Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

4,4 '-Dipyridyl-N,N '-dioxide complexes of metal-thiocyanate/selenocyanate: pi-stacked molecular rods as three-dimensional support for two-dimensional polymeric sheets and intra/interchain S center dot center dot center dot S interaction dependent architecture of the R-2(2)(8) synthon driven assembly of one-dimensional polymeric chains

Three supramolecular complexes of Co(II) using SCN-/SeCN- in combination with 4,4'-dipyridyl-N,N'-dioxide (dpyo), i.e., {[Co(SCN)(2)(dpyo)(2)].(dpyo)}(n) ( 1), {[Co(SCN)(2)(dpyo)(H2O)(2)].(H2O)}(n) ( 2), {[Co(SeCN)(2)(dpyo)(H2O)(2)]center dot(H2O)}(n) ( 3), have been synthesized and characterized by single-crystal X-ray analysis. Complex 1 is a rare example of a dpyo bridged two-dimensional (2D) coordination polymer, and pi-stacked dpyo supramolecular rods are generated by the lattice dpyo, passing through the rhombic grid of stacked layers, resulting in a three-dimensional (3D) superstructure. Complexes 2 and 3 are isomorphous one-dimensional (1D) coordination polymers [-Co-dpyo-Co-] that undergo self-assembly leading to a bilayer architecture derived through an R-2(2)(8) H-bonding synthon between coordinated water and dpyo oxygen. A reinvestigation of coordination polymers [Mn(SCN)(2)(dpyo)( H2O)(MeOH)](n) ( 4) and {[Fe(SCN)(2)(dpyo)(H2O)(2)]center dot(H2O)}(n) ( 5) reported recently by our group [ Manna et al. Indian J. Chem. 2006, 45A, 1813] reveals brick wall topology rather than bilayer architecture is due to the decisive role of S center dot center dot center dot S/Se center dot center dot center dot Se interactions in determining the helical nature in 4 and 5 as compared to zigzag polymeric chains in 2 and 3, although the same R-2(2)(8) synthon is responsible for supramolecular assembly in these complexes.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Riemann solvers with primitive parameter vectors for two-dimensional compressible flows of a real gas

An analysis of averaging procedures is presented for an approximate Riemann solver for the equations governing the compressible flow of a real gas. This study extends earlier work for the Euler equations with ideal gases.

Dynamic stall for a Vertical Axis Wind Turbine in a two-dimensional study

The last few years have proved that Vertical Axis Wind Turbines (VAWTs) are more suitable for urban areas than Horizontal Axis Wind Turbines (HAWTs). To date, very little has been published in this area to assess good performance and lifetime of VAWTs either in open or urban areas. At low tip speed ratios (TSRs<5), VAWTs are subjected to a phenomenon called 'dynamic stall'. This can really affect the fatigue life of a VAWT if it is not well understood. The purpose of this paper is to investigate how CFD is able to simulate the dynamic stall for 2-D flow around VAWT blades. During the numerical simulations different turbulence models were used and compared with the data available on the subject. In this numerical analysis the Shear Stress Transport (SST) turbulence model seems to predict the dynamic stall better than the other turbulence models available. The limitations of the study are that the simulations are based on a 2-D case with constant wind and rotational speeds instead of considering a 3-D case with variable wind speeds. This approach was necessary for having a numerical analysis at low computational cost and time. Consequently, in the future it is strongly suggested to develop a more sophisticated model that is a more realistic simulation of a dynamic stall in a three-dimensional VAWT.

Wave-breaking characteristics of Northern Hemisphere winter blocking: a two-dimensional approach

This paper generalises and applies recently developed blocking diagnostics in a two- dimensional latitude-longitude context, which takes into consideration both mid- and high-latitude blocking. These diagnostics identify characteristics of the associated wave-breaking as seen in the potential temperature (θ) on the dynamical tropopause, in particular the cyclonic or anticyclonic Direction of wave-Breaking (DB index), and the Relative Intensity (RI index) of the air masses that contribute to blocking formation. The methodology is extended to a 2-D domain and a cluster technique is deployed to classify mid- and high-latitude blocking according to the wave-breaking characteristics. Mid-latitude blocking is observed over Europe and Asia, where the meridional gradient of θ is generally weak, whereas high-latitude blocking is mainly present over the oceans, to the north of the jet-stream, where the meridional gradient of θ is much stronger. They occur respectively on the equatorward and poleward flank of the jet- stream, where the horizontal shear ∂u/∂y is positive in the first case and negative in the second case. A regional analysis is also conducted. It is found that cold-anticyclonic and cyclonic blocking divert the storm-track respectively to the south and to the north over the East Atlantic and western Europe. Furthermore, warm-cyclonic blocking over the Pacific and cold-anticyclonic blocking over Europe are identified as the most persistent types and are associated with large amplitude anomalies in temperature and precipitation. Finally, the high-latitude, cyclonic events seem to correlate well with low- frequency modes of variability over the Pacific and Atlantic Ocean.

The stability of a two-dimensional vorticity filament under uniform strain

The quantitative effects of uniform strain and background rotation on the stability of a strip of constant vorticity (a simple shear layer) are examined. The thickness of the strip decreases in time under the strain, so it is necessary to formulate the linear stability analysis for a time-dependent basic flow. The results show that even a strain rate γ (scaled with the vorticity of the strip) as small as 0.25 suppresses the conventional Rayleigh shear instability mechanism, in the sense that the r.m.s. wave steepness cannot amplify by more than a certain factor, and must eventually decay. For γ < 0.25 the amplification factor increases as γ decreases; however, it is only 3 when γ e 0.065. Numerical simulations confirm the predictions of linear theory at small steepness and predict a threshold value necessary for the formation of coherent vortices. The results help to explain the impression from numerous simulations of two-dimensional turbulence reported in the literature that filaments of vorticity infrequently roll up into vortices. The stabilization effect may be expected to extend to two- and three-dimensional quasi-geostrophic flows.

Rossby waves and two-dimensional turbulence in a large-scale zonal jet

The theory of homogeneous barotropic beta-plane turbulence is here extended to include effects arising from spatial inhomogeneity in the form of a zonal shear flow. Attention is restricted to the geophysically important case of zonal flows that are barotropically stable and are of larger scale than the resulting transient eddy field. Because of the presumed scale separation, the disturbance enstrophy is approximately conserved in a fully nonlinear sense, and the (nonlinear) wave-mean-flow interaction may be characterized as a shear-induced spectral transfer of disturbance enstrophy along lines of constant zonal wavenumber k. In this transfer the disturbance energy is generally not conserved. The nonlinear interactions between different disturbance components are turbulent for scales smaller than the inverse of Rhines's cascade-arrest scale κβ[identical with] (β0/2urms)½ and in this regime their leading-order effect may be characterized as a tendency to spread the enstrophy (and energy) along contours of constant total wavenumber κ [identical with] (k2 + l2)½. Insofar as this process of turbulent isotropization involves spectral transfer of disturbance enstrophy across lines of constant zonal wavenumber k, it can be readily distinguished from the shear-induced transfer which proceeds along them. However, an analysis in terms of total wavenumber K alone, which would be justified if the flow were homogeneous, would tend to mask the differences. The foregoing theoretical ideas are tested by performing direct numerical simulation experiments. It is found that the picture of classical beta-plane turbulence is altered, through the effect of the large-scale zonal flow, in the following ways: (i) while the turbulence is still confined to K Kβ, the disturbance field penetrates to the largest scales of motion; (ii) the larger disturbance scales K < Kβ exhibit a tendency to meridional rather than zonal anisotropy, namely towards v2 > u2 rather than vice versa; (iii) the initial spectral transfer rate away from an isotropic intermediate-scale source is significantly enhanced by the shear-induced transfer associated with straining by the zonal flow. This last effect occurs even when the large-scale shear appears weak to the energy-containing eddies, in the sense that dU/dy [double less-than sign] κ for typical eddy length and velocity scales.

Spectral nonlocality, absolute equilibria and Kraichnan-Leith-Batchelor phenomenology in two-dimensional turbulent energy cascades

We study the degree to which Kraichnan–Leith–Batchelor (KLB) phenomenology describes two-dimensional energy cascades in α turbulence, governed by ∂θ/∂t+J(ψ,θ)=ν∇2θ+f, where θ=(−Δ)α/2ψ is generalized vorticity, and ψ^(k)=k−αθ^(k) in Fourier space. These models differ in spectral non-locality, and include surface quasigeostrophic flow (α=1), regular two-dimensional flow (α=2) and rotating shallow flow (α=3), which is the isotropic limit of a mantle convection model. We re-examine arguments for dual inverse energy and direct enstrophy cascades, including Fjørtoft analysis, which we extend to general α, and point out their limitations. Using an α-dependent eddy-damped quasinormal Markovian (EDQNM) closure, we seek self-similar inertial range solutions and study their characteristics. Our present focus is not on coherent structures, which the EDQNM filters out, but on any self-similar and approximately Gaussian turbulent component that may exist in the flow and be described by KLB phenomenology. For this, the EDQNM is an appropriate tool. Non-local triads contribute increasingly to the energy flux as α increases. More importantly, the energy cascade is downscale in the self-similar inertial range for 2.5<α<10. At α=2.5 and α=10, the KLB spectra correspond, respectively, to enstrophy and energy equipartition, and the triad energy transfers and flux vanish identically. Eddy turnover time and strain rate arguments suggest the inverse energy cascade should obey KLB phenomenology and be self-similar for α<4. However, downscale energy flux in the EDQNM self-similar inertial range for α>2.5 leads us to predict that any inverse cascade for α≥2.5 will not exhibit KLB phenomenology, and specifically the KLB energy spectrum. Numerical simulations confirm this: the inverse cascade energy spectrum for α≥2.5 is significantly steeper than the KLB prediction, while for α<2.5 we obtain the KLB spectrum.

Unsteady flow simulation of a vertical axis augmented wind turbine: a two-dimensional study

As the integration of vertical axis wind turbines in the built environment is a promising alternative to horizontal axis wind turbines, a 2D computational investigation of an augmented wind turbine is proposed and analysed. In the initial CFD analysis, three parameters are carefully investigated: mesh resolution; turbulence model; and time step size. It appears that the mesh resolution and the turbulence model affect result accuracy; while the time step size examined, for the unsteady nature of the flow, has small impact on the numerical results. In the CFD validation of the open rotor with secondary data, the numerical results are in good agreement in terms of shape. It is, however, observed a discrepancy factor of 2 between numerical and experimental data. Successively, the introduction of an omnidirectional stator around the wind turbine increases the power and torque coefficients by around 30–35% when compared to the open case; but attention needs to be given to the orientation of the stator blades for optimum performance. It is found that the power and torque coefficients of the augmented wind turbine are independent of the incident wind speed considered.

A frequency-independent boundary element method for scattering by two-dimensional screens and apertures

We propose and analyse a hybrid numerical–asymptotic hp boundary element method (BEM) for time-harmonic scattering of an incident plane wave by an arbitrary collinear array of sound-soft two-dimensional screens. Our method uses an approximation space enriched with oscillatory basis functions, chosen to capture the high-frequency asymptotics of the solution. We provide a rigorous frequency-explicit error analysis which proves that the method converges exponentially as the number of degrees of freedom N increases, and that to achieve any desired accuracy it is sufficient to increase N in proportion to the square of the logarithm of the frequency as the frequency increases (standard BEMs require N to increase at least linearly with frequency to retain accuracy). Our numerical results suggest that fixed accuracy can in fact be achieved at arbitrarily high frequencies with a frequency-independent computational cost, when the oscillatory integrals required for implementation are computed using Filon quadrature. We also show how our method can be applied to the complementary ‘breakwater’ problem of propagation through an aperture in an infinite sound-hard screen.