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

Real-Time Visualization and Quantitation of Vascular Permeability In Vivo: Implications for Drug Delivery

The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors

A Search for Dark Higgs Bosons

Recent astrophysical and terrestrial experiments have motivated the proposal of a dark sector with GeV-scale gauge boson force carriers and new Higgs bosons. We present a search for a dark Higgs boson using 516 fb-1 of data collected with the BABAR detector. We do not observe a significant signal and we set 90% confidence level upper limits on the product of the Standard Model-dark sector mixing angle and the dark sector coupling constant.Comment: 7 pages, 5 postscript figures, published version with improved plots for b/w printin

Infection of Anopheles gambiae mosquitoes with entomopathogenic fungi: effect of host age and blood-feeding status

Physiological characteristics of insects can influence their susceptibility to fungal infection of which age and nutritional status are among the most important. An understanding of host–pathogen interaction with respect to these physiological characteristics of the host is essential if we are to develop fungal formulations capable of reducing malaria transmission under field conditions. Here, two independent bioassays were conducted to study the effect of age and blood-feeding status on fungal infection and survival of Anopheles gambiae s.s. Giles. Mosquitoes were exposed to 2 × 1010 conidia m−2 of oil-formulated Metarhizium anisopliae ICIPE-30 and of Beauveria bassiana I93-825, respectively, and their survival was monitored daily. Three age groups of mosquitoes were exposed, 2–4, 5–8, and 9–12 days since emergence. Five groups of different feeding status were exposed: non-blood-fed, 3, 12, 36, and 72 h post-blood feeding. Fungal infection reduced the survival of mosquitoes regardless of their age and blood-feeding status. Although older mosquitoes died relatively earlier than younger ones, age did not tend to affect mosquito susceptibility to fungal infection. Non-blood-fed mosquitoes were more susceptible to fungus infection compared to all categories of blood-fed mosquitoes, except for those exposed to B. bassiana 72 h post-blood feeding. In conclusion, formulations of M. anisopliae and B. bassiana can equally affect mosquitoes of different age classes, with them being relatively more susceptible to fungus infection when non-blood-fed

A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors

Host Responses in Life-History Traits and Tolerance to Virus Infection in Arabidopsis thaliana

Knowing how hosts respond to parasite infection is paramount in understanding the effects of parasites on host populations and hence host–parasite co-evolution. Modification of life-history traits in response to parasitism has received less attention than other defence strategies. Life-history theory predicts that parasitised hosts will increase reproductive effort and accelerate reproduction. However, empirical analyses of these predictions are few and mostly limited to animal-parasite systems. We have analysed life-history trait responses in 18 accessions of Arabidopsis thaliana infected at two different developmental stages with three strains of Cucumber mosaic virus (CMV). Accessions were divided into two groups according to allometric relationships; these groups differed also in their tolerance to CMV infection. Life-history trait modification upon virus infection depended on the host genotype and the stage at infection. While all accessions delayed flowering, only the more tolerant allometric group modified resource allocation to increase the production of reproductive structures and progeny, and reduced the length of reproductive period. Our results are in agreement with modifications of life-history traits reported for parasitised animals and with predictions from life-history theory. Thus, we provide empirical support for the general validity of theoretical predictions. In addition, this experimental approach allowed us to quantitatively estimate the genetic determinism of life-history trait plasticity and to evaluate the role of life-history trait modification in defence against parasites, two largely unexplored issues

The Relationship of Within-Host Multiplication and Virulence in a Plant-Virus System

Background. Virulence does not represent any obvious advantage to parasites. Most models of virulence evolution assume that virulence is an unavoidable consequence of within-host multiplication of parasites, resulting in trade-offs between within-host multiplication and between-host transmission fitness components. Experimental support for the central assumption of this hypothesis, i.e., for a positive correlation between within-host multiplication rates and virulence, is limited for plant-parasite systems. Methodology/Principal Findings. We have addressed this issue in the system Arabidopsis thaliana-Cucumber mosaic virus (CMV). Virus multiplication and the effect of infection on plant growth and on viable seed production were quantified for 21 Arabidopsis wild genotypes infected by 3 CMV isolates. The effect of infection on plant growth and seed production depended of plant architecture and length of postembryonic life cycle, two genetically-determined traits, as well as on the time of infection in the plant's life cycle. A relationship between virus multiplication and virulence was not a general feature of this host-parasite system. This could be explained by tolerance mechanisms determined by the host genotype and operating differently on two components of plant fitness, biomass production and resource allocation to seeds. However, a positive relationship between virus multiplication and virulence was detected for some accessions with short life cycle and high seed weight to biomass ratio, which show lower levels of tolerance to infection. Conclusions/Significance. These results show that genotype-specific tolerance mechanisms may lead to the absence of a clear relationship between parasite multiplication and virulence. Furthermore, a positive correlation between parasite multiplication and virulence may occur only in some genotypes and/or environmental conditions for a given host-parasite system. Thus, our results challenge the general validity of the trade-off hypothesis for virulence evolution, and stress the need of considering the effect of both the host and parasite genotypes in analyses of host-parasite interactions. © 2007 Pagán et al.Ministerio de Educación y Ciencia, Spain.Peer Reviewe

The Relationship of Within-Host Multiplication and Virulence in a Plant-Virus System

Background. Virulence does not represent any obvious advantage to parasites. Most models of virulence evolution assume that virulence is an unavoidable consequence of within-host multiplication of parasites, resulting in trade-offs between within-host multiplication and between-host transmission fitness components. Experimental support for the central assumption of this hypothesis, i.e., for a positive correlation between within-host multiplication rates and virulence, is limited for plant-parasite systems. Methodology/Principal Findings. We have addressed this issue in the system Arabidopsis thaliana-Cucumber mosaic virus (CMV). Virus multiplication and the effect of infection on plant growth and on viable seed production were quantified for 21 Arabidopsis wild genotypes infected by 3 CMV isolates. The effect of infection on plant growth and seed production depended of plant architecture and length of postembryonic life cycle, two genetically-determined traits, as well as on the time of infection in the plant's life cycle. A relationship between virus multiplication and virulence was not a general feature of this host-parasite system. This could be explained by tolerance mechanisms determined by the host genotype and operating differently on two components of plant fitness, biomass production and resource allocation to seeds. However, a positive relationship between virus multiplication and virulence was detected for some accessions with short life cycle and high seed weight to biomass ratio, which show lower levels of tolerance to infection. Conclusions/Significance. These results show that genotype-specific tolerance mechanisms may lead to the absence of a clear relationship between parasite multiplication and virulence. Furthermore, a positive correlation between parasite multiplication and virulence may occur only in some genotypes and/or environmental conditions for a given host-parasite system. Thus, our results challenge the general validity of the trade-off hypothesis for virulence evolution, and stress the need of considering the effect of both the host and parasite genotypes in analyses of host-parasite interactions. © 2007 Pagán et al.Ministerio de Educación y Ciencia, Spain.Peer Reviewe