Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

BACKGROUND: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells

Combined laboratory and numerical studies of the interaction between buoyant and plate-driven upwelling beneath segmented spreading centers

A combination of laboratory and numerical models are used to examine the mantle flow beneath a segmented ridge generated by the interaction of a linear, buoyant upwelling source with plate-driven flow. In the absence of plate spreading, the linear buoyant source creates a very narrow (across-axis), two-dimensional upwelling pattern. The plate-driven flow consists of a quasi-linear sheet-like upwelling that cuts beneath ridge-transform inside corners and is not centered beneath the spreading segments. When buoyant and plate-driven flows are combined, material rises beneath the inside corners and flows away from the axis asymmetrically. Near the ends of segments, this results in a geometrical misfit between the center of mantle upwelling and the ridge axis. If a similar pattern of mantle flow occurs beneath a segmented mid-ocean ridge, the result will be a thinner crust toward segment ends and possibly a negative correlation between extent of mantle melting and average depth of melting. These results indicate that even with an essentially two-dimensional source, in cases where it is oblique to the actual spreading segments, the upwelling beneath a segmented ridge will appear to be three-dimensional along axis. Since slow spreading ridges are generally more segmented than fast spreading ridges, this effect is likely to be more important at slow spreading ridges. Copyright 1996 by the American Geophysical Union

Supporting Runtime Tool Interaction for Parallel Simulations

: Scientists from many disciplines now routinely use modeling and simulation techniques to study physical and biological phenomena. Advances in high-performance architectures and networking have made it possible to build complex simulations with parallel and distributed interacting components. Unfortunately, the software needed to support such complex simulations has lagged behind hardware developments. We focus here on one aspect of such support: runtime program interaction. We have developed a runtime interaction framework and we have implemented a specific instance of it for an application in seismic tomography. That instance, called TierraLab, extends the geoscientists' existing (legacy) tomography code with runtime interaction capabilities which they access through a MATLAB interface. The scientist can stop a program, retrieve data, analyze and visualize that data with existing MATLAB routines, modify the data, and resume execution. They can do this all within a familiar MATLAB..