CFP and YFP photostabilities are differentially affected by common mounting fluids

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking and tracing of specific proteins or cells or to determine protein interactions. In the later case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is visualized by specific microscopy techniques. When we applied a commonly used FRET microscopy technique - the increase in CFP-fluorescence after bleaching of YFP, we noticed that it worked well for live cells, but that most of the FRET-signal was lost in fixed cells mounted in commercial microscopy mounting fluids. Subsequently, we could show that CFP bleached much faster in the mounting medium than in live cells, while the opposite effect was observed for YFP. This change in photostability was not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increased in live cells after illumination at the YFP-excitation wavelength – a phenomenon, which might cause a false-positive signal in the FRET-microscopy technique that is based on bleaching of YFP. All together our results show that it is problematic to use commercially available mounting fluids for fluorescent proteins due to their differential effects on the bleaching kinetics and that the FRET microscopy technique based on bleaching of the acceptor is prone to artefacts at least for the CFP/YFP pair

Interaction of the TNFR-receptor associated factor TRAF1 with I-kappa B kinase 2 (IKK2, IKK-beta, IKBKB) and TRAF2 indicating a dose dependent regulatory function of TRAF1 for NF-kappa B signaling

IKK2 is one of the most crucial signaling kinases for activation of the transcription factor NF-kappa B. Since many NF-kappa B activating pathways converge at the level of IKK2, we searched for interaction partners of this kinase using the C-terminal part (aa 466-756) as bait in a yeast two-hybrid system. We identified the N-terminal part (aa 1-228) of the TNF-receptor associated factor TRAF1 as putative interaction partner, which was subsequently confirmed in mammalian cells by coimmunoprecipitation experiments. However, this interaction seemed weaker than the interaction between TRAF1 and TRAF2, an important activating adapter molecule of NF-kappa B signaling indicating that relative levels of IKK2, TRAF1 and TRAF2 might be important for the final biological readout. Reporter gene and kinase assays using ectopic expression of TRAF1 indicated that it can have both activating and inhibiting functions for IKK2 and NF-kappa B. Co-expression of fluorescently tagged TRAF1 and TRAF2 at different ratios implied that TRAF1 can affect clustering and presumably the activating function of TRAF2 in a dose dependent manner

Cost-efficient wireless mobile backhaul topologies: an analytical study

Proceedings of: 2010 IEEE Global Telecommunications Conference (GLOBECOM 2010), 6-10 December 2010, Miami, Florida, USAWireless communication technologies such as microwave radios are used to provide high-speed mobile backhaul connectivity for radio access networks in cases in which wirebased alternatives, e.g. cable or fiber, are not readily available and cannot be deployed in an economic or timely manner. Current mobile backhauls are predominantly deployed in tree or ring topologies, which simplify traffic management. Yet, with the increasing demand on backhaul capacity and the immense cost pressure on mobile backhaul solutions, meshed wireless mobile backhauls have been identified as a promising evolution. While traffic management in wireless mesh networks have been studied extensively in the literature, so far there is no quantitative analysis comparing the different topology options, i.e. mesh, ring and tree, regarding network performance and deployment cost. This paper fills this gap by studying the minimum cost problem of connecting a set of base station/gateway sites using different topologies while supporting both time- and space-varying traffic demands. Furthermore, we consider the additional constraint of resilience to single link failures. The evaluation results show that meshed wireless backhaul topologies are a cost-effective alternative to trees and rings, in particular in the face of spatial and temporal fluctuation of traffic demand and protection against link failuresEuropean Community's Seventh Framework ProgramPublicad

Distributed intelligence in pedestrian simulations

In order to accurately simulate pedestrian behaviour in complex situations, one is required to model both the physical environment and the strategic decision-making of individuals We present a method for integrating both of these model requirements, by distributing the computational complexity across discrete modules. These modules communicate with each other via XML messages. The approach also provides considerable flexibility for changing and evolving the model. The model is explained using an example of simulating hikers in the Swiss Alps.SNF, NFP 48, Habitats and Landscapes of the Alp

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

Background
The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings
When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance
Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions

Explosive Disintegration of a Massive Young Stellar System in Orion

Young massive stars in the center of crowded star clusters are expected to undergo close dynamical encounters that could lead to energetic, explosive events. However, there has so far never been clear observational evidence of such a remarkable phenomenon. We here report new interferometric observations made with the Submillimeter Array (SMA) that indicate the well known enigmatic wide-angle outflow located in the Orion BN/KL star-forming region to have been produced by such a violent explosion during the disruption of a massive young stellar system, and that this was caused by a close dynamical interaction about 500 years ago. This outflow thus belongs to a totally different family of molecular flows which is not related to the classical bipolar flows that are generated by stars during their formation process. Our molecular data allow us to create a 3D view of the debris flow and to link this directly to the well known Orion H$_2$ "fingers" farther outComment: Accepted by ApJ Letters The 3D movie can be found in: ftp://ftp.mpifr-bonn.mpg.de/outgoing/lzapata/movie.gi

Effects of hyperoxia and hyperoxic oscillations on the proteome of murine lung microvascular endothelium

Patients presenting with insufficient tissue oxygenation and impaired lung function as in acute respiratory distress syndrome (ARDS) frequently require mechanical ventilation with supplemental oxygen. Despite the lung being used to experiencing the highest partial pressure of oxygen during healthy breathing, the organ is susceptible to oxygen-induced injury at supraphysiological concentrations. Hyperoxia-induced lung injury (HALI) has been regarded as a second hit to pre-existing lung injury and ventilator-induced lung injury (VILI) attributed to oxidative stress. The injured lung has a tendency to form atelectasis, a cyclic collapse and reopening of alveoli. The affected lung areas experience oxygen conditions that oscillate between hyperoxia and hypoxia rather than remaining in a constant hyperoxic state. Mechanisms of HALI have been investigated in many animal models previously. These studies provided insights into the effects of hyperoxia on the whole organism. However, cell type-specific responses have not been dissected in detail, but are necessary for a complete mechanistic understanding of ongoing pathological processes. In our study, we investigated the effects of constant and intermittent hyperoxia on the lung endothelium from a mouse by an in vitro proteomic approach. We demonstrate that these oxygen conditions have characteristic effects on the pulmonary endothelial proteome that underlie the physiological (patho)mechanisms