41 research outputs found

    Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%

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    Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein

    Chemotactic activity of extracellular nucleotideson human immune cells.

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    Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5–triphosphate (UTP) and uridine 5–diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation

    P2X7 receptor: Death or life?

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    The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis

    NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana

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    We thank Dr. Ben Smith (University of Oklahoma) for assistance with FLIM-FRET measurements and Dr. Min Ni (University of Minnesota) for critical reading of the manuscript. The cop1-4 mutant allele and cop1-4 co-9 cross were kindly provided by George Coupland (Max Planck Institute).Author Summary Light perception is critically important for the fitness of plants in both natural and agricultural settings. Plants not only use light for photosynthesis, but also as a cue for proper development. As a seedling emerges from soil it must determine the light environment and adopt an appropriate growth habit. When blue and red wavelengths are the dominant sources of light, plants will undergo photomorphogenesis. Photomorphogenesis describes a number of developmental responses initiated by light in a seedling, and includes shortened stems and establishing the ability to photosynthesize. The genes regulating photomorphogenesis have been studied extensively, but a complete picture remains elusive. Here we describe the finding that NUCLEAR FACTOR-Y (NF-Y) genes are positive regulators of photomorphogenesis—i.e., in plants where NF-Y genes are mutated, they display some characteristics of dark grown plants, even though they are in the light. Our data suggests that the roles of NF-Y genes in light perception do not fit in easily with those of other described pathways. Thus, studying these genes promises to help develop a more complete picture of how light drives plant development.Yeshttp://www.plosgenetics.org/static/editorial#pee

    First multi-wavelength campaign on the gamma-ray-loud active galaxy IC 310

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    Context. The extragalactic very-high-energy gamma-ray sky is rich in blazars. These are jetted active galactic nuclei that are viewed at a small angle to the line-of-sight. Only a handful of objects viewed at a larger angle are so far known to emit above 100 GeV. Multi-wavelength studies of such objects up to the highest energies provide new insights into the particle and radiation processes of active galactic nuclei.Aims. We aim to report the results from the first multi-wavelength campaign observing the TeV detected nucleus of the active galaxy IC 310, whose jet is observed at a moderate viewing angle of 10 degrees-20 degrees.Methods. The multi-instrument campaign was conducted between 2012 November and 2013 January, and involved observations with MAGIC, Fermi, INTEGRAL, Swift, OVRO, MOJAVE and EVN. These observations were complemented with archival data from the AllWISE and 2MASS catalogs. A one-zone synchrotron self-Compton model was applied to describe the broadband spectral energy distribution.Results. IC 310 showed an extraordinary TeV flare at the beginning of the campaign, followed by a low, but still detectable TeV flux. Compared to previous measurements in this energy range, the spectral shape was found to be steeper during the low emission state. Simultaneous observations in the soft X-ray band showed an enhanced energy flux state and a harder-when-brighter spectral shape behavior. No strong correlated flux variability was found in other frequency regimes. The broadband spectral energy distribution obtained from these observations supports the hypothesis of a double-hump structure.Conclusions. The harder-when-brighter trend in the X-ray and VHE emission, observed for the first time during this campaign, is consistent with the behavior expected from a synchrotron self-Compton scenario. The contemporaneous broadband spectral energy distribution is well described with a one-zone synchrotron self-Compton model using parameters that are comparable to those found for other gamma-ray-emitting misaligned blazars

    Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity

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    Epac-based FRET sensors have been widely used for the detection of cAMP concentrations in living cells. Originally developed by us as well as others, we have since then reported several important optimizations that make these sensors favourite among many cell biologists. We here report cloning and characterization of our fourth generation of cAMP sensors, which feature outstanding photostability, dynamic range and signal-to-noise ratio. The design is based on mTurquoise2, currently the brightest and most bleaching-resistant donor, and a new acceptor cassette that consists of a tandem of two cp173Venus fluorophores. We also report variants with a single point mutation, Q270E, in the Epac moiety, which decreases the dissociation constant of cAMP from 9.5 to 4 ÎĽM, and thus increases the affinity ~ 2.5-fold. Finally, we also prepared and characterized dedicated variants with non-emitting (dark) acceptors for single-wavelength FLIM acquisition that display an exceptional near-doubling of fluorescence lifetime upon saturation of cAMP levels. We believe this generation of cAMP outperforms all other sensors and therefore recommend these sensors for all future studies
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