2,023 research outputs found

    Effect of Ca2+ on the Steady-State and Time-Resolved Emission Properties of the Genetically Encoded Fluorescent Sensor CatchER

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    We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ

    Application of Designed Calcium Sensors with Fast Kinetic Responses

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    GPU-based Iterative Cone Beam CT Reconstruction Using Tight Frame Regularization

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    X-ray imaging dose from serial cone-beam CT (CBCT) scans raises a clinical concern in most image guided radiation therapy procedures. It is the goal of this paper to develop a fast GPU-based algorithm to reconstruct high quality CBCT images from undersampled and noisy projection data so as to lower the imaging dose. For this purpose, we have developed an iterative tight frame (TF) based CBCT reconstruction algorithm. A condition that a real CBCT image has a sparse representation under a TF basis is imposed in the iteration process as regularization to the solution. To speed up the computation, a multi-grid method is employed. Our GPU implementation has achieved high computational efficiency and a CBCT image of resolution 512\times512\times70 can be reconstructed in ~5 min. We have tested our algorithm on a digital NCAT phantom and a physical Catphan phantom. It is found that our TF-based algorithm is able to reconstrct CBCT in the context of undersampling and low mAs levels. We have also quantitatively analyzed the reconstructed CBCT image quality in terms of modulation-transfer-function and contrast-to-noise ratio under various scanning conditions. The results confirm the high CBCT image quality obtained from our TF algorithm. Moreover, our algorithm has also been validated in a real clinical context using a head-and-neck patient case. Comparisons of the developed TF algorithm and the current state-of-the-art TV algorithm have also been made in various cases studied in terms of reconstructed image quality and computation efficiency.Comment: 24 pages, 8 figures, accepted by Phys. Med. Bio

    C2 Continuous Blending of Time-Dependent Parametric Surfaces.

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    Surface blending is widely applied in mechanical engineering. Creating a smooth transition surface of C2 continuity between time-dependent parametric surfaces that change their positions and shapes with time is an important and unsolved topic in surface blending. In order to address this issue, this paper develops a new approach to unify both time-dependent and time-independent surface blending with C2 continuity. It proposes a new surface blending mathematical model consisting of a vector-valued sixth-order partial differential equation and blending boundary constraints and investigates a simple and efficient approximate analytical solution of the mathematical model. A number of examples are presented to demonstrate the effectiveness and applications. The proposed approach has the advantages of (1) unifying time-independent and time-dependent surface blending, (2) always maintaining C2 continuity at trimlines when parametric surfaces change their positions and shapes with time, (3) providing effective shape control handles to achieve the expected shapes of blending surfaces but still exactly satisfy the given blending boundary constraints, and (4) quickly generating C2 continuous blending surfaces from the approximate analytical solution with easiness, good accuracy, and high efficiency

    Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

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    <p>Abstract</p> <p>Background</p> <p>Fumarase catalyzes the reversible hydration of fumarate to <smcaps>L</smcaps>-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of <smcaps>L</smcaps>-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications.</p> <p>Results</p> <p>A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from <it>Bacteroides </it>sp. 2_1_33B and <it>Parabacteroides distasonis </it>ATCC 8503 (26% identical and 43% similar). The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in <it>E. coli </it>BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form <smcaps>L</smcaps>-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg<sup>2+</sup>. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: <it>K</it><sub>m</sub>= 0.48 mM, <it>V</it><sub>max </sub>= 827 μM/min/mg, and <it>k</it><sub>cat</sub>/<it>K</it><sub>m </sub>= 1900 mM/s.</p> <p>Conclusions</p> <p>We isolated a novel fumarase gene, <it>fumF</it>, from a sequence-based screen of a plasmid metagenomic library from uncultivated marine microorganisms. The properties of FumF protein may be ideal for the industrial production of <smcaps>L</smcaps>-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.</p

    4Pipe4-A 454 data analysis pipeline for SNP detection in datasets with no reference sequence or strain information

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    This work was fully supported by projects SOBREIRO/0036/2009 (under the framework of the Cork Oak ESTs Consortium), PTDC/BIA-BEC/098783/2008 and PTDC/AGR-GPL/119943/2010 from Fundação para a Ciência e Tecnologia (FCT) – Portugal. F. Pina-Martins was funded by FCT grant SFRH/BD/51411/2011, under the PhD program “Biology and Ecology of Global Changes”, Univ. Aveiro & Univ. Lisbon, Portugal. D. Batista was funded by FCT grant SFRH/BPD/104629/2014
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