the first time, inorganic and organic linkages between supertetrahedral clusters coexist.

Flow structure and near-field dispersion in arrays of building-like obstacles

Dispersion in the near-field region of localised releases in urban areas is difficult to predict because of the strong influence of individual buildings. Effects include upstream dispersion, trapping of material into building wakes and enhanced concentration fluctuations. As a result, concentration patterns are highly variable in time and mean profiles in the near field are strongly non-Gaussian. These aspects of near-field dispersion are documented by analysing data from direct numerical simulations in arrays of building-like obstacles and are related to the underlying flow structure. The mean flow structure around the buildings is found to exert a strong influence over the dispersion of material in the near field. Diverging streamlines around buildings enhance lateral dispersion. Entrainment of material into building wakes in the very near field gives rise to secondary sources, which then affect the subsequent dispersion pattern. High levels of concentration fluctuations are also found in this very near field; the fluctuation intensity is of order 2 to 5.

Chemistry and structure by design: ordered CuNi(CN)4 sheets with copper(II) in a square-planar environment

Layered copper–nickel cyanide, CuNi(CN)4, a 2-D negative thermal expansion material, is one of a series of copper(II)-containing cyanides derived from Ni(CN)2. In CuNi(CN)4, unlike in Ni(CN)2, the cyanide groups are ordered generating square-planar Ni(CN)4 and Cu(NC)4 units. The adoption of square-planar geometry by Cu(II) in an extended solid is very unusual.

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

C(k)-solvability near the characteristic set for a class of planar complex vector fields of infinite type

This paper deals with semi-global C(k)-solvability of complex vector fields of the form L = partial derivative/partial derivative t + x(r) (a(x) + ib(x))partial derivative/partial derivative x, r >= 1, defined on Omega(epsilon) = (-epsilon, epsilon) x S(1), epsilon > 0, where a and b are C(infinity) real-valued functions in (-epsilon, epsilon). It is shown that the interplay between the order of vanishing of the functions a and b at x = 0 influences the C(k)-solvability at Sigma = {0} x S(1). When r = 1, it is permitted that the functions a and b of L depend on the x and t variables, that is, L = partial derivative/partial derivative t + x(a(x, t) + ib(x, t))partial derivative/partial derivative x, where (x, t) is an element of Omega(epsilon).

Gevrey solvability near the characteristic set for a class of planar complex vector fields of infinite type

We study the Gevrey solvability of a class of complex vector fields, defined on Omega(epsilon) = (-epsilon, epsilon) x S(1), given by L = partial derivative/partial derivative t + (a(x) + ib(x))partial derivative/partial derivative x, b not equivalent to 0, near the characteristic set Sigma = {0} x S(1). We show that the interplay between the order of vanishing of the functions a and b at x = 0 plays a role in the Gevrey solvability. (C) 2008 Elsevier Inc. All rights reserved.

Searching for planar signatures in WMAP

We search for planar deviations of statistical isotropy in the Wilkinson Microwave Anisotropy Probe (WMAP) data by applying a recently introduced angular-planar statistics both to full-sky and to masked temperature maps, including in our analysis the effect of the residual foreground contamination and systematics in the foreground removing process as sources of error. We confirm earlier findings that full-sky maps exhibit anomalies at the planar (l) and angular (l) scales (l; l) = (2; 5); (4; 7); and (6; 8), which seem to be due to unremoved foregrounds since this features are present in the full-sky map but not in the masked maps. On the other hand, our test detects slightly anomalous results at the scales (l; l) = (10; 8) and (2; 9) in the masked maps but not in the full-sky one, indicating that the foreground cleaning procedure (used to generate the full-sky map) could not only be creating false anomalies but also hiding existing ones. We also find a significant trace of an anomaly in the full-sky map at the scale (l; l) = (10; 5), which is still present when we consider galactic cuts of 18.3% and 28.4%. As regards the quadrupole (l = 2), we find a coherent over-modulation over the whole celestial sphere, for all full-sky and cut-sky maps. Overall, our results seem to indicate that current CMB maps derived from WMAP data do not show significant signs of anisotropies, as measured by our angular-planar estimator. However, we have detected a curious coherence of planar modulations at angular scales of the order of the galaxy`s plane, which may be an indication of residual contaminations in the full-and cut-sky maps.

Elaboration and optimization of (Y,Er)Al(3)(BO(3))(4) glassy planar waveguides through the sol-gel process

In this work, a sol-gel route was used to prepare Y(0.9)Er(0.1)Al(3)(BO(3))(4) glassy thin films by spin-coating technique looking for the preparation and optimization of planar waveguides for integrated optics. The films were deposited on silica and silicon substrates using stable sols synthesized by the sol-gel process. Deposits with thicknesses ranging between 520 and 720 nm were prepared by a multi-layer process involving heat treatments at different temperatures from glass transition to the film crystallization and using heating rates of 2 degrees C/min. The structural characterization of the layers was performed by using grazing incidence X-ray diffraction and Raman spectroscopy as a function of the heat treatment. Microstructural evolution in terms of annealing temperatures was followed by high resolution scanning electron microscopy and atomic force microscopy. Optical transmission spectra were used to determine the refractive index and the film thicknesses through the envelope method. The optical and guiding properties of the films were studied by m-line spectroscopy. The best films were monomode with 620 nm thickness and a refractive index around 1.664 at 980 nm wavelength. They showed good waveguiding properties with high light-coupling efficiency and low propagation loss at 632.8 and 1550 nm of about 0.88 dB/cm. (C) 2009 Elsevier B.V. All rights reserved.