Sample records for analogue fret acceptor

  1. Anomalous surplus energy transfer observed with multiple FRET acceptors.

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    Srinagesh V Koushik

    Full Text Available BACKGROUND: Förster resonance energy transfer (FRET is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k(D-->A can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates. METHODOLOGY/PRINCIPAL FINDINGS: The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean and either two or three FRET acceptors (Venus. FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates. CONCLUSIONS/SIGNIFICANCE: We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is

  2. A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth. (United States)

    Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F


    FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.

  3. Phanta: a non-fluorescent photochromic acceptor for pcFRET.

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    Craig Don Paul

    Full Text Available We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003 we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.

  4. Rise-time of FRET-acceptor fluorescence tracks protein folding

    NARCIS (Netherlands)

    Lindhoud, S.; Westphal, A.H.; Van Mierlo, C.P.M.; Visser, A.J.W.G.; Borst, J.W.


    Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no correspon

  5. Studying Z-DNA and B- to Z-DNA transitions using a cytosine analogue FRET-pair. (United States)

    Dumat, Blaise; Larsen, Anders Foller; Wilhelmsson, L Marcus


    Herein, we report on the use of a tricyclic cytosine FRET pair, incorporated into DNA with different base pair separations, to study Z-DNA and B-Z DNA junctions. With its position inside the DNA structure, the FRET pair responds to a B- to Z-DNA transition with a distinct change in FRET efficiency for each donor/acceptor configuration allowing reliable structural probing. Moreover, we show how fluorescence spectroscopy and our cytosine analogues can be used to determine rate constants for the B- to Z-DNA transition mechanism. The modified cytosines have little influence on the transition and the FRET pair is thus an easily implemented and virtually non-perturbing fluorescence tool to study Z-DNA. This nucleobase analogue FRET pair represents a valuable addition to the limited number of fluorescence methods available to study Z-DNA and we suggest it will facilitate, for example, deciphering the B- to Z-DNA transition mechanism and investigating the interaction of DNA with Z-DNA binding proteins.

  6. AccPbFRET: An ImageJ plugin for semi-automatic, fully corrected analysis of acceptor photobleaching FRET images

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    Vereb György


    Full Text Available Abstract Background The acceptor photobleaching fluorescence resonance energy transfer (FRET method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios. Results AccPbFRET is a user-friendly, efficient ImageJ plugin which allows fully corrected, pixel-wise calculation and detailed, ROI (region of interest-based analysis of FRET efficiencies in microscopic images. Furthermore, automatic registration and semi-automatic analysis of large image sets is provided, which are not available in any existing FRET evaluation software. Conclusion Despite of the widespread applicability of the acceptor photobleaching FRET technique, this is the first paper where all possible sources of major errors of the measurement and analysis are considered, and AccPbFRET is the only program which provides the complete suite of corrections – for registering image pairs, for unwanted photobleaching of the donor, for cross-talk of the acceptor and/or its photoproduct to the donor channel and for partial photobleaching of the acceptor. The program efficiently speeds up the analysis of large image sets even for novice users and is freely available.

  7. FRET study in oligopeptide-linked donor–acceptor system in PVA matrix (United States)

    Shah, Sunil; Mandecki, Wlodek; Li, Ji; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal


    An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.

  8. Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.

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    Viviane Devauges

    Full Text Available We present a novel imaging system combining total internal reflection fluorescence (TIRF microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.

  9. Protein-protein interactions in the plant Golgi apparatus, studied with FRET acceptor photobleaching technique

    DEFF Research Database (Denmark)

    Poulsen, Christian Peter

    to the plant Golgi apparatus and involved mainly in arabinogalactan protein (AGP) biosynthesis. Co-expression analysis identified 4 GTs and 4 NSTs possibly involved in AGP biosynthesis. As part of the method development, the cytoskeleton-acting agent Cytochalasin D was tested as an inhibitor...... of the actinomyosin based movement of Golgi vesicles, and was proved to be superior to commonly used fixatives such as the cross-linking agent paraformaldehyde which causes quenching of the fluorophores. According to FRET analysis, the results showed association between two galactosyltransferases, AtGALT29A and At...

  10. A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example.

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    Gerard N M van der Krogt

    Full Text Available 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.

  11. Single molecule FRET detection in CdSe-QD donor and Cy5-labeled molecular chaperone acceptor complex by imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tani, Toshiro, E-mail: [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Institute of Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Oda, Masaru [Division of Advanced Applied Physics, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Institute of Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Sakai, Hiroshi; Araki, Daisuke; Itoh, Yoshinori [Department of Applied Physics, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Ohtaki, Akashi; Yohda, Masafumi [Division of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan); Institute of Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Kogane-i, Tokyo 184-8588 (Japan)


    We report single molecule spectroscopic evidence of FRET in CdSe quantum dot (QD) conjugated with Cy5-labeled molecular chaperone systems in buffer solutions. Donor QDs are core-shell type nanocrystals covered with organic surfactants on their outermost surfaces, i.e. CdSe/ZnS/TOPO's. As prototype molecular chaperones, we adopt prefoldins (PFDs), on which Cy5's are labeled as acceptors. Donor QDs possess two-fold degenerate emission dipoles perpendicular to the c-axis, due to their Wurtzite crystal structures, while acceptor Cy5's possess linear absorption and emission dipoles. Thus, their combination provides novel features to those in conventional FRET systems. PFDs are jellyfish-shaped hexameric co-chaperones of group II chaperonins, which recognize hydrophobic portions of denatured proteins and encapsulate them within their central cavities. Hence, PFDs will also capture the CdSe/ZnS/TOPO QDs due to its surface similarity to the denatured proteins. By introducing simple microscope setup for single QD-PFD-Cy5 spectroscopy, we have successfully captured the emission spectra in FRET regime. We also have observed peculiar features in time evolution profiles of single QD emissions conjugated with Cy5-labeled PFDs under polarization modulation measurements. Notable point of our hybrid conjugates is that they are biochemically in living action. We describe our present results in relation to possible protein reactions.

  12. Characterization of Nucleobase Analogue FRET Acceptor tCnitro

    DEFF Research Database (Denmark)

    Preus, Søren; Börjesson, Karl; Kilså, Kristine;


    , the UV-vis absorption of tCnitro is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pKa of 11.1. The structure and electronic spectrum of tCnitrois further characterized by density functional theory calculations....

  13. Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching (United States)

    Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael


    Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

  14. IIem-spFRET: improved Iem-spFRET method for robust FRET measurement (United States)

    Zhang, Jiang; Lin, Fangrui; Chai, Liuying; Wei, Lichun; Chen, Tongsheng


    We recently developed a quantitative Förster resonance energy transfer (FRET) measurement method based on emission-spectral unmixing (Iem-spFRET). We here developed an improved Iem-spFRET method (termed as IIem-spFRET) for more robust FRET measurement in living cells. First, two background (BG) spectral fingerprints measured from blank living cells are introduced to remove BG and autofluorescence. Second, we introduce a ρ factor denoting the ratio of two molar extinction coefficient ratios (γ) of acceptor to donor at two excitations into IIem-spFRET for direct measurement of the γ values using a tandem construct with unknown FRET efficiency (E). We performed IIem-spFRET on our microscope-spectrometer platform to measure the γ values of Venus (V) to Cerulean (C) and the E values of C32V, CVC, VCV, and VCVV constructs, respectively, in living Huh7 cells. For the C32V or CVC cells, the Iem-spFRET and IIem-spFRET methods measured consistent E values. However, for the cells especially with low expressing levels of VCV or VCVV, the E values measured by Iem-spFRET showed large deviations and fluctuations, whereas the IIem-spFRET method greatly improved the measured E values. Collectively, IIem-spFRET is a powerful and robust tool for quantitatively measuring FRET signal in living cells.

  15. Binomial distribution-based quantitative measurement of multiple-acceptors fluorescence resonance energy transfer by partially photobleaching acceptor (United States)

    Zhang, Lili; Yu, Huaina; Zhang, Jianwei; Chen, Tongsheng


    We report that binomial distribution depending on acceptor photobleaching degree can be used to characterize the proportions of various kinds of FRET (Fluorescence Resonance Energy Transfer) constructs resulted from partial acceptor photobleaching of multiple-acceptors FRET system. On this basis, we set up a rigorous quantitation theory for multiple-acceptors FRET construct named as Mb-PbFRET which is not affected by the imaging conditions and fluorophore properties. We experimentally validate Mb-PbFRET with FRET constructs consisted of one donor and two or three acceptors inside living cells on confocal and wide-field microscopes.

  16. Identification of Different Donor-Acceptor Structures via Förster Resonance Energy Transfer (FRET in Quantum-Dot-Perylene Bisimide Assemblies

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    Christian von Borczyskowski


    Full Text Available Nanoassemblies are formed via self-assembly of ZnS capped CdSe quantum dots (QD and perylene bisimide (PBI dyes. Upon assembly formation the QD photoluminescence is quenched, as can be detected both via single particle detection and ensemble experiments in solution. Quenching has been assigned to FRET and NON-FRET processes. Analysis of FRET allows for a distinction between different geometries of the QD dye assemblies. Time-resolved single molecule spectroscopy reveals intrinsic fluctuations of the PBI fluorescence lifetime and spectrum, caused by rearrangement of the phenoxy side groups. The distribution of such molecular conformations and their changed dynamics upon assembly formation are discussed in the scope of FRET efficiency and surface ligand density.

  17. Spectral Fine Tuning of Cyanine Dyes: Electron Donor-Acceptor Substituted Analogues of Thiazole Orange† (United States)

    Rastede, Elizabeth E.; Tanha, Matteus; Yaron, David; Watkins, Simon C.; Waggoner, Alan S.; Armitage, Bruce A.


    The introduction of electron donor and acceptor groups at strategic locations on a fluorogenic cyanine dye allows fine-tuning of the absorption and emission spectra while preserving the ability of the dye to bind to biomolecular hosts such as double-stranded DNA and a single-chain antibody fragment originally selected for binding to the parent unsubstituted dye, thiazole orange (TO). The observed spectral shifts are consistent with calculated HOMO-LUMO energy gaps and reflect electron density localization on the quinoline half of TO in the LUMO. A dye bearing donating methoxy and withdrawing trifluoromethyl groups on the benzothiazole and quinoline rings, respectively, shifts the absorption spectrum to sufficiently longer wavelengths to allow excitation at green wavelengths as opposed to the parent dye, which is optimally excited in the blue. PMID:26171668

  18. A Guide to Fluorescent Protein FRET Pairs (United States)

    Bajar, Bryce T.; Wang, Emily S.; Zhang, Shu; Lin, Michael Z.; Chu, Jun


    Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. PMID:27649177

  19. FRET in membrane biophysics: an overview

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    Luis MS Loura


    Full Text Available Förster resonance energy transfer (FRET, in most applications used as a spectroscopicruler, allows an easy determination of the donor-acceptor intermolecular distance.However, the situation becomes complex in membranes, since around each donor there isan ensemble of acceptors at non-correlated distances. In this review, state-of-the-artmethodologies for this situation are presented, usually involving time-resolved data andmodel fitting. This powerful approach can be used to study the occurrence of phaseseparation (rafts or other type of domains, allowing their detection as well as sizeevaluation. Formalisms for studying lipid-protein and protein-protein interactionsaccording to specific topologies are also addressed. The advantages and addedcomplexity of a specific type of FRET (energy homo-transfer or energy migration aredescribed, as well as applications of FRET under the microscope.

  20. Pulse-shaping based two-photon FRET stoichiometry. (United States)

    Flynn, Daniel C; Bhagwat, Amar R; Brenner, Meredith H; Núñez, Marcos F; Mork, Briana E; Cai, Dawen; Swanson, Joel A; Ogilvie, Jennifer P


    Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

  1. Fluorescence Resonance Energy Transfer Using Spiropyran and Diarylethene Photochromic Acceptors

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    E. A. Jares-Erijman


    Full Text Available We describe the preparation and photophysical characterization of two model compounds designed to test a new approach for the quantitative determination of Fluorescence Resonance Energy Transfer (FRET in biological systems. The method enables modulation of FRET by exploiting the unique reversible spectral properties of photochromic diarylethenes and spiropyrans to create switchable energy acceptors.

  2. CTAB enhancement of FRET in DNA structures. (United States)

    Oh, Taeseok; Takahashi, Tsukasa; Kim, Sejung; Heller, Michael J


    The effect of cetyl-trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye-conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye-conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

  3. FRET and Non-FRET processes in quantum dot - dye nanoassemblies

    Energy Technology Data Exchange (ETDEWEB)

    Kowerko, Danny; Schuster, Joerg; Borczyskowski, Christian von [Center of Nanostructured Materials and Analytics, TU-Chemnitz, 09107 Chemnitz (Germany)


    Assemblies of semiconductor quantum dots (QDs - donor) and organic (dye - acceptor) molecules are of increasing interest for numerous applications in science and technology. Recent publications on such systems report on charge and fluorescence resonant energy transfer (FRET), prevalently neglecting an exact quantitative spectroscopic analysis of the acceptor. Recently, calculations of Seifert et al. and Blaudeck and coworkers demonstrated the crucial dependence of trap state emission from the surface coverage. In our contribution we emphasize the role of dye induces surface trap states as one mechanism - besides FRET - for QD quenching. Thus detailed quantitative analysis of titration experiments of differently functionalized perylenebismide molecules and CdSe/ZnS quantum dots will be discussed. By means of single particle spectroscopy we may even compare spectroscopic properties of the free and bound species on one and the same sample under identical conditions. Furthermore, analysis of FRET allows for a distinction between different geometries of the dye-QD assemblies.

  4. PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs. (United States)

    Feige, Jérôme N; Sage, Daniel; Wahli, Walter; Desvergne, Béatrice; Gelman, Laurent


    Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed-throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set-up with different photomultipliers for the donor and FRET channels, a set-up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug-in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug-in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells.

  5. Evaluating the Relationship between FRET Changes and Distance Changes Using DNA Length and Restriction Enzyme Specificity (United States)

    Pazhani, Yogitha; Horn, Abigail E.; Grado, Lizbeth; Kugel, Jennifer F.


    FRET (Fo¨rster resonance energy transfer) involves the transfer of energy from an excited donor fluorophore to an acceptor molecule in a manner that is dependent on the distance between the two. A biochemistry laboratory experiment is described that teaches students how to use FRET to evaluate distance changes in biological molecules. Students…

  6. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging. (United States)

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen En Henegouwen, Paul M P; Jennings, Travis L; Susumu, Kimihiro; Medintz, Igor L; Hildebrandt, Niko; Miller, Lawrence W


    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress sample autofluorescence and background fluorescence from directly excited FRET acceptors, Tb-to-QD FRET has rarely been exploited for biomolecular imaging. We demonstrate Tb-to-QD time-gated FRET nanoassemblies that can be applied for intra- and extracellular imaging. Immunostaining of different epitopes of the epidermal growth factor receptor (EGFR) with Tb- and QD-conjugated antibodies and nanobodies allowed for efficient Tb-to-QD FRET on A431 cell membranes. The broad usability of Tb-to-QD FRET was further demonstrated by intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET using microinjection as well as cell-penetrating peptide-mediated endocytosis with HeLa cells. Effective brightness enhancement by FRET from several Tb to the same QD, the use of low nanomolar concentrations, and the quick and sensitive detection void of FRET acceptor background fluorescence are important advantages for advanced intra- and extracellular imaging of biomolecular interactions.

  7. Understanding and modeling Förster-type resonance energy transfer (FRET)

    CERN Document Server

    Hernández Martínez, Pedro Ludwig; Demir, Hilmi Volkan


    This Brief presents a complete study of the generalized theory of Förster-type energy transfer in nanostructures with mixed dimensionality. Here the aim is to obtain a generalized theory of FRET including a comprehensive set of analytical equations for all combinations and configurations of nanostructures and deriving generic expressions for the dimensionality involved. In this brief, the modification of FRET mechanism with respect to the nanostructure serving as the donor vs. the acceptor will be included, focusing on the rate’s distance dependency and the role of the effective dielectric function in FRET, which will be a unique, useful source for those who study and model FRET.

  8. Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins. (United States)

    Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert


    This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

  9. Single cell FRET analysis for the identification of optimal FRET-pairs in Bacillus subtilis using a prototype MEM-FLIM system.

    Directory of Open Access Journals (Sweden)

    Ruud G J Detert Oude Weme

    Full Text Available Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET. Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM. For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.

  10. Borromean three-body FRET in frozen Rydberg gases (United States)

    Faoro, R.; Pelle, B.; Zuliani, A.; Cheinet, P.; Arimondo, E.; Pillet, P.


    Controlling the interactions between ultracold atoms is crucial for quantum simulation and computation purposes. Highly excited Rydberg atoms are considered in this prospect for their strong and controllable interactions known in the dipole-dipole case to induce non-radiative energy transfers between atom pairs, similarly to fluorescence resonance energy transfer (FRET) in biological systems. Here we predict few-body FRET processes in Rydberg atoms and observe the first three-body resonance energy transfer in cold Rydberg atoms using cold caesium atoms. In these resonances, additional relay atoms carry away an energy excess preventing the two-body resonance, leading thus to a Borromean type of energy transfer. These few-body processes present strong similarities with multistep FRET between chromophores sometimes called donor-bridge-acceptor or superexchange. Most importantly, they generalize to any Rydberg atom and could lead to new implementations of few-body quantum gates or entanglement.

  11. Fretting of AISI 9310 and selected fretting resistant surface treatments (United States)

    Bill, R. C.


    Fretting wear experiments were conducted with uncoated AISI 9310 mating surfaces, and with combinations incorporating a selected coating to one of the mating surfaces. Wear measurements and SEM observations indicated that surface fatigue, as made evident by spallation and surface crack formation, is an important mechanism in promoting fretting wear to uncoated 9310. Increasing humidity resulted in accelerated fretting, and a very noticeable difference in nature of the fretting debris. Of the coatings evaluated, aluminum bronze with a polyester additive was most effective at reducing wear and minimizing fretting damage to the mating uncoated surface, by means of a selflubricating film that developed on the fretting surfaces. Chromium plate performed as an effective protective coating, itself resisting fretting and not accelerating damage to the uncoated surface.

  12. FRET and colocalization analyzer--a method to validate measurements of sensitized emission FRET acquired by confocal microscopy and available as an ImageJ Plug-in. (United States)

    Hachet-Haas, Muriel; Converset, Noël; Marchal, Olivier; Matthes, Hans; Gioria, Sophie; Galzi, Jean-Luc; Lecat, Sandra


    Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

  13. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins. (United States)

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A


    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells.

  14. Proposal of a New Method for Measuring Förster Resonance Energy Transfer (FRET Rapidly, Quantitatively and Non-Destructively

    Directory of Open Access Journals (Sweden)

    Paul Johannes Helm


    Full Text Available The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor” to another chromophore (an “acceptor”, in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET. The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle ½ and frequencies ƒ1 and ƒ2 by electro-optic modulators. In an ensemble exposed to these laser beams, the donor excitation is modulated at ƒ1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at ƒ2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at ƒ1 but also at the beat frequency Δƒ: = |ƒ1 − ƒ2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.

  15. Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lan, Tien-Hung; Wu, Guangyu; Lambert, Nevin A., E-mail:


    Diffusion can enhance Förster resonance energy transfer (FRET) when donors or acceptors diffuse distances that are similar to the distances separating them during the donor's excited state lifetime. Lanthanide donors remain in the excited state for milliseconds, which makes them useful for time-resolved FRET applications but also allows time for diffusion to enhance energy transfer. Here we show that diffusion dramatically enhances FRET between membrane proteins labeled with lanthanide donors. This phenomenon complicates interpretation of experiments that use long-lived donors to infer association or proximity of mobile membrane proteins, but also offers a method of monitoring diffusion in membrane domains in real time in living cells. - Highlights: • Diffusion enhances TR-FRET from membrane proteins labeled with lanthanide donors. • Diffusion-dependent FRET can overshadow FRET due to oligomerization or clustering. • FRET studies using lanthanide-tagged membrane proteins should consider diffusion. • FRET from lanthanide donors can be used to monitor membrane protein diffusion.

  16. Engineering of weak helper interactions for high-efficiency FRET probes. (United States)

    Grünberg, Raik; Burnier, Julia V; Ferrar, Tony; Beltran-Sastre, Violeta; Stricher, François; van der Sloot, Almer M; Garcia-Olivas, Raquel; Mallabiabarrena, Arrate; Sanjuan, Xavier; Zimmermann, Timo; Serrano, Luis


    Fluorescence resonance energy transfer (FRET)-based detection of protein interactions is limited by the very narrow range of FRET-permitting distances. We show two different strategies for the rational design of weak helper interactions that co-recruit donor and acceptor fluorophores for a more robust detection of bimolecular FRET: (i) in silico design of electrostatically driven encounter complexes and (ii) fusion of tunable domain-peptide interaction modules based on WW or SH3 domains. We tested each strategy for optimization of FRET between (m)Citrine and mCherry, which do not natively interact. Both approaches yielded comparable and large increases in FRET efficiencies with little or no background. Helper-interaction modules can be fused to any pair of fluorescent proteins and could, we found, enhance FRET between mTFP1 and mCherry as well as between mTurquoise2 and mCitrine. We applied enhanced helper-interaction FRET (hiFRET) probes to study the binding between full-length H-Ras and Raf1 as well as the drug-induced interaction between Raf1 and B-Raf.

  17. APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

    Directory of Open Access Journals (Sweden)

    Heidi J Chial

    Full Text Available BACKGROUND: Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported. METHODOLOGY: Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species. CONCLUSIONS: All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes

  18. Experimental verification of the kinetic theory of FRET using optical microspectroscopy and obligate oligomers. (United States)

    Patowary, Suparna; Pisterzi, Luca F; Biener, Gabriel; Holz, Jessica D; Oliver, Julie A; Wells, James W; Raicu, Valerică


    Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells.

  19. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples.

    Directory of Open Access Journals (Sweden)

    Joachim Goedhart

    Full Text Available 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.

  20. Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors

    Directory of Open Access Journals (Sweden)

    Kenny F. Chou


    Full Text Available Förster (or fluorescence resonance energy transfer amongst semiconductor quantum dots (QDs is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The brightness and photostability of QDs make them attractive for highly sensitive sensing and long-term, repetitive imaging applications, respectively, but the overlapping donor and acceptor excitation signals that arise when QDs serve as both the donor and acceptor lead to high background signals from direct excitation of the acceptor. The fundamentals of FRET within a nominally homogeneous QD population as well as energy transfer between two distinct colors of QDs are discussed. Examples of successful sensors are highlighted, as is cascading FRET, which can be used for solar harvesting.

  1. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

    Full Text Available Fluorescence Resonance Energy Transfer (FRET microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  2. Highly Sensitive Homogeneous Immunoassays Based on Construction of Silver Triangular Nanoplates-Quantum Dots FRET System (United States)

    Zeng, Qinghui; Li, Qin; Ji, Wenyu; Bin, Xue; Song, Jie


    With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D-A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form.

  3. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging (United States)

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger


    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02-04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

  4. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications. (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo


    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C3N4) and transition metal dichalcogenides (e.g. MoS2, MnO2, and WS2). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring.

  5. Fluorescence resonance energy transfer (FRET) in chemistry and biology: Non-Förster distance dependence of the FRET rate

    Indian Academy of Sciences (India)

    Sangeeta Saini; Harjinder Singh; Biman Bagchi


    Fluorescence resonance energy transfer (FRET) is a popular tool to study equilibrium and dynamical properties of polymers and biopolymers in condensed phases and is now widely used in conjunction with single molecule spectroscopy. In the data analysis, one usually employs the Förster expression which predicts (1/6) distance dependence of the energy transfer rate. However, critical analysis shows that this expression can be of rather limited validity in many cases. We demonstrate this by explicitly considering a donor-acceptor system, polyfluorene (PF6)-tetraphenylporphyrin (TPP), where the size of both donor and acceptor is comparable to the distance separating them. In such cases, one may expect much weaker distance (as 1/2 or even weaker) dependence. We have also considered the case of energy transfer from a dye to a nanoparticle. Here we find 1/4 distance dependence at large separations, completely different from Förster. We also discuss recent application of FRET to study polymer conformational dynamics.

  6. Ratiometric FRET-based detection of DNA and micro-RNA in solution

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, Evgenia G., E-mail: ematveev@hsc.unt.ed [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States); Stewart, Donald R. [Omm Scientific, Inc., 2600 N. Stemmons Freeway, Suite 129, Dallas, TX 75207 (United States); Gryczynski, Ignacy [Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107 (United States)


    A ratiometric method for detecting DNA oligomers in bulk solution based on Foerster resonance energy transfer (FRET) is described. The two fluorescence signals (green and red), originating from Cy3 (donor, green) and Cy5 (acceptor, red) labels, are simultaneously detected from the pre-hybridized Cy3oligomerY:Cy5oligomerX system. The ratio of red to green intensities is sensitive to the presence of the single-stranded complimentary oligomer, which replaces single-stranded Cy3oligomerY in the donor:acceptor complex and perturbs the FRET. The detection scheme is generally applicable to the detection of DNA and RNA, and particularly micro-RNA. The proposed method is applicable to various double-stranded various lengths targets (manipulation of the sample preparation conditions, such as temperature, incubation time, denaturizing agent, may be needed).

  7. Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes (United States)

    Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.


    A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

  8. FRET-based modified graphene quantum dots for direct trypsin quantification in urine

    Energy Technology Data Exchange (ETDEWEB)

    Poon, Chung-Yan; Li, Qinghua [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Zhang, Jiali; Li, Zhongping [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Dong, Chuan [Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006 (China); Lee, Albert Wai-Ming; Chan, Wing-Hong [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong); Li, Hung-Wing, E-mail: [Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong Special Administrative Region (Hong Kong)


    A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening. - Highlights: • A FRET-based biosensor was developed for direct quantification of trypsin. • Fast and sensitive screening of pancreatic disease was facilitated. • The direct quantification of trypsin in urine samples was demonstrated.

  9. DNA quantification based on FRET realized by combination with surfactant CPB. (United States)

    Liu, Chunxia; Wang, Lei; Jiang, Wei


    In this work, we developed a novel DNA quantitative analysis based on fluorescence resonance energy transfer (FRET) realized by combination with a surfactant CPB. The approach was capable of detecting long-stranded DNA in a separation-free format. A sandwich-type FAM-c-DNA-t-DNA-r-DNA-TAMRA conjugate was first formed by the capture probe tagged with FAM, the reporter probe tagged with TAMRA and the target DNA through hybridization. The donor (FAM) and the acceptor (TAMRA) were bridged to afford a FRET system. Subsequently, an addition of the cationic surfactant CPB to the system resulted in a substantial change of the microenvironment and an effective condensation of DNA strands. Consequently, without altering the component of the double strands, an enhanced acceptor fluorescence signal from FRET was achieved and a quantification of the target DNA containing 30 bases was enabled. Under the optimal experimental conditions, an excellent linear relationship between the increase of acceptor fluorescent peak area and the target DNA concentration was obtained over the range from 1.0 x 10(-7) to 3.0 x 10(-9) mol L(-1). The proposed approach offered adequate sensitivity for the detection of the target DNA at 1.0 x 10(-9) mol L(-1).

  10. Significant FRET between SWNT/DNA and rare earth ions: a signature of their spatial correlations. (United States)

    Ignatova, Tetyana; Najafov, Hikmat; Ryasnyanskiy, Aleksandr; Biaggio, Ivan; Zheng, Ming; Rotkin, Slava V


    Significant acceleration of the photoluminescence (PL) decay rate was observed in water solutions of two rare earth ions (REIs), Tb and Eu. We propose that the time-resolved PL spectroscopy data are explained by a fluorescence resonance energy transfer (FRET) between the REIs. FRET was directly confirmed by detecting the induced PL of the energy acceptor, Eu ion, under the PL excitation of the donor ion, Tb, with FRET efficiency reaching 7% in the most saturated solution, where the distance between the unlike REIs is the shortest. Using this as a calibration experiment, a comparable FRET was measured in the mixed solution of REIs with single-wall nanotubes (SWNTs) wrapped with DNA. From the FRET efficiency of 10% and 7% for Tb and Eu, respectively, the characteristic distance between the REI and SWNT/DNA was obtained as 15.9 ± 1.3 Å, suggesting that the complexes are formed because of Coulomb attraction between the REI and the ionized phosphate groups of the DNA.

  11. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes. (United States)

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis


    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application.

  12. Fretting fatigue in AISI 1015 steel

    Indian Academy of Sciences (India)

    R Gnanamoorthy; R Rosi Reddy


    A small osillatory movement between two contacting surfaces is termed as fretting and on many occasions it acts as the crack initiation site leading to catastrophic failure of the overall structure. The occurrence of fretting is observed in many engineering structures such as shaft flanges, gas turbines, steel ropes etc. An experimental facility, which can simulate the fretting fatigue in many engineering applications, is the primary requirement of the research program. A laboratory fretting fatigue test facility capable of varying many influencing parameters of fretting fatigue such as slip amplitutde, frequency, contact pressure, etc is designed and developed. Preliminary investigations on plain and fretting fatigue behaviour of AISI 1015 structural steel are reported in this paper. A strength reduction factor of about 1.30 was obtained due to fretting for the test material under the present experimental conditions. Influence of contact load on fretting was also studied. Increasing fretting contact load decreased the fatigue life in the range investigated. Failure analysis showed typical stage I oblique crack growth followed by stage II straight crack perpedicular to the fretting zone.


    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI


    Full Text Available Fretting is now fully identified as a small amplitude oscilatory motion which induces a harmonic tangential force between two surfaces in contact.It is related to three main loadings, i.e. fretting-wear, fretting-fatigue and fretting corrosion.Fretting regimes were first mapped by Vingsbo. In a similar way, three fretting regimes will be considered: stick regime,slip regime and mixed regime. The mixed regime was made up of initial gross slip followed by partial slip condition after a few hundred cycles. Obviously the partial slip transition develops the highest stress levels which can induce fatigue crack nucleation depending on the fatigue properties of the two contacting first bodies. Therefore prediction of the frontier between partial slip and gross slip is required.

  14. FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET. (United States)

    Ingargiola, Antonino; Lerner, Eitan; Chung, SangYoon; Weiss, Shimon; Michalet, Xavier


    Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to

  15. Fluorescence Resonance Energy Transfer (FRET) sensor

    CERN Document Server

    Hussain, Syed Arshad; Chakraborty, Sekhar; Saha, Jaba; Roy, Arpan Datta; Chakraborty, Santanu; Debnath, Pintu; Bhattacharjee, D


    The applications of Fluorescence resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. FRET can be used as spectroscopic ruler in various areas such as structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting, and metallic nanomaterials etc. Based on the mechanism of FRET a variety of novel chemical sensors and Biosensors have been developed. This review highlights the recent applications of sensitive and selective ratiometric FRET based sensors.

  16. FRET enhancement close to gold nanoparticles positioned in DNA origami constructs. (United States)

    Aissaoui, Nesrine; Moth-Poulsen, Kasper; Käll, Mikael; Johansson, Peter; Wilhelmsson, L Marcus; Albinsson, Bo


    Here we investigate the energy transfer rates of a Förster resonance energy transfer (FRET) pair positioned in close proximity to a 5 nm gold nanoparticle (AuNP) on a DNA origami construct. We study the distance dependence of the FRET rate by varying the location of the donor molecule, D, relative to the AuNP while maintaining a fixed location of the acceptor molecule, A. The presence of the AuNP induces an alteration in the spontaneous emission of the donor (including radiative and non-radiative rates) which is strongly dependent on the distance between the donor and AuNP surface. Simultaneously, the energy transfer rates are enhanced at shorter D-A (and D-AuNP) distances. Overall, in addition to the direct influence of the acceptor and AuNP on the donor decay there is also a significant increase in decay rate not explained by the sum of the two interactions. This leads to enhanced energy transfer between donor and acceptor in the presence of a 5 nm AuNP. We also demonstrate that the transfer rate in the three "particle" geometry (D + A + AuNP) depends approximately linearly on the transfer rate in the donor-AuNP system, suggesting the possibility to control FRET process with electric field induced by 5 nm AuNPs close to the donor fluorophore. It is concluded that DNA origami is a very versatile platform for studying interactions between molecules and plasmonic nanoparticles in general and FRET enhancement in particular.

  17. Time-Resolved Analysis of a Highly Sensitive Förster Resonance Energy Transfer Immunoassay Using Terbium Complexes as Donors and Quantum Dots as Acceptors

    Directory of Open Access Journals (Sweden)

    Niko Hildebrandt


    Full Text Available CdSe/ZnS core/shell quantum dots (QDs are used as efficient Förster Resonance Energy Transfer (FRET acceptors in a time-resolved immunoassays with Tb complexes as donors providing a long-lived luminescence decay. A detailed decay time analysis of the FRET process is presented. QD FRET sensitization is evidenced by a more than 1000-fold increase of the QD luminescence decay time reaching ca. 0.5 milliseconds, the same value to which the Tb donor decay time is quenched due to FRET to the QD acceptors. The FRET system has an extremely large Förster radius of approx. 100 Å and more than 70% FRET efficiency with a mean donor-acceptor distance of ca. 84 Å, confirming the applied biotin-streptavidin binding system. Time-resolved measurement allows for suppression of short-lived emission due to background fluorescence and directly excited QDs. By this means a detection limit of 18 attomol QDs within the immunoassay is accomplished, an improvement of more than two orders of magnitude compared to commercial systems.

  18. Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA. (United States)

    Bag, Subhendu Sekhar; Jana, Subhashis; Pradhan, Manoj Kumar


    The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO-LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor-acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II β-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor-acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide-protein interaction.

  19. Development of an AP-FRET based analysis for characterizing RNA-protein interactions in myotonic dystrophy (DM1.

    Directory of Open Access Journals (Sweden)

    Shagufta Rehman

    Full Text Available Förster Resonance Energy Transfer (FRET microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1 is caused by a (CTGn repeat expansion in the 3' UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1. In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

  20. Axisymmetric fretting analysis in coated cylinder

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha


    Fretting is essentially a contact fatigue phenomenon, although bulk stresses and material properties contribute to final failure. The near surface state of stress developed under oscillatory contact between machine elements plays a major role in deciding the severity of fretting. It is possible to enhance tribological properties by coating the surface. There is rather scanty literature available on fretting analysis of coated components. Presence of such coatings has a large influence on the near surface state of stress. The effect of coatings on the severity of fretting is the focus of this paper. Results obtained for both hard and soft coatings are compared with the results obtained for the homogeneous case. The component geometry and loading are chosen to be cylindrical to enable 3D elastic axisymmetric fretting analysis. The results are compared with 2D models (strip and half-plane) to examine their utility and validity for understanding axisymmetric fretting. Contact pressure and frictional shear loading cases are solved separately and superposed appropriately depending on the coefficient of friction considered. Results for different values of coefficient of friction and elastic mismatch are illustrated through contour plots of stresses and strains. These results are expected to be helpful for identifying fretting failure zones and fracture mechanisms in coated components. Analytical results presented here could serve as useful benchmarks for calibrating numerical codes and experimental techniques.

  1. Understanding and modeling Förster-type resonance energy transfer (FRET) introduction to FRET

    CERN Document Server

    Govorov, Alexander; Demir, Hilmi Volkan


    This Brief presents a historical overview of the Förster-type nonradiative energy transfer and a compilation of important progress in FRET research, starting from Förster until today, along with a summary of the current state-of-the-art. Here the objective is to provide the reader with a complete account of important milestones in FRET studies and FRET applications as well as a picture of the current status.

  2. Coverage-dependent changes of cytochrome c transverse location in phospholipid membranes revealed by FRET. (United States)

    Domanov, Yegor A; Molotkovsky, Julian G; Gorbenko, Galyna P


    The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.

  3. A graphene quantum dot-based FRET system for nuclear-targeted and real-time monitoring of drug delivery. (United States)

    Chen, Hui; Wang, Zhuyuan; Zong, Shenfei; Chen, Peng; Zhu, Dan; Wu, Lei; Cui, Yiping


    A graphene quantum dot-based FRET system is demonstrated for nuclear-targeted drug delivery, which allows for real-time monitoring of the drug release process through FRET signals. In such a system, graphene quantum dots (GQDs) simultaneously serve as the carriers of drugs and donors of FRET pairs. Additionally, a peptide TAT as the nuclear localization signal is conjugated to GQDs, which facilitates the transportation of the delivery system to the nucleus. We have demonstrated that: (a) both the conjugated TAT and small size of GQDs contribute to targeting the nucleus, which results in a significantly enhanced intranuclear accumulation of drugs; (b) FRET signals being extremely sensitive to the distance between donors and acceptors are capable of real-time monitoring of the separation process of drugs and GQDs, which is more versatile in tracking the drug release dynamics. Our strategy for the assembly of a FRET-based drug delivery system may be unique and universal for monitoring the dynamic release process. This study may give more exciting new opportunities for improving the therapeutic efficacy and tracking precision.

  4. Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements.

    Directory of Open Access Journals (Sweden)

    Gil Rahamim

    Full Text Available Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM, and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.

  5. FRET Sensor for Erythrosine Dye Based on Organic Nanoparticles: Application to Analysis of Food Stuff. (United States)

    Mahajan, Prasad G; Bhopate, Dhanaji P; Kolekar, Govind B; Patil, Shivajirao R


    An aqueous suspension of fluorescent nanoparticles (PHNNPs) of naphthol based fluorescent organic compound 1-[(Z)-(2-phenylhydrazinylidene) methyl] naphthalene -2-ol (PHN) were prepared using reprecipitation method shows bathochromically shifted aggregation induced enhanced emission (AIEE) in the spectral region where erythrosine (ETS) food dye absorbs strongly. The average size of 72.6 nm of aqueous suspension of PHNNPs obtained by Dynamic light scattering results shows a narrow particle size distribution. The negative zeta potential of nano probe (-22.6 mV) responsible to adsorb oppositely charged analyte on its surface and further permit to bind nano probe and analyte within the close distance proximity required for efficient fluorescence resonance energy transfer (FRET) to take place from donor (PHNNPs) to acceptor (ETS). Systematic FRET experiments performed by measuring fluorescence quenching of PHNNPs with successive addition of ETS solution exploited the use of the PHNNPs as a novel nano probe for the detection of ETS in aqueous solution with extremely lower limit of detection equal to 3.6 nM (3.1 ng/mL). The estimation of photo kinetic and thermodynamic parameters such as quenching rate constant, enthalpy change (∆H), Gibbs free energy change (∆G) and entropy change (∆S) was obtained by the quenching results obtained at different constant temperatures which were found to fit the well-known Stern-Volmer relation. The mechanism of binding and fluorescence quenching of PHNNPs by ETS food dye is proposed on the basis of results obtained in photophysical studies, thermodynamic parameter, energy transfer efficiency, critical energy transfer distance (R0) and distance of approach between donor-acceptor molecules (r). The proposed FRET method based on fluorescence quenching of PHNNPs was successfully applied to develop an analytical method for estimation of ETS from food stuffs without interference of other complex ingredients. Graphical Abstract A

  6. Improving Photoconductance of Fluorinated Donors with Fluorinated Acceptors

    Energy Technology Data Exchange (ETDEWEB)

    Garner, Logan E.; Larson, Bryon; Oosterhout, Stefan; Owczarczyk, Zbyslaw; Olson, Dana C.; Kopidakis, Nikos; Boltalina, Olga V.; Strauss, Steven H.; Braunecker, Wade A.


    This work investigates the influence of fluorination of both donor and acceptor materials on the generation of free charge carriers in small molecule donor/fullerene acceptor BHJ OPV active layers. A fluorinated and non-fluorinated small molecule analogue were synthesized and their optoelectronic properties characterized. The intrinsic photoconductance of blends of these small molecule donors was investigated using time-resolved microwave conductivity. Blends of the two donor molecules with a traditional non-fluorinated fullerene (PC70BM) as well as a fluorinated fullerene (C60(CF3)2-1) were investigated using 5% and 50% fullerene loading. We demonstrate for the first time that photoconductance in a 50:50 donor:acceptor BHJ blend using a fluorinated fullerene can actually be improved relative to a traditional non-fluorinated fullerene by fluorinating the donor molecule as well.

  7. Förster Resonance Energy Transfer (FRET between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

    Directory of Open Access Journals (Sweden)

    Radek Šachl


    Full Text Available The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D and acceptor (A molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10 for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

  8. Action-FRET of a Gaseous Protein (United States)

    Daly, Steven; Knight, Geoffrey; Halim, Mohamed Abdul; Kulesza, Alexander; Choi, Chang Min; Chirot, Fabien; MacAleese, Luke; Antoine, Rodolphe; Dugourd, Philippe


    Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question—and thus further develop mass spectrometry as a tool for structural biology—structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state—where a systematic decrease is observed—and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization.

  9. Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes. (United States)

    Briggman, Kevin L; Kristan, William B; González, Jesús E; Kleinfeld, David; Tsien, Roger Y


    Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

  10. Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases. (United States)

    Bienert, Roland; Zimmermann, Boris; Rombach-Riegraf, Verena; Gräber, Peter


    H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

  11. Imaging of caspase-3 activation by a novel FRET probe composed of CFP and DsRed (United States)

    Lin, Juquiang; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming


    Caspases-3 is a kind of cysteine proteases and plays an important role in cell apoptosis. It has been reported that caspase-3 activation can be real-time detected in living cells by fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein and enhanced yellow fluorescent protein. However, the large spectral overlap between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) emission and the highly sensitivity to pH of YFP restricted their detecting sensitivity and reliability. CFP and red fluorescent protein (DsRed) possess superb wavelength separation of donor and acceptor emission spectra and DsRed was insensitive to pH, so the FRET probe composed of CFP and DsRed would be more suitable for imaging caspase-3 activation than the FRET probe composed of CFP and YFP. We constructed a vector that encoded CRS (caspase-3 recognition site) fused with CFP and DsRed (CFP-CRS-DsRed). In CFP-CRS-DsRed expressing tumor cells, FRET from CFP to DsRed could be detected. In the Clinical applications of cancer chemotherapy, cisplatin is one of the most broadly used drugs. It was already confirmed that caspase-3 was activated in HeLa cell treated by cisplatin. When the cells were stimulated with cisplatin, we found that the FRET efficient was remarkably decreased and then disappeared. It indicated that actived caspase-3 cleaved the CFP-CRS-DsRed fusion protein at CRS site. Thus, the FRET probe of CFP-CRS-DsRed could sensitively and reliably monitor caspase-3 activation in living cell. This probe will be highly useful for rapid-screening potential drugs that may target the apoptotic process and for imaging tumors in vivo.

  12. Single Molecule Sensitive FRET in Attoliter Droplets

    CERN Document Server

    Milas, Peker; Gamari, Ben D; Goldner, Lori S


    Single molecular-pair fluorescence resonance energy transfer (spFRET) has become an cross-disciplinary tool for understanding molecular folding and interactions. While providing detailed information about the individual members of a molecular ensemble, this technique is always limited by fluorophore brightness and stability. In the case of diffusing molecules, the experiment is further limited by the number of photons that can be collected during the time it takes for a molecule to diffuse across the detection volume. To maximize the number of photons it is common to either increase the detection volume at the expense of increased background, or increase the diffusion time by adding glycerol or sucrose to increase viscosity. Here we demonstrate that FRET from attoliter volume (100 nm radius) aqueous droplets in perfluorinated oil has significantly higher signal-to-noise and a much wider dynamic range than FRET from molecules diffusing in solution. However, our measurements also reveal a droplet environment th...

  13. The 1974 National Acceptors Survey. (United States)

    Laing, J; Phillips, J; Zablan, Z; Llorente, R; Cabigon, J


    The 1974 National Acceptors Survey in the Philippines studied 4 methods of contraception: pill, IUD, rhythm, and condom. After 1 year, 72% of IUD acceptors had an IUD in place but only 29% of condom acceptors were still using condoms. Pills and rhythm were equally effective in terms of continuation and pregnancy rates. Continuation rates were higher among acceptors at postpartum clinics than at other clnics, higher among urban respondents than rural, and higher among older respondents. Those with more children had higher continuation rates, whereas those who wanted more children had lower continuation rates. Continuation rates also increased 1) as the duration of marriage lengthened; 2) with a later age at marriage; 3) with higher educational attainment; 4) among income-contributing respondents with higher incomes; 5) among previous contraceptive users; and 6) when physicians provided the services rather than nonmedical personnel, including medical screenings. Clinic attendance, husband's occupation, whether or not there was payment, and husband's support are other factors that seemed to affect continuation rates. Contraceptive effectiveness values were higher among pill and IUD acceptors. The percentage of reduction in fertility following acceptance of a method was 74% for IUDs and 27% for condoms. Fertility reduction was great among acceptors at postpartum clinics, acceptors in central visayas, and the highly educated. Future births averted ranged, for every 100 acceptors, 208 with the IUD, 32 with the condom, and 1 each pill and rhythm. Factors affecting method selection were administrative, beliefs, and preferences. The most common complaint about provision of clinical services was that the staff should spend more time in home visits (86%).

  14. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  15. Comparative Fluorescence Resonance Energy-Transfer Study in Pluronic Triblock Copolymer Micelle and Niosome Composed of Biological Component Cholesterol: An Investigation of Effect of Cholesterol and Sucrose on the FRET Parameters. (United States)

    Roy, Arpita; Kundu, Niloy; Banik, Debasis; Sarkar, Nilmoni


    The formation of pluronic triblock copolymer (F127)-cholesterol-based niosome and its interaction with sugar (sucrose) molecules have been investigated. The morphology of F127-cholesterol -based niosome in the presence of sucrose has been successfully demonstrated using dynamic light scattering (DLS) and transmission electron microscopic (TEM) techniques. The DLS profiles and TEM images clearly suggest that the size of the niosome aggregates increases significantly in the presence of sucrose. In addition to structural characterization, a detailed comparative fluorescence resonance energy transfer (FRET) study has been carried out in these F127-containing aggregates, involving coumarin 153 (C153) as donor (D) and rhodamine 6G (R6G) as an acceptor (A) to monitor the dynamic heterogeneity of the systems. Besides, time-resolved anisotropy and fluorescence correlation spectroscopy measurements have been carried out to monitor the rotational and lateral diffusion motion in these F127-cholesterol-based aggregates using C153 and R6G, respectively. During the course of FRET study, we have observed multiple time constants of FRET inside the F127-cholesterol-based niosomes in contrast with the F127 micelle. This corresponds to the presence of more than one preferential donor-acceptor (D-A) distance in niosomes than in F127 micelle. FRET has also been successfully used to probe the effect of sucrose on the morphology of F127-cholesterol-based niosome. In the presence of sucrose, the time constant of FRET further increases as the D-A distances increase in sucrose-decorated niosome. Finally, the excitation-wavelength-dependent FRET studies have indicated that as the excitation of donor molecules varies from 408 to 440 nm the contribution of the faster rise component of the acceptor enhances considerably, which clearly establishes the dynamics heterogeneity of both systems. Our findings also indicate that FRET is completely intravesicular in nature in these block copolymer

  16. A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

    Directory of Open Access Journals (Sweden)

    Jakobus van Unen

    Full Text Available G-protein coupled receptors (GPCRs can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

  17. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

    Directory of Open Access Journals (Sweden)

    Dipankar Bhattacharya

    Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

  18. Acceptors in ZnO

    Energy Technology Data Exchange (ETDEWEB)

    Mccluskey, Matthew D.; Corolewski, Caleb; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T.; Walter, Eric D.; Norton, M. G.; Harrison, Kale W.; Ha, Su Y.


    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence shows that these point defects have acceptor levels 3.2, 1.5, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO2 contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals has been attributed to an acceptor, which may involve a zinc vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g = 2.0033 and g = 2.0075, along with an isotropic center at g = 2.0053.

  19. Acceptors in ZnO

    Energy Technology Data Exchange (ETDEWEB)

    McCluskey, Matthew D., E-mail:; Corolewski, Caleb D.; Lv, Jinpeng; Tarun, Marianne C.; Teklemichael, Samuel T. [Department of Physics and Astronomy, Washington State University, Pullman, Washington 99164-2814 (United States); Walter, Eric D. [Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 (United States); Norton, M. Grant; Harrison, Kale W. [School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164-2920 (United States); Ha, Su [Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164-6515 (United States)


    Zinc oxide (ZnO) has potential for a range of applications in the area of optoelectronics. The quest for p-type ZnO has focused much attention on acceptors. In this paper, Cu, N, and Li acceptor impurities are discussed. Experimental evidence indicates these point defects have acceptor levels 3.2, 1.4, and 0.8 eV above the valence-band maximum, respectively. The levels are deep because the ZnO valence band is quite low compared to conventional, non-oxide semiconductors. Using MoO{sub 2} contacts, the electrical resistivity of ZnO:Li was measured and showed behavior consistent with bulk hole conduction for temperatures above 400 K. A photoluminescence peak in ZnO nanocrystals is attributed to an acceptor, which may involve a Zn vacancy. High field (W-band) electron paramagnetic resonance measurements on the nanocrystals revealed an axial center with g{sub ⊥} = 2.0015 and g{sub //} = 2.0056, along with an isotropic center at g = 2.0035.

  20. Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters (United States)

    Prajapati, Roopali; Chatterjee, Surajit; Kannaujiya, Krishna K.; Mukherjee, Tushar Kanti


    The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence of cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) and Calf Thymus DNA (CT-DNA) have been demonstrated using steady-state fluorescence and femtosecond fluorescence upconversion techniques. The synthesized Ag NCs were characterized using FTIR, mass spectrometry, XPS, HRTEM, DLS, UV-Vis and PL spectroscopy. Mass spectrometric analysis reveals the formation of ultrasmall Ag4 NCs with a small amount of Ag5 NCs. UV-Vis and PL spectra reveal distinct molecular-like optoelectronic behaviour of these ultrasmall Ag NCs. The dihydrolipoic acid-capped Ag NCs strongly quench the fluorescence of DAPI with concomitant increase in its photoluminescence (PL) intensity at 675 nm. This steady-state fluorescence quenching proceeds with a significant shortening of the fluorescence lifetime of DAPI in the presence of Ag NCs, signifying the nonradiative Förster resonance energy transfer (FRET) from DAPI to Ag NCs. Various energy transfer parameters have been estimated from FRET theory. The present FRET pair shows a characteristic Förster distance of 2.45 nm and can be utilized as a reporter of short-range distances in various FRET based applications. Moreover, this nonradiative FRET is completely suppressed in the presence of both 0.2 wt% PDADMAC and CT-DNA. Our results reveal selective compartmentalization of Ag NCs and DAPI in the presence of 0.2 wt% PDADMAC and CT-DNA, respectively. This selective compartmentalization of donor and acceptor and the subsequent modification of the FRET process may find application in various sensing, photovoltaic, and light harvesting applications.The mechanism and dynamics of excitation energy transfer (EET) from photo-excited 4',6-diamidino-2-phenylindole (DAPI) to silver nanoclusters (Ag NCs) and its subsequent modulation in the presence


    Directory of Open Access Journals (Sweden)

    Szilárd Tamás Vezér


    Full Text Available Due to the industrial demand, the determination of the wear behaviour of polymeric materials is an important research task. Rubbers and elastomers are used widely in contacts, where wear is the dominant failure mechanisms. Furthermore, only the material properties under large displacements were investigated in the majority of existing studies. Depending on the input physical parameters of the tribological systems small oscillations are also observed on the measured signals (due to stick-slip like effect in the contact zone of the elastomers. To describe the failure behaviour under this special condition, a novel fretting fatigue test system was developed and built on a electro-dynamical shaker in this study. The contact area were defined with some additional test, like 2D full field strain analysis, compression and creep tests. Based on the methodology developed and applied for steels and polymer composites, Running Condition Fretting Maps for two elastomer grades (HNBR and TPU were determined.

  2. The role of oxidation in the fretting wear process (United States)

    Bill, R. C.


    Fretting experiments were conducted on titanium, a series of Ni-Cr-Al alloys and on some high temperature turbine alloys at room temperature and at elevated temperatures in air and in various inert environments. It was found that, depending on temperature and environment, the fretting behavior of the materials examined could be classified according to four general types of behavior. Briefly, these types of behavior were: (1) the complete absence of oxidation, as in inert environments, generally leading to low rates of fretting wear but high fretting friction; (2) gradual attrition of surface oxide with each fretting stroke, found in these experiments to operate in concert with other dominating mechanisms; (3) rapid oxidation at surface fatigue damage sites, resulting in undermining and rapid disintegration of the load bearing surface; and (4) the formation of coherent, protective oxide film, resulting in low rates of fretting wear. An analytical model predicting conditions favorable to the fourth type of behavior was outlined.

  3. Fretting wear behavior of AZ91D magnesium alloy

    Institute of Scientific and Technical Information of China (English)

    CHEN An-hua; HUANG Wei-jiu; LI Zhao-feng


    The fretting behaviour of the AZ91D magnesium alloy was investigated. The influence of the number of cycles, normal load (contact pressure) and the amplitude of slip on the fretting behavior of the material were focused. Fretting tests were performed under various running conditions with regard to normal load levels and slip amplitudes. The friction coefficient between the surfaces at the fretting junction was continuously recorded. The fretting damage on the magnesium specimens was studied by SEM. The results show that the wear volume increases with the increase of slip amplitude, and linearly increases with the increase of normal load in the mixed and gross slip regime, but the normal load has no obvious effect on the wear volume in the partial slip regime. The predominant fretting wear mechanism of magnesium alloy in the slip regime is the oxidation wear, delaminated wear and abrasive wear.

  4. Discrimination between FRET and non-FRET quenching in a photochromic CdSe quantum dot/dithienylethene dye system (United States)

    Dworak, Lars; Reuss, Andreas J.; Zastrow, Marc; Rück-Braun, Karola; Wachtveitl, Josef


    A photochromic Förster resonance energy transfer (FRET) system was employed to disentangle the fluorescence quenching mechanisms in quantum dot/photochromic dye hybrids. In the off-state of the dye the main quenching mechanism is FRET whereas the moderate quenching in the on-state is due to non-FRET pathways opened up upon assembly.A photochromic Förster resonance energy transfer (FRET) system was employed to disentangle the fluorescence quenching mechanisms in quantum dot/photochromic dye hybrids. In the off-state of the dye the main quenching mechanism is FRET whereas the moderate quenching in the on-state is due to non-FRET pathways opened up upon assembly. Electronic supplementary information (ESI) available: QD and DTE synthesis, preparation of the DTE/QD coupled system, TEM image of the nanocrystals and experimental details. See DOI: 10.1039/c4nr05144k

  5. Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, Evgenia G., E-mail: [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States); Gryczynski, Zygmunt [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States); Stewart, Donald R. [Omm Scientific, Inc., 2600 N. Stemmons Freeway, Suite 129, Dallas, TX 75207 (United States); Gryczynski, Ignacy [Center for Commercialization of Fluorescence Technologies, University of North Texas, Health Science Center, Department of Molecular Biology and Immunology and Department of Cell Biology and Genetics, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (United States)


    A new FRET-based method for the ratiometric detection of DNA oligomers on a surface using TIRF detection mode is reported. The dual-labeled system consisting of two hybridized oligomers, Cy3oligoY:Cy5oligoX was immobilized on the surface, and the total internal reflection fluorescence (TIRF) was used to detect emission signals from the surface. Two signals, green and red, which originated from the green donor Cy3 and the red acceptor Cy5, have been simultaneously detected. When the target single-stranded complimentary oligomer was present in the solution, this oligomer replaced the Cy3oligoY in the donor:acceptor complex on the surface and the ratio of red-to-green signal was dramatically changed. This detection scheme is generally applicable to the detection of DNA or RNA on a surface.

  6. Analysis of caspase-3 in ASTC-a-1 cells treated with mitomycin C using acceptor photobleaching techniques (United States)

    Wang, Huiying; Chen, Tongsheng; Sun, Lei


    Caspase-3 is a key activated death protease, which catalyzes the specific cleavage of many cellular proteins and induces DNA cleavage eventually. In this report, cells were treated with mitomycin C (MMC) at different concentration and its activity was detected by cell counting kit (CCK-8). Based on results of CCK-8, cells were treated with 10μg/mL MMC and Hoechst 33258 has been used to observe cell apoptosis. Fluorescence resonance energy transfer (FRET) and confocal microscopy have been used to the effect of MMC on the caspase3 activation in living cells. Human lung adenocarcinoma cells (ASTC-a-1) was transfected with plasmid SCAT3 (pSCAT3)/CKAR FRET receptor. Acceptor photobleaching techniques of FRET plasmid has been used to destruct fluorophore of cells stably expressing SCAT3 reporter on a fluorescence confocal microscope. The activity of caspase3 can be analyzed by FRET dynamics of SCAT3 in living cells. Our results show that MM C can induce ASTC-a-1 cell apoptosis through activation of caspase3.

  7. Photoswitchable semiconductor nanocrystals with self-regulating photochromic Förster resonance energy transfer acceptors (United States)

    Díaz, Sebastián A.; Gillanders, Florencia; Jares-Erijman, Elizabeth A.; Jovin, Thomas M.


    Photoswitchable molecules and nanoparticles constitute superior biosensors for a wide range of industrial, research and biomedical applications. Rendered reversible by spontaneous or deterministic means, such probes facilitate many of the techniques in fluorescence microscopy that surpass the optical resolution dictated by diffraction. Here we have devised a family of photoswitchable quantum dots (psQDs) in which the semiconductor core functions as a fluorescence donor in Förster resonance energy transfer (FRET), and multiple photochromic diheteroarylethene groups function as acceptors upon activation by ultraviolet light. The QDs were coated with a polymer bearing photochromic groups attached via linkers of different length. Despite the resulting nominal differences in donor-acceptor separation and anticipated FRET efficiencies, the maximum quenching of all psQD preparations was 38±2%. This result was attributable to the large ultraviolet absorption cross-section of the QDs, leading to preferential cycloreversion of photochromic groups situated closer to the nanoparticle surface and/or with a more favourable orientation.

  8. Photoswitchable semiconductor nanocrystals with self-regulating photochromic Förster resonance energy transfer acceptors. (United States)

    Díaz, Sebastián A; Gillanders, Florencia; Jares-Erijman, Elizabeth A; Jovin, Thomas M


    Photoswitchable molecules and nanoparticles constitute superior biosensors for a wide range of industrial, research and biomedical applications. Rendered reversible by spontaneous or deterministic means, such probes facilitate many of the techniques in fluorescence microscopy that surpass the optical resolution dictated by diffraction. Here we have devised a family of photoswitchable quantum dots (psQDs) in which the semiconductor core functions as a fluorescence donor in Förster resonance energy transfer (FRET), and multiple photochromic diheteroarylethene groups function as acceptors upon activation by ultraviolet light. The QDs were coated with a polymer bearing photochromic groups attached via linkers of different length. Despite the resulting nominal differences in donor-acceptor separation and anticipated FRET efficiencies, the maximum quenching of all psQD preparations was 38±2%. This result was attributable to the large ultraviolet absorption cross-section of the QDs, leading to preferential cycloreversion of photochromic groups situated closer to the nanoparticle surface and/or with a more favourable orientation.

  9. Single molecule FRET data analysis procedures for FRET efficiency determination: probing the conformations of nucleic acid structures. (United States)

    Krüger, Asger Christian; Birkedal, Victoria


    Single molecule FRET microscopy is an attractive technique for studying structural dynamics and conformational diversity of nucleic acid structures. Some of its strengths are that it can follow structural changes on a fast time scale and identify conformation distributions arising from dynamic or static population heterogeneity. Here, we give a description of the experiment and data analysis procedures of this method and detail what parameters are needed for FRET efficiency calculation. Using single molecule FRET data obtained on G-quadruplex DNA structures that exhibit large conformation diversity, we illustrate that the shape of the FRET distribution changes depending on what parameters are included in the data analysis procedure.

  10. In silico FRET from simulated dye dynamics (United States)

    Hoefling, Martin; Grubmüller, Helmut


    Single molecule fluorescence resonance energy transfer (smFRET) experiments probe molecular distances on the nanometer scale. In such experiments, distances are recorded from FRET transfer efficiencies via the Förster formula, E=1/(1+(). The energy transfer however also depends on the mutual orientation of the two dyes used as distance reporter. Since this information is typically inaccessible in FRET experiments, one has to rely on approximations, which reduce the accuracy of these distance measurements. A common approximation is an isotropic and uncorrelated dye orientation distribution. To assess the impact of such approximations, we present the algorithms and implementation of a computational toolkit for the simulation of smFRET on the basis of molecular dynamics (MD) trajectory ensembles. In this study, the dye orientation dynamics, which are used to determine dynamic FRET efficiencies, are extracted from MD simulations. In a subsequent step, photons and bursts are generated using a Monte Carlo algorithm. The application of the developed toolkit on a poly-proline system demonstrated good agreement between smFRET simulations and experimental results and therefore confirms our computational method. Furthermore, it enabled the identification of the structural basis of measured heterogeneity. The presented computational toolkit is written in Python, available as open-source, applicable to arbitrary systems and can easily be extended and adapted to further problems. Catalogue identifier: AENV_v1_0 Program summary URL: Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GPLv3, the bundled SIMD friendly Mersenne twister implementation [1] is provided under the SFMT-License. No. of lines in distributed program, including test data, etc.: 317880 No. of bytes in distributed program, including test data, etc.: 54774217 Distribution format: tar.gz Programming language

  11. Structural dynamics of potassium-channel gating revealed by single-molecule FRET. (United States)

    Wang, Shizhen; Vafabakhsh, Reza; Borschel, William F; Ha, Taekjip; Nichols, Colin G


    Crystallography has provided invaluable insights regarding ion-channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites and then examined structural dynamics within lipid membranes by single-molecule fluorescence resonance energy transfer (FRET). We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states, both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and in cation channels in general.

  12. Structural dynamics of potassium channel gating revealed by single molecule FRET (United States)

    Borschel, William F.; Ha, Taekjip; Nichols, Colin G.


    Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general. PMID:26641713

  13. Development of FRET-Based Assays in the Far-Red Using CdTe Quantum Dots

    Directory of Open Access Journals (Sweden)

    E. Z. Chong


    Full Text Available Colloidal quantum dots (QDs are now commercially available in a biofunctionalized form, and Förster resonance energy transfer (FRET between bioconjugated dots and fluorophores within the visible range has been observed. We are particularly interested in the far-red region, as from a biological perspective there are benefits in pushing to ∼700 nm to minimize optical absorption (ABS within tissue and to avoid cell autofluorescence. We report on FRET between streptavidin- (STV- conjugated CdTe quantum dots, Qdot705-STV, with biotinylated DY731-Bio fluorophores in a donor-acceptor assay. We also highlight the changes in DY731-Bio absorptivity during the streptavidin-biotin binding process which can be attributed to the structural reorientation. For fluorescence beyond 700 nm, different alloy compositions are required for the QD core and these changes directly affect the fluorescence decay dynamics producing a marked biexponential decay with a long-lifetime component in excess of 100 nanoseconds. We compare the influence of the two QD relaxation routes upon FRET dynamics in the presence of DY731-Bio.

  14. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM (United States)

    Demeautis, Claire; Sipieter, François; Roul, Julien; Chapuis, Catherine; Padilla-Parra, Sergi; Riquet, Franck B.; Tramier, Marc


    Monitoring of different signalling enzymes in a single assay using multiplex biosensing provides a multidimensional workspace to elucidate biological processes, signalling pathway crosstalk, and determine precise sequence of events at the single living cell level. In this study, we interrogate the complexity in cAMP/PKA-MAPK/ERK1&2 crosstalk by using multi-parameter biosensing experiments to correlate biochemical activities simultaneously in time and space. Using a single excitation wavelength dual colour FLIM method we are able to detect fluorescence lifetime images of two donors to simultaneously measure PKA and ERK1&2 kinase activities in the same cellular localization by using FRET biosensors. To this end, we excite two FRET donors mTFP1 and LSSmOrange with a 440 nm wavelength and we alleviate spectral bleed-through associated limitations with the very dim-fluorescent acceptor ShadowG for mTFP1 and the red-shifted mKate2 for LSSmOrange. The simultaneous recording of PKA and ERK1&2 kinase activities reveals concomitant EGF-mediated activations of both kinases in HeLa cells. Under these conditions the subsequent Forskolin-induced cAMP release reverses the transient increase of EGF-mediated ERK1&2 kinase activity while reinforcing PKA activation. Here we propose a validated methodology for multiparametric kinase biosensing in living cells using FRET-FLIM. PMID:28106114

  15. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

    Directory of Open Access Journals (Sweden)

    Potzkei Janko


    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  16. Fret Replica Inspection Laser Scanner (FRILS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretz, S.; Hanley, K., E-mail:, E-mail: [Ontario Power Generation, Inspection Maintenance and Commercial Services, Pickering, Ontario (Canada)


    In the stress analysis of flaws and artifacts found in pressure tubes, it is crucial to have detailed knowledge of the flaw geometry. Fuel channel inspections by ultrasonic or eddy current inspection methods alone cannot provide the complete required geometry information. Replicas, which are a negative impression of surface pressure tube indications, are scanned with a laser system which will provide the additional detail required. FRILS was initially developed in 1993 to establish in-house capability of profiling indications on the inside diameter surface of pressure tubes. The need of this profiling was initially a response to the discovery of fuel bundle bearing pad fretting (FBBPF) caused by flow induced fuel bundle vibration. The benefits of the system were soon realized as a tool for profiling debris type indications. Although the primary use of FRILS is to profile FBBBF and Debris Fretting, since its inception the FRILS inspection system has become an instrumental tool in flaw assessment for: Fuel Bundle Bearing Pad Frets (FBBPF); Debris Frets; Scratches; Crevice Corrosion; Oxide Jacking; Areas of surface roughness; and, Weld Profiling. Replicas are collected via acquisition from tooling on both the Channel and Gauging Apparatus for Reactors (CIGAR) and the Advanced Non-Destructive Examination (ANDE) systems. The ANDE system is a high speed data acquisition system which includes both an ultrasonic inspection tool and a replication tool. Although both of these tools were designed to be delivered with the UDM, the platform for these tools was built with flexibility allowing for adoption to other delivery systems. These tools were based on the experience of the CIGAR inspection system. The CIGAR system has also undergone many system upgrades resulting in reduced inspection times. The FRILS system - Fret Replication Inspection Laser Scanner system was developed and has been upgraded to meet the demands of the improved inspection and replication systems. FRILS

  17. Förster Resonance Energy Transfer (FRET Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2

    Directory of Open Access Journals (Sweden)

    Dennis A. Dougherty


    Full Text Available We provide a theory for employing Förster resonance energy transfer (FRET measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET for the two predominant stoichiometries, (α43(β22 vs. (α42(β23. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α43(β22 exceeds that of (α42(β23. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α43(β22 stoichiometry to mostly (α42(β23. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α42(β23 and (α43(β22 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.


    Directory of Open Access Journals (Sweden)

    Stefan GHIMIȘI


    Full Text Available The fretting phenomenon represents particulary and complex form of wear who is; generaly, and/or weary of fretting who is produced on the load contact in a relative oscialatory movement lay small amplitude.A simultaneoustly applied tangential force and normal into contact appears a adhesion force

  19. BOBA FRET: bootstrap-based analysis of single-molecule FRET data.

    Directory of Open Access Journals (Sweden)

    Sebastian L B König

    Full Text Available Time-binned single-molecule Förster resonance energy transfer (smFRET experiments with surface-tethered nucleic acids or proteins permit to follow folding and catalysis of single molecules in real-time. Due to the intrinsically low signal-to-noise ratio (SNR in smFRET time traces, research over the past years has focused on the development of new methods to extract discrete states (conformations from noisy data. However, limited observation time typically leads to pronounced cross-sample variability, i.e., single molecules display differences in the relative population of states and the corresponding conversion rates. Quantification of cross-sample variability is necessary to perform statistical testing in order to assess whether changes observed in response to an experimental parameter (metal ion concentration, the presence of a ligand, etc. are significant. However, such hypothesis testing has been disregarded to date, precluding robust biological interpretation. Here, we address this problem by a bootstrap-based approach to estimate the experimental variability. Simulated time traces are presented to assess the robustness of the algorithm in conjunction with approaches commonly used in thermodynamic and kinetic analysis of time-binned smFRET data. Furthermore, a pair of functionally important sequences derived from the self-cleaving group II intron Sc.ai5γ (d3'EBS1/IBS1 is used as a model system. Through statistical hypothesis testing, divalent metal ions are shown to have a statistically significant effect on both thermodynamic and kinetic aspects of their interaction. The Matlab source code used for analysis (bootstrap-based analysis of smFRET data, BOBA FRET, as well as a graphical user interface, is available via

  20. Near surface stress analysis strategies for axisymmetric fretting

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha


    Fretting is essentially a surface phenomenon, but bulk stresses and material properties contribute to subsequent failure. This feature of fretting demands a thorough understanding of near surface stresses under the joint action of normal, shear and thermal loading. Axisymmetric fretting is of great concern in piping and coupling design. In this paper, we develop design tools for Near Surface Analysis (NSA) for understanding axisymmetric fretting. Axisymmetric Fretting Analysis (AFA) becomes formidable owing to localised tractions that call for Fourier transform techniques. We develop two different NSA strategies based on two-dimensional plane strain models: 2D strip model (2DS) and half-plane Flamant model (2DF). We compare the results of 2DS and 2DF with the exact results for AFA obtained using Love’s stress function in conjunction with Fourier transform. There is a good correspondence between stress components obtained from 2D-models.

  1. Ice and debris in the fretted terrain, Mars (United States)

    Lucchitta, B. K.


    Viking moderate and high resolution images along the northern highland margin have been monoscopically and stereoscopically examined in order to study the development of fretted terrain. Young debris aprons around mesas and debris in tributary channels create typical fretted morphologies identical to ancient fretted morphologies. This suggests that the debris-apron process operating relatively recently also shaped the fretted terrain of the past. The debris aprons were lubricated by interstitial ice derived from ground ice. Abundant collapse features suggest that ground ice existed and may have flowed in places. The fretting process has been active for a long period and may be active today. The location of debris aprons in two latitudinal belts may be controlled by atmospheric conditions that permit ice in the region to remain in the ground below depths of about one meter and temperatures warm enough for ice to flow.

  2. Large Scale Bacterial Colony Screening of Diversified FRET Biosensors.

    Directory of Open Access Journals (Sweden)

    Julia Litzlbauer

    Full Text Available Biosensors based on Förster Resonance Energy Transfer (FRET between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.

  3. FRET based quantification and screening technology platform for the interactions of leukocyte function-associated antigen-1 (LFA-1 with intercellular adhesion molecule-1 (ICAM-1.

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    Full Text Available The interaction between leukocyte function-associated antigen-1(LFA-1 and intercellular adhesion molecule-1 (ICAM-1 plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple 'in solution' steady state fluorescence resonance energy transfer (FRET technique to obtain the dissociation constant (Kd of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.

  4. Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

    Directory of Open Access Journals (Sweden)

    Lina Ma


    Full Text Available This work reports on a simple and feasible fluorescence resonance energy transfer (FRET assay for detecting reactive oxygen species (ROS both in solution and living cell using polydopamine nanoparticle (PDA NP as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.

  5. Understanding FRET as a Research Tool for Cellular Studies

    Directory of Open Access Journals (Sweden)

    Dilip Shrestha


    Full Text Available Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET. FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types.

  6. Intonation and Compensation of Fretted String Instruments

    CERN Document Server

    Varieschi, Gabriele U


    In this paper we present mathematical models and we analyze the physics related to the problem of intonation of musical instruments such as guitars, mandolins and similar, i.e., we study how to produce perfectly in tune notes on these instruments. This analysis begins with the correct fret placement on the instrument fingerboard, following precise mathematical laws, but then it becomes increasingly complicated due to the geometrical deformation of the strings when these instruments are played, and also due to the inharmonic characteristics of the same strings. As a consequence of these factors, perfect intonation of all the notes on the instrument can never be achieved, but complex compensation procedures are introduced and studied to minimize the problem. To test the validity of these compensation procedures, we have performed extensive measurements using standard monochord sonometers and other basic acoustical devices, which confirm the correctness of our theoretical models. In particular, these experimenta...

  7. Analogue computing methods

    CERN Document Server

    Welbourne, D


    Analogue Computing Methods presents the field of analogue computation and simulation in a compact and convenient form, providing an outline of models and analogues that have been produced to solve physical problems for the engineer and how to use and program the electronic analogue computer. This book consists of six chapters. The first chapter provides an introduction to analogue computation and discusses certain mathematical techniques. The electronic equipment of an analogue computer is covered in Chapter 2, while its use to solve simple problems, including the method of scaling is elaborat

  8. An Experimental Study of Fretting of Gear Teeth (United States)

    Krantz, Timothy L.


    Experiments were conducted to study fretting of gears. The gears were made from case-carburized AISI 9310 alloy to match the material of a flight actuator gearbox of interest. The objective of the testing was to produce damage representative of that observed on flight hardware. The following correlations and observations were noted. The amplitude of dithering motion very strongly influenced the type and magnitude of damage. Sliding amounts on the order of 30% of the width of the line contact were judged to most readily produce fretting damage. There was observed an incubation period on the order of tens-of-thousands of cycles, and the incubation period was influenced by surface roughness, torque, and the motion extent. Fretting damage could be produced for any of the torques tested, and the severity of damage increased slightly with torque. Gear teeth having surface roughness of 0.7-0.8 micrometer were somewhat more resistant to fretting than were smoother surfaces.

  9. QD-Based FRET Probes at a Glance

    Directory of Open Access Journals (Sweden)

    Armen Shamirian


    Full Text Available The unique optoelectronic properties of quantum dots (QDs give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided.

  10. Fluorescent proteins as genetically encoded FRET biosensors in life sciences. (United States)

    Hochreiter, Bernhard; Garcia, Alan Pardo; Schmid, Johannes A


    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  11. FRET-based identification of mRNAs undergoing translation.

    Directory of Open Access Journals (Sweden)

    Benjamin Stevens

    Full Text Available We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET. With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  12. FRET-based identification of mRNAs undergoing translation. (United States)

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E


    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  13. Indicators based on fluorescence resonance energy transfer (FRET). (United States)

    Tsien, Roger Y


    One of the major new trends in the design of indicators for optically imaging biochemical and physiological functions of living cells has been the exploitation of fluorescence resonance energy transfer (FRET). FRET is a well-known spectroscopic technique for monitoring changes in the proximity and mutual orientation of pairs of chromophores. It has long been used in biochemistry and cell biology to assess distances and orientations between specific labeling sites within a single macromolecule or between two separate molecules. More recently, macromolecules or molecular pairs have been engineered to change their FRET in response to biochemical and physiological signals such as membrane potential, cyclic AMP (cAMP), protease activity, free Ca(2+) and Ca(2+)-calmodulin (CaM) concentrations, protein-protein heterodimerization, phosphorylation, and reporter-gene expression. Because FRET is general, nondestructive, and easily imaged, it has proven to be one of the most versatile spectroscopic readouts available to the designer of new probes. FRET is particularly amenable to emission ratioing, which is more reliably quantifiable than single-wavelength monitoring and better suited than excitation ratioing to high-speed and laser-excited imaging. This article summarizes the photophysical principles of FRET and the types of indicators used.

  14. a Study on the Fretting Fatigue Life of Zircaloy Alloys (United States)

    Kwon, Jae-Do; Park, Dae-Kyu; Woo, Seung-Wan; Chai, Young-Suck

    Studies on the strength and fatigue life of machines and structures have been conducted in accordance with the development of modern industries. In particular, fine and repetitive cyclic damage occurring in contact regions has been known to have an impact on fretting fatigue fractures. The main component of zircaloy alloy is Zr, and it possesses good mechanical characteristics at high temperatures. This alloy is used in the fuel rod material of nuclear power plants because of its excellent resistance. In this paper, the effect of the fretting damage on the fatigue behavior of the zircaloy alloy is studied. Further, various types of mechanical tests such as tension and plain fatigue tests are performed. Fretting fatigue tests are performed with a flat-flat contact configuration using a bridge-type contact pad and plate-type specimen. Through these experiments, it is found that the fretting fatigue strength decreases by about 80% as compared to the plain fatigue strength. Oblique cracks are observed in the initial stage of the fretting fatigue, in which damaged areas are found. These results can be used as the basic data for the structural integrity evaluation of corrosion-resisting alloys considering the fretting damages.

  15. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences (United States)

    Hochreiter, Bernhard; Pardo Garcia, Alan; Schmid, Johannes A.


    Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. PMID:26501285

  16. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

    Directory of Open Access Journals (Sweden)

    Bernhard Hochreiter


    Full Text Available Fluorescence- or Förster resonance energy transfer (FRET is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a cleavage; (b conformational-change; (c mechanical force and (d changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them.

  17. Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements.

    Directory of Open Access Journals (Sweden)

    Bobin George Abraham

    Full Text Available Fluorescence Resonance Energy Transfer (FRET using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM.

  18. Analogue MIMO Detection

    Directory of Open Access Journals (Sweden)

    McNamara Darren


    Full Text Available In this contribution we propose an analogue receiver that can perform turbo detection in MIMO systems. We present the case for a receiver that is built from nonlinear analogue devices, which perform detection in a "free-flow" network (no notion of iterations. This contribution can be viewed as an extension of analogue turbo decoder concepts to include MIMO detection. These first analogue implementations report reductions of few orders of magnitude in the number of required transistors and in consumed energy, and the same order of improvement in processing speed. It is anticipated that such analogue MIMO decoder could bring about the same advantages, when compared to traditional digital implementations.

  19. Hole-transfer induced energy transfer in perylene diimide dyads with a donor-spacer-acceptor motif. (United States)

    Kölle, Patrick; Pugliesi, Igor; Langhals, Heinz; Wilcken, Roland; Esterbauer, Andreas J; de Vivie-Riedle, Regina; Riedle, Eberhard


    We investigate the photoinduced dynamics of perylene diimide dyads based on a donor-spacer-acceptor motif with polyyne spacers of varying length by pump-probe spectroscopy, time resolved fluorescence, chemical variation and quantum chemistry. While the dyads with pyridine based polyyne spacers undergo energy transfer with near-unity quantum efficiency, in the dyads with phenyl based polyyne spacers the energy transfer efficiency drops below 50%. This suggests the presence of a competing electron transfer process from the spacer to the energy donor as the excitation sink. Transient absorption spectra, however, reveal that the spacer actually mediates the energy transfer dynamics. The ground state bleach features of the polyyne spacers appear due to the electron transfer decay with the same time constant present in the rise of the ground state bleach and stimulated emission of the perylene energy acceptor. Although the electron transfer process initially quenches the fluorescence of the donor it does not inhibit energy transfer to the perylene energy acceptor. The transient signatures reveal that electron and energy transfer processes are sequential and indicate that the donor-spacer electron transfer state itself is responsible for the energy transfer. Through the introduction of a Dexter blocker unit into the spacer we can clearly exclude any through bond Dexter-type energy transfer. Ab initio calculations on the donor-spacer and the donor-spacer-acceptor systems reveal the existence of a bright charge transfer state that is close in energy to the locally excited state of the acceptor. Multipole-multipole interactions between the bright charge transfer state and the acceptor state enable the energy transfer. We term this mechanism coupled hole-transfer FRET. These dyads represent a first example that shows how electron transfer can be connected to energy transfer for use in novel photovoltaic and optoelectronic devices.

  20. Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique. (United States)

    Lee, Jiung-De; Chang, Yu-Fen; Kao, Fu-Jen; Kao, Lung-Sen; Lin, Chung-Chih; Lu, Ai-Chu; Shyu, Bai-Chuang; Chiou, Shih-Hwa; Yang, De-Ming


    Exocytosis has been proposed to contain four sequential steps, namely docking, priming, fusion, and recycling, and to be regulated by various proteins-protein interactions. Synaptosomal-associated protein of 25 kDa (SNAP25) has recently been found to bind rabphilin, the Rab3A specific binding protein, in vitro. However, it is still unclear whether SNAP25 and rabphilin interact during exocytosis within cells in vivo. This problem was addressed by the integration of fluorescence resonance energy transfer (FRET) with high sensitivity fluorescence lifetime imaging microscopy (FLIM) to observe this protein-protein interaction. Enhanced green fluorescence protein-labeled SNAP25 (donor) and red fluorescence protein-labeled rabphilin (acceptor) were expressed in neuroendocrine PC12 cells as a FRET pair and ATP stimulation was carried out for various durations. With 10 s stimulation, a 0.17-ns left shift of the lifetime peak was found when compared with donor only. Analysis of the lifetime image further suggested that the lifetime recovered to a similar level as the donor only in a time dependent manner. Four-dimensional (4D) images by FLIM provided useful information indicating that the interaction of SNAP25 and rabphilin occurred particularly within optical sections near cell membrane. Together the results suggest that SNAP25 bound rabphilin loosely at docking step before exocytosis and the binding became tighter at the very start of exocytosis. Finally, these two proteins dissociated after stimulation. To our knowledge, this is the first report to demonstrate the interaction of SNAP25 and rabphilin in situ using the FLIM-FRET technique within neuroendocrine cells.

  1. Broadband Light-Harvesting Molecular Triads with High FRET Efficiency Based on the Coumarin-Rhodamine-BODIPY Platform. (United States)

    He, Longwei; Zhu, Sasa; Liu, Yong; Xie, Yinan; Xu, Qiuyan; Wei, Haipeng; Lin, Weiying


    Broadband capturing and FRET-based light-harvesting molecular triads, CRBs, based on the coumarin-rhodamine-BODIPY platform were rationally designed and synthesized. The absorption band of CRBs starts from blue-green to yellow-orange regions (330-610 nm), covering the strong radiation scope of sunlight. The peripheral coumarin and BODIPY chromophore energy could transfer to the central acceptor rhodamine by a one-step direct way. The energy of the coumarin moiety could also transfer to the BODIPY unit, subsequently transferring to the rhodamine core by two-step sequential ways. Both the efficiencies of the coumarin moiety and the BODIPY unit to the rhodamine core in CRBs, determined by two different ways, are very high.

  2. Determining protein complex structures based on a Bayesian model of in vivo Förster resonance energy transfer (FRET) data. (United States)

    Bonomi, Massimiliano; Pellarin, Riccardo; Kim, Seung Joong; Russel, Daniel; Sundin, Bryan A; Riffle, Michael; Jaschob, Daniel; Ramsden, Richard; Davis, Trisha N; Muller, Eric G D; Sali, Andrej


    The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking.

  3. FRET energy transfer via Pdots improves the efficiency of photodynamic therapy and leads to rapid cell death. (United States)

    Haupt, Sara; Lazar, Itay; Weitman, Hana; Shav-Tal, Yaron; Ehrenberg, Benjamin


    Photodynamic therapy (PDT) is well established as a clinical treatment modality for various diseases, including cancer and especially for the treatment of superficial tumors. However, one of the disadvantages of the photoactivatable molecules is their low absorbance in the optical window for photosensitizer excitation. The use of nanoparticles in photodynamic therapy can address this deficiency and improve treatment efficiency. Pdots are nano-sized particles, composed of conjugated chromophoric polymers. By mixing them with PEGylated phospholipids they can become soluble and stable colloids. They exhibit a broad absorption band with a strong and narrow emission band. In this study, we examined two types of Pdots (MEH-PPV and CN-PPV) with two different lengths of the PEGylated lipids coating, 350 and 2000. When a photosensitizer, such as mTHPC, comes in close contact with the amphiphilic coating of the Pdots, a very efficient fluorescence resonance energy transfer (FRET) occurs between the donor, the Pdots and the acceptor, the sensitizer. This process, together with the significant uptake of the Pdots-sensitizer pair by MCF-7 cancerous cells causes irreversible damage to the cells. This damage is greater when the Pdots are comprised from the CN-PPV polymer and coated with the PEG2000-PE lipid. Altogether, we demonstrate that implementing FRET energy transfer in the PDT protocol leads to quicker and more aggressive cell death, thus improving the efficacy of the photodynamic therapy.

  4. Rational design of a thermalresponsive-polymer-switchable FRET system for enhancing the temperature sensitivity of upconversion nanophosphors (United States)

    Xiao, Qingbo; Li, Yanfang; Li, Fujin; Zhang, Mengxin; Zhang, Zhijun; Lin, Hongzhen


    Here we propose a thermoresponsive polymer PNIPAM modulated fluorescence resonance energy transfer (FRET) system to enhance the temperature sensitivity of upconversion nanophosphors (UCNPs). By utilizing red/near-infrared dual emitting NaLuF4:Mn2+,Ln3+ (Ln3+ = Yb3+, Er3+, Tm3+) UCNPs as the energy donor and Au nanoparticles as the acceptor, the temperature resolution of the UCNPs is significantly increased from 3.1 °C to 0.9 °C in the physiological temperature range. Conjugating the UCNPs and acceptors into discrete nanocomposites in our samples facilitates reversible regulation of the emission intensity of UCNPs, which thus would extend their application range in biosensing, especially for probing the dynamic changes of local micro-environments in biological tissues. As there are a broad variety of stimuli to which smart polymers can reversibly respond, our experiments are also extendable to various external conditions in local micro-environments, such as pH values, metal ions, glucose, and tissue-specific enzymes.Here we propose a thermoresponsive polymer PNIPAM modulated fluorescence resonance energy transfer (FRET) system to enhance the temperature sensitivity of upconversion nanophosphors (UCNPs). By utilizing red/near-infrared dual emitting NaLuF4:Mn2+,Ln3+ (Ln3+ = Yb3+, Er3+, Tm3+) UCNPs as the energy donor and Au nanoparticles as the acceptor, the temperature resolution of the UCNPs is significantly increased from 3.1 °C to 0.9 °C in the physiological temperature range. Conjugating the UCNPs and acceptors into discrete nanocomposites in our samples facilitates reversible regulation of the emission intensity of UCNPs, which thus would extend their application range in biosensing, especially for probing the dynamic changes of local micro-environments in biological tissues. As there are a broad variety of stimuli to which smart polymers can reversibly respond, our experiments are also extendable to various external conditions in local micro

  5. Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap (United States)

    Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Deckers-Hebestreit, Gabriele; Mayer, Günter; Henkel, Thomas; Heitkamp, Thomas; Börsch, Michael


    Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap -- a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using Förster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.

  6. Fluorescence characterization of co-immobilization-induced multi-enzyme aggregation in a polymer matrix using Förster resonance energy transfer (FRET): toward the metabolon biomimic. (United States)

    Wu, Fei; Minteer, Shelley D


    Sequential metabolic enzymes can form supramolecular complexes named metabolons in vivo through enzyme-enzyme association or aggregation to facilitate efficient substrate channeling. By separately labeling enzymes with lysine-targeting carboxylic acid succinimidyl ester fluorophores of distinct excitation wavelengths, this research presents a quantitative study of polymer-entrapment-induced in vitro multi-enzyme aggregation from three Krebs cycle enzymes using Förster resonance energy transfer (FRET) to find potential polymer materials for immobilizing enzyme cascades and inducing the metabolon biomimic formation on electrodes. The effect of hydrophobic modification of linear polyethylenimine, Nafion, and chitosan polymers on metabolon formation has been investigated through photobleaching FRET imaging in addition to traditional steady-state fluorescence spectroscopy. By partially destroying FRET acceptors of longer excitation wavelength, increased fluorescence from dequenched donors of shorter excitation wavelength was measured and enzyme interactions in terms of energy-transfer efficiencies were mapped point by point. Results show that trimethyloctadecylammonium-modified Nafion works best in inducing multi-enzyme aggregation and exhibits a promising future in immobilized metabolon biomimics with the most uniform enzyme organization, as indicated by the protein distance distribution.

  7. Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study (United States)

    Greife, Annemarie; Felekyan, Suren; Ma, Qijun; Gertzen, Christoph G. W.; Spomer, Lina; Dimura, Mykola; Peulen, Thomas O.; Wöhler, Christina; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena; Seidel, Claus A. M.


    TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers – likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

  8. Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer. (United States)

    Hounsou, Candide; Margathe, Jean-François; Oueslati, Nadia; Belhocine, Abderazak; Dupuis, Elodie; Thomas, Cécile; Mann, André; Ilien, Brigitte; Rognan, Didier; Trinquet, Eric; Hibert, Marcel; Pin, Jean-Philippe; Bonnet, Dominique; Durroux, Thierry


    G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.

  9. Expectation-maximization of the potential of mean force and diffusion coefficient in Langevin dynamics from single molecule FRET data photon by photon. (United States)

    Haas, Kevin R; Yang, Haw; Chu, Jhih-Wei


    The dynamics of a protein along a well-defined coordinate can be formally projected onto the form of an overdamped Lagevin equation. Here, we present a comprehensive statistical-learning framework for simultaneously quantifying the deterministic force (the potential of mean force, PMF) and the stochastic force (characterized by the diffusion coefficient, D) from single-molecule Förster-type resonance energy transfer (smFRET) experiments. The likelihood functional of the Langevin parameters, PMF and D, is expressed by a path integral of the latent smFRET distance that follows Langevin dynamics and realized by the donor and the acceptor photon emissions. The solution is made possible by an eigen decomposition of the time-symmetrized form of the corresponding Fokker-Planck equation coupled with photon statistics. To extract the Langevin parameters from photon arrival time data, we advance the expectation-maximization algorithm in statistical learning, originally developed for and mostly used in discrete-state systems, to a general form in the continuous space that allows for a variational calculus on the continuous PMF function. We also introduce the regularization of the solution space in this Bayesian inference based on a maximum trajectory-entropy principle. We use a highly nontrivial example with realistically simulated smFRET data to illustrate the application of this new method.

  10. Wave propagation in coated cylinders with reference to fretting fatigue

    Indian Academy of Sciences (India)

    M Ramesh; Satish V Kailas; K R Y Simha


    Fretting fatigue is the phenomenon of crack initiation due to dynamic contact loading, a situation which is commonly encountered in mechanical couplings subjected to vibration. The study of fretting fatigue in high frequency regime has gained importance in recent years. However the stress wave effects at high frequency y loading is scanty in the literature. The objective of present investigation is to study stress wave propagation in cylinders with reference to high frequency fretting. The case of a coated cylinder is considered since coating is often provided to improve tribological properties of the component. Rule of mixtures is proposed to understand the dispersion phenomenon in coated or layered cylinder knowing the dispersion relation for the cases of homogeneous cylinders made of coating and substrate materials separately. The possibility of stress wave propagation at the interface with a particular phase velocity without dispersion is also discussed. Results are given for two different thicknesses of coating.

  11. A portable FRET analyzer for rapid detection of sugar content. (United States)

    Kim, Haseong; Kim, Hyo Sang; Ha, Jae-Seok; Lee, Seung-Goo


    Fluorescence resonance energy transfer (FRET) is widely used as a core process in biometric sensors to detect small molecules such as sugars, calcium ions, or amino acids. However, FRET based biosensors with innate weak signal intensity require the use of expensive, high-sensitive equipment. In the present study, these shortcomings were overcome with the fabrication of a sensitive, inexpensive, and portable analyzer which provides quantitative detection of small molecules in a liquid sample. The usability of the developed analyzer was successfully tested by measuring sucrose and maltose contents in commercially available beverage samples, with better performance than the conventional monochromator-type spectrofluorometer. It is anticipated that miniaturization of the equipment and improving the FRET based biosensors will contribute to the practical use of this hand-held analyzer in conditions where high-end equipment is not available.

  12. Fretting wear behavior of nitrogen implanted Zircaloy-4 alloy

    Institute of Scientific and Technical Information of China (English)


    Zircaloy-4 was implanted with nitrogen at 120keV with various ion doses between 1 × 1013 and 1 × 1014 ions/m2. Fretting wear tests were performed at various cycles and loads under water immersion condition by the fretting simulator, The implanted surfaces were analyzed by Auger electron spectroscopy (AES) and transmission electron microscope(TEM). Micro-hardnees tester measured surface hardness. It is shown that nitrogen imphantation produced Zirconium nitride oxide and high density dislocations in implanted ltyer, surface hardness was enhanced from HK280 for unimplantedspecimen to HK1800 for a total ion dose of 1 × 1014 ions/m2. The nitrogen ion implantation treatment provided significantimprovements in the resistance of fretting damage.

  13. Intravital FRET: Probing Cellular and Tissue Function in Vivo

    Directory of Open Access Journals (Sweden)

    Helena Radbruch


    Full Text Available The development of intravital Förster Resonance Energy Transfer (FRET is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.

  14. Beyond fullerenes: design of nonfullerene acceptors for efficient organic photovoltaics. (United States)

    Li, Haiyan; Earmme, Taeshik; Ren, Guoqiang; Saeki, Akinori; Yoshikawa, Saya; Murari, Nishit M; Subramaniyan, Selvam; Crane, Matthew J; Seki, Shu; Jenekhe, Samson A


    New electron-acceptor materials are long sought to overcome the small photovoltage, high-cost, poor photochemical stability, and other limitations of fullerene-based organic photovoltaics. However, all known nonfullerene acceptors have so far shown inferior photovoltaic properties compared to fullerene benchmark [6,6]-phenyl-C60-butyric acid methyl ester (PC60BM), and there are as yet no established design principles for realizing improved materials. Herein we report a design strategy that has produced a novel multichromophoric, large size, nonplanar three-dimensional (3D) organic molecule, DBFI-T, whose π-conjugated framework occupies space comparable to an aggregate of 9 [C60]-fullerene molecules. Comparative studies of DBFI-T with its planar monomeric analogue (BFI-P2) and PC60BM in bulk heterojunction (BHJ) solar cells, by using a common thiazolothiazole-dithienosilole copolymer donor (PSEHTT), showed that DBFI-T has superior charge photogeneration and photovoltaic properties; PSEHTT:DBFI-T solar cells combined a high short-circuit current (10.14 mA/cm(2)) with a high open-circuit voltage (0.86 V) to give a power conversion efficiency of 5.0%. The external quantum efficiency spectrum of PSEHTT:DBFI-T devices had peaks of 60-65% in the 380-620 nm range, demonstrating that both hole transfer from photoexcited DBFI-T to PSEHTT and electron transfer from photoexcited PSEHTT to DBFI-T contribute substantially to charge photogeneration. The superior charge photogeneration and electron-accepting properties of DBFI-T were further confirmed by independent Xenon-flash time-resolved microwave conductivity measurements, which correctly predict the relative magnitudes of the conversion efficiencies of the BHJ solar cells: PSEHTT:DBFI-T > PSEHTT:PC60BM > PSEHTT:BFI-P2. The results demonstrate that the large size, multichromophoric, nonplanar 3D molecular design is a promising approach to more efficient organic photovoltaic materials.

  15. Carcinogenicity of insulin analogues

    NARCIS (Netherlands)

    Braak, Sebastiaan Johannes ter


    There is epidemiological evidence that the use of some insulin analogues by diabetic patients is correlated with an increased cancer risk. In vitro exposure experiments revealed that insulin glargine (LANTUS) was the only commercial insulin analogue with an increased mitogenic potential. In the huma

  16. A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV. (United States)

    Liang, Caishuang; Wang, Huan; He, Kui; Chen, Chunyan; Chen, Xiaoming; Gong, Hang; Cai, Changqun


    Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960pM, with a limit of detection (LOD, 3σ) of 9.6pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost.

  17. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy. (United States)

    Valieva, Maria E; Gerasimova, Nadezhda S; Kudryashova, Kseniya S; Kozlova, Anastasia L; Kirpichnikov, Mikhail P; Hu, Qi; Botuyan, Maria Victoria; Mer, Georges; Feofanov, Alexey V; Studitsky, Vasily M


    A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

  18. Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

    Directory of Open Access Journals (Sweden)

    Maria E. Valieva


    Full Text Available A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD and the high mobility group (HMG domain of the structure-specific recognition protein 1 (SSRP1 subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16 and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3. Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

  19. Survey of analogue spacetimes

    CERN Document Server

    Visser, Matt


    Analogue spacetimes, (and more boldly, analogue models both of and for gravity), have attracted significant and increasing attention over the last decade and a half. Perhaps the most straightforward physical example, which serves as a template for most of the others, is Bill Unruh's model for a dumb hole, (mute black hole, acoustic black hole), wherein sound is dragged along by a moving fluid --- and can even be trapped behind an acoustic horizon. This and related analogue models for curved spacetimes are useful in many ways: Analogue spacetimes provide general relativists with extremely concrete physical models to help focus their thinking, and conversely the techniques of curved spacetime can sometimes help improve our understanding of condensed matter and/or optical systems by providing an unexpected and countervailing viewpoint. In this introductory chapter, I shall provide a few simple examples of analogue spacetimes as general background for the rest of the contributions.

  20. Intramolecular Charge-Transfer Interaction of Donor-Acceptor-Donor Arrays Based on Anthracene Bisimide. (United States)

    Iwanaga, Tetsuo; Ogawa, Marina; Yamauchi, Tomokazu; Toyota, Shinji


    We designed anthracene bisimide (ABI) derivatives having two triphenylamine (TPA) groups as donor units at the 9,10-positions to form a novel π-conjugated donor-acceptor system. These compounds and their analogues with ethynylene linkers were synthesized by Suzuki-Miyaura and Sonogashira coupling reactions, respectively. In UV-vis spectra, the linker-free derivatives showed broad absorption bands arising from intramolecular charge-transfer interactions. Introducing ethynylene linkers resulted in a considerable red shift of the absorption bands. In fluorescence spectra, the ethynylene derivatives showed intense emission bands at 600-650 nm. Their photophysical and electrochemical properties were compared with those of the corresponding mono TPA derivatives on the basis of theoretical calculations and cyclic voltammetry to evaluate the intramolecular electronic interactions between the donor and acceptor units.

  1. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Directory of Open Access Journals (Sweden)

    Shin-Rong Lee


    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  2. Alternating-laser excitation : single-molecule FRET and beyond

    NARCIS (Netherlands)

    Hohlbein, Johannes; Craggs, Timothy D.; Cordes, Thorben


    The alternating-laser excitation (ALEX) scheme continues to expand the possibilities of fluorescence-based assays to study biological entities and interactions. Especially the combination of ALEX and single-molecule Forster Resonance Energy Transfer (smFRET) has been very successful as ALEX enables

  3. Plant-based FRET biosensor discriminates enviornmental zinc levels (United States)

    Heavy metal accumulation in the environment poses great risks to flora and fauna. However, monitoring sites prone to accumulation poses scale and economic challenges. In this study, we present and test a method for monitoring these sites using fluorescent resonance energy transfer (FRET) change in r...

  4. Fretting Fatigue of Gamma TiAl Studied (United States)

    Miyoshi, Kazuhisa; Lerch, Bradley A.; Draper, Susan L.


    Gamma titanium-aluminum alloy (g-TiAl) is an attractive new material for aerospace applications because of its low density and high specific strength in comparison to currently used titanium and nickel-base alloys. Potential applications for this material are compressor and low-pressure turbine blades. These blades are fitted into either the compressor or turbine disks via a dovetail connection. The dovetail region experiences a complex stress state due to the alternating centrifugal force and the natural high-frequency vibration of the blade. Because of the dovetail configuration and the complex stress state, fretting is often a problem in this area. Furthermore, the local stress state becomes more complex when the influence of the metal-metal contact and the edge of the contact is evaluated. Titanium and titanium-based alloys in the clean state exhibit strong adhesive bonds when in contact with themselves and other materials (refs. 1 and 2). This adhesion causes heavy surface damage and high friction in practical cases. Although the wear produced by fretting may be mild, the reduction in fatigue life can be substantial. Thus, there is the potential for fretting problems with these TiAl applications. Since TiAl is an emerging material, there has been limited information about its fretting behavior.

  5. Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity

    Directory of Open Access Journals (Sweden)

    Alexander S. Goryashchenko


    Full Text Available This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds, pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.

  6. Double Acceptor Interaction in Semimagnetic Quantum Dot

    Directory of Open Access Journals (Sweden)

    A. Merwyn Jasper D. Reuben


    Full Text Available The effect of geometry of the semimagnetic Quantum Dot on the Interaction energy of a double acceptor is computed in the effective mass approximation using the variational principle. A peak is observed at the lower dot sizes as a magnetic field is increased which is attributed to the reduction in confinement.

  7. Syntheses of donor-acceptor-functionalized dihydroazulenes

    DEFF Research Database (Denmark)

    Broman, Søren Lindbæk; Jevric, Martyn; Bond, Andrew


    The dihydroazulene (DHA)/vinylheptafulvene (VHF) photo/thermoswitch has been of interest for use in molecular electronics and advanced materials. The switching between the two isomers has previously been found to depend strongly on the presence of donor and acceptor groups. The fine-tuning of opt...

  8. Electron Donor Acceptor Interactions. Final Progress Report

    Energy Technology Data Exchange (ETDEWEB)



    The Gordon Research Conference (GRC) on Electron Donor Acceptor Interactions was held at Salve Regina University, Newport, Rhode Island, 8/11-16/02. Emphasis was placed on current unpublished research and discussion of the future target areas in this field.

  9. Single-Molecule Pull-down FRET (SiMPull-FRET) to dissect the mechanisms of biomolecular machines (United States)

    Kahlscheuer, Matthew L.; Widom, Julia; Walter, Nils G.


    Spliceosomes are multi-megadalton RNA-protein complexes responsible for the faithful removal of non-coding segments (introns) from pre-messenger RNAs (pre-mRNAs), a process critical for the maturation of eukaryotic mRNAs for subsequent translation by the ribosome. Both the spliceosome and ribosome, as well as many other RNA and DNA processing machineries, contain central RNA components that endow biomolecular complexes with precise, sequence-specific nucleic acid recognition and versatile structural dynamics. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) microscopy is a powerful tool for the study of local and global conformational changes of both simple and complex biomolecular systems involving RNA. The integration of biochemical tools such as immunoprecipitation with advanced methods in smFRET microscopy and data analysis has opened up entirely new avenues towards studying the mechanisms of biomolecular machines isolated directly from complex biological specimens such as cell extracts. Here we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy in exemplary single molecule pull-down FRET (SiMPull-FRET) studies of the yeast spliceosome and discuss the broad application potential of this technique. PMID:26068753

  10. rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments. (United States)

    Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János


    Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry.

  11. Synthesis of Tonghaosu Analogues

    Institute of Scientific and Technical Information of China (English)

    SUN Hai; LIN Yingjie; WU Yulin; WU Yikang


    Several new analogues of natural antifeedant tonghaosu were synthesized via m-CPBA (m-chloroperoxybenzoic acid) oxidation of corresponding 3-(a-furyl)propanols, Luche reduction of the resulting enone, epoxidation, acid-mediated spiroketalization, and radical mediated dehydration.

  12. Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc (United States)


    Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time. PMID:27547982

  13. Interference Assembly and Fretting Wear Analysis of Hollow Shaft


    Chuanjun Han; Jie Zhang


    Fretting damage phenomenon often appears in the interference fit assembly. The finite element model of hollow shaft and shaft sleeve was established, and the equivalent stress and contact stress were computed after interference assembly. The assembly body of hollow shaft and shaft sleeve was in whirling bending load, and the contact status (sticking, sliding, and opening) and the distribution of stress along one typical contact line were computed under different loads, interferences, hollow d...

  14. Fretting corrosion in power contacts: Electrical and thermal analysis


    El Mossouess, S; Benjemâa, N; Carvou, E; El Abdi, R; Benmamas, L; Doublet, L


    International audience; -Fretting corrosion phenomenon is known as the main cause of contact resistance increasing in signal contact. But for power connectors this undesirable phenomenon in embarked systems is more complex because high current induces high voltages and subsequent Joule overheating can be expected during vibration. Our study deals with contact similar of power connectors ones submitted to vibration am-plitudes up to 25 micrometres at frequencies of a few 10Hz under a current r...

  15. Distinguishing the cross-beta spine arrangements in amyloid fibrils using FRET analysis. (United States)

    Deng, Wei; Cao, Aoneng; Lai, Luhua


    The recently published microcrystal structures of amyloid fibrils from small peptides greatly enhanced our understanding of the atomic-level structure of the amyloid fibril. However, only a few amyloid fibrils can form microcrystals. The dansyl-tryptophan fluorescence resonance energy transfer (FRET) pair was shown to be able to detect the inter-peptide arrangement of the Transthyretin (105-115) amyloid fibril. In this study, we combined the known microcrystal structures with the corresponding FRET efficiencies to build a model for amyloid fibril structure classification. We found that fibrils with an antiparallel structural arrangement gave the largest FRET signal, those with a parallel arrangement gave the lowest FRET signal, and those with a mixed arrangement gave a moderate FRET signal. This confirms that the amyloid fibril structure patterns can be classified based on the FRET efficiency.

  16. Fretting fatigue crack propagation rate under variable loading conditions

    Directory of Open Access Journals (Sweden)

    C. Gandiolle


    Full Text Available Fretting fatigue experiments aim to represent industrial problems and most of them endure variable loading. Being able to assess lifetime of assemblies, especially for low propagation rate conditions, is essential as experimental validation is often too expensive. Both experimental and numerical approaches are proposed to follow the crack propagation rate of steel on steel cylinder/plane fretting fatigue contact submitted to variable loading conditions. An original experimental monitoring has been implemented on the fretting-fatigue test device to observe crack propagation using a potential drop technique. A calibration curve relating crack length and electrical potential was established for the studied contact. It allows direct knowledge of the crack length and crack propagation rate. It was applied to mixed load test showing crack arrest for the last loading condition. To explain this behavior, a 2-dimensional FE modeling was implemented to simulate the complexes multi-axial contact stressing. The crack propagation rate was formalized using an effective stress intensity factor amplitude ΔKeff coupled with Paris law of the material. The crack arrest condition for a given loading was related to ΔKeff along the expected crack path crossing the material crack arrest threshold ΔK0. The failure was related to ΔKeff reaching the critical stress intensity factor KIC. A good correlation with experiments was observed allowing to predict the crack arrest condition although the model tends to overestimate the final crack length extension.

  17. Considerations for sensitivity analysis, uncertainty quantification, and data assimilation for grid-to-rod fretting

    Energy Technology Data Exchange (ETDEWEB)

    Michael Pernice


    Grid-to-rod fretting is the leading cause of fuel failures in pressurized water reactors, and is one of the challenge problems being addressed by the Consortium for Advanced Simulation of Light Water Reactors to guide its efforts to develop a virtual reactor environment. Prior and current efforts in modeling and simulation of grid-to-rod fretting are discussed. Sources of uncertainty in grid-to-rod fretting are also described.

  18. pyFRET: A Python Library for Single Molecule Fluorescence Data Analysis


    Murphy, Rebecca R.; Jackson, Sophie E.; Klenerman, David


    Single molecule F\\"orster resonance energy transfer (smFRET) is a powerful experimental technique for studying the properties of individual biological molecules in solution. However, as adoption of smFRET techniques becomes more widespread, the lack of available software, whether open source or commercial, for data analysis, is becoming a significant issue. Here, we present pyFRET, an open source Python package for the analysis of data from single-molecule fluorescence experiments from freely...

  19. A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously. (United States)

    Götz, M; Wortmann, P; Schmid, S; Hugel, T


    Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

  20. Fretting Fatigue Experiment and Analysis of AlSi9Cu2Mg Alloy

    Directory of Open Access Journals (Sweden)

    Jun Wang


    Full Text Available An investigation was carried out in order to study the fretting fatigue behavior of an AlSi9Cu2Mg aluminum alloy. The fretting fatigue tests of AlSi9Cu2Mg were performed using a specially designed testing machine. The failure mechanism of fretting fatigue was explored by studying the fracture surfaces, fretting scars, fretting debris, and micro-hardness of fretting fatigue specimens using scanning electron microscopy (SEM, energy-dispersive X-ray spectroscopy (EDX, and micro Vickers hardness test techniques. The experimental results show that the fretting fatigue limit (42 MPa is significantly reduced to approximately 47% of the plain fatigue limit (89 MPa under 62.5 MPa contact pressure. Furthermore, the fretting fatigue life decreases with increasing alternating stress and increasing contact pressure. The examination results suggest that the stress concentrates induced by oxidation-assisted wear on the contact interface led to the earlier initiation and propagation of crack under the fretting condition.

  1. A communication theoretical analysis of FRET-based mobile ad hoc molecular nanonetworks. (United States)

    Kuscu, Murat; Akan, Ozgur B


    Nanonetworks refer to a group of nanosized machines with very basic operational capabilities communicating to each other in order to accomplish more complex tasks such as in-body drug delivery, or chemical defense. Realizing reliable and high-rate communication between these nanomachines is a fundamental problem for the practicality of these nanonetworks. Recently, we have proposed a molecular communication method based on Förster Resonance Energy Transfer (FRET) which is a nonradiative excited state energy transfer phenomenon observed among fluorescent molecules, i.e., fluorophores. We have modeled the FRET-based communication channel considering the fluorophores as single-molecular immobile nanomachines, and shown its reliability at high rates, and practicality at the current stage of nanotechnology. In this study, for the first time in the literature, we investigate the network of mobile nanomachines communicating through FRET. We introduce two novel mobile molecular nanonetworks: FRET-based mobile molecular sensor/actor nanonetwork (FRET-MSAN) which is a distributed system of mobile fluorophores acting as sensor or actor node; and FRET-based mobile ad hoc molecular nanonetwork (FRET-MAMNET) which consists of fluorophore-based nanotransmitter, nanoreceivers and nanorelays. We model the single message propagation based on birth-death processes with continuous time Markov chains. We evaluate the performance of FRET-MSAN and FRET-MAMNET in terms of successful transmission probability and mean extinction time of the messages, system throughput, channel capacity and achievable communication rates.


    DEFF Research Database (Denmark)


    The present invention relates to new compounds being structurally and functionally similar to Actinomycin D and to combinatorial libraries of such compounds. The Actinomycin D analogues according to the present invention comprise two linear or cyclic peptide moieties constituted by $g...

  3. Mining the Sinorhizobium meliloti transportome to develop FRET biosensors for sugars, dicarboxylates and cyclic polyols.

    Directory of Open Access Journals (Sweden)

    Alexandre Bourdès

    Full Text Available Förster resonance energy transfer (FRET biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens. To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors.Two new vectors were developed for cloning genes for solute-binding proteins (SBPs between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2 and red fluorescent protein (mKate2 FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose, D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate. To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP transport systems.FRET based on orange (mOrange2 and red fluorescent protein (mKate2 partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i cyclic polyols, (ii L-deoxy sugars, (iii β-linked disaccharides and (iv C4-dicarboxylates could be developed to study metabolism in vivo.

  4. Synthesis and evaluation of new NIR-fluorescent probes for cathepsin B: ICT versus FRET as a turn-ON mode-of-action. (United States)

    Kisin-Finfer, Einat; Ferber, Shiran; Blau, Rachel; Satchi-Fainaro, Ronit; Shabat, Doron


    Recent years have seen tremendous progress in the design and study of molecular imaging geared towards biological and biomedical applications. The expression or activity of specific enzymes including proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has been attracting significant attention in cancer research, and many imaging probes targeting its activity have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared (NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One probe is based on an ICT activation mechanism of a donor-two-acceptor π-electron dye system, while the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast.

  5. Multifunctional Concentric FRET-Quantum Dot Probes for Tracking and Imaging of Proteolytic Activity. (United States)

    Massey, Melissa; Li, Jia Jun; Algar, W Russ


    Proteolysis has many important roles in physiological regulation. It is involved in numerous cell signaling processes and the pathogenesis of many diseases, including cancers. Methods of visualizing and assaying proteolytic activity are therefore in demand. Förster resonance energy transfer (FRET) probes offer several advantages in this respect. FRET supports end-point or real-time measurements, does not require washing or separation steps, and can be implemented in various assay or imaging formats. In this chapter, we describe methodology for preparing self-assembled concentric FRET (cFRET) probes for multiplexed tracking and imaging of proteolytic activity. The cFRET probe comprises a green-emitting semiconductor quantum dot (QD) conjugated with multiple copies of two different peptide substrates for two target proteases. The peptide substrates are labeled with different fluorescent dyes, Alexa Fluor 555 and Alexa Fluor 647, and FRET occurs between the QD and both dyes, as well as between the two dyes. This design enables a single QD probe to track the activity of two proteases simultaneously. Fundamental cFRET theory is presented, and procedures for using the cFRET probe for quantitative measurement of the activity of two model proteases are given, including calibration, fluorescence plate reader or microscope imaging assays, and data analysis. Sufficient detail is provided for other researchers to adapt this method to their specific requirements and proteolytic systems of interest.

  6. FRET Imaging Trackable Long-Circulating Biodegradable Nanomedicines for Ovarian Cancer Therapy (United States)


    Award Number: W81XWH-13-1-0160 TITLE: FRET Imaging Trackable Long-Circulating Biodegradable ...TITLE AND SUBTITLE FRET Imaging Trackable Long-Circulating Biodegradable Nanomedicines for Ovarian Cancer Therapy 5a. CONTRACT NUMBER...strategy, which permits visualizing the biodegradation of copolymer-drug conjugates at the body, tissue and cell levels in real time. The information

  7. π-Bridge-Independent 2-(Benzo[c][1,2,5]thiadiazol-4-ylmethylene)malononitrile-Substituted Nonfullerene Acceptors for Efficient Solar Cells

    KAUST Repository

    Wang, Kai


    Molecular acceptors are promising alternatives to fullerenes (e.g. PC61/71BM) in the fabrication of high-efficiency bulk-heterojunction (BHJ) solar cells. While solution-processed polymer-fullerene BHJ devices have recently met the 10% efficiency threshold, molecular acceptors have yet to prove comparably efficient with polymer donors. At this point in time, it is important to forge a better understanding of the design parameters that directly impact small-molecule (SM) acceptor performance in BHJ solar cells. In this report, we show that 2-(benzo[c][1,2,5]thiadiazol-4-ylmethylene)malononitrile (BM)-terminated SM acceptors can achieve efficiencies as high as 5.3% in BHJ solar cells with the polymer donor PCE10. Through systematic device optimization and characterization studies, we find that the nonfull-erene analogues (FBM, CBM and CDTBM) all perform comparably well, independent of the molecular structure and electronics of the π-bridge that links the two electron-deficient BM end groups. With estimated electron affinities within range of those of common fullerenes (4.0-4.3 eV), and a wider range of ionization potentials (6.2-5.6 eV), the SM acceptors absorb in the visible spectrum and effectively contribute to the BHJ device photocurrent. BM-substituted SM acceptors are promising alterna-tives to fullerenes in solution-processed BHJ solar cells.

  8. pyFRET: A Python Library for Single Molecule Fluorescence Data Analysis

    CERN Document Server

    Murphy, Rebecca R; Klenerman, David


    Single molecule F\\"orster resonance energy transfer (smFRET) is a powerful experimental technique for studying the properties of individual biological molecules in solution. However, as adoption of smFRET techniques becomes more widespread, the lack of available software, whether open source or commercial, for data analysis, is becoming a significant issue. Here, we present pyFRET, an open source Python package for the analysis of data from single-molecule fluorescence experiments from freely diffusing biomolecules. The package provides methods for the complete analysis of a smFRET dataset, from burst selection and denoising, through data visualisation and model fitting. We provide support for both continuous excitation and alternating laser excitation (ALEX) data analysis. pyFRET is available as a package downloadable from the Python Package Index (PyPI) under the open source three-clause BSD licence, together with links to extensive documentation and tutorials, including example usage and test data. Additio...

  9. A Versatile Multiple Target Detection System Based on DNA Nano-assembled Linear FRET Arrays. (United States)

    Li, Yansheng; Du, Hongwu; Wang, Wenqian; Zhang, Peixun; Xu, Liping; Wen, Yongqiang; Zhang, Xueji


    DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as inconstant programmable templates for assembly of biosensors. In this paper, a versatile, scalable and multiplex detection system is reported based on an extending fluorescent resonance energy transfer (FRET) cascades on a linear DNA assemblies. Seven combinations of three kinds of targets are successfully detected through the changes of fluorescence spectra because of the three-steps FRET or non-FRET continuity mechanisms. This nano-assembled FRET-based nanowire is extremely significant for the development of rapid, simple and sensitive detection system. The method used here could be extended to a general platform for multiplex detection through more-step FRET process.

  10. Fretting damage behavior and mechanism of tin coated zircaloy-4 tube

    Institute of Scientific and Technical Information of China (English)

    Tae-HyungKim; Ji-HyunSung; Seock-SamKim


    The fretting characteristics of TiN coated Zircaloy-4 tube were investigated experimen-tally. The fretting experiment was performed using TIN coated Zircaloy-4 tube as the fuel rod clad-ding material and uncoated Zircaloy-4 tube as one of grids. TIN coating is probably one of the mostfrequently and successfully used PVD coatings for the mitigation of fretting. In this study, TiN coat-ing by PVD was employed for improvement of Zircaloy-4 tube fretting characteristics. The frettingtester was designed and manufactured for this experiment. The number of cycles, slip amplitudeand normal load were selected as main factors of fretting. The results of this research showed thatthe wear volume of TiN coated Zircaloy-4 tube increased as number of cycles, normal load andslip amplitude increase but the quantity of volume was lower than the case of uncoated Zircaloy-4tube pairs.

  11. Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

    Directory of Open Access Journals (Sweden)

    George Chennell


    Full Text Available We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP Activated Protein Kinase (AMPK FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR, for fluorescence lifetime imaging (FLIM readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP, in the original FRET biosensor, AMPK activity reporter (AMPKAR, with mTurquoise2 (mTq2FP, increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.

  12. The reaction of choline dehydrogenase with some electron acceptors. (United States)

    Barrett, M C; Dawson, A P


    1. The choline dehydrogenase (EC WAS SOLUBILIZED FROM ACETONE-DRIED POWDERS OF RAT LIVER MITOCHONDRIA BY TREATMENT WITH Naja naja venom. 2. The kinetics of the reaction of enzyme with phenazine methosulphate and ubiquinone-2 as electron acceptors were investigated. 3. With both electron acceptors the reaction mechanism appears to involve a free, modified-enzyme intermediate. 4. With some electron acceptors the maximum velocity of the reaction is independent of the nature of the acceptor. With phenazine methosulphate and ubiquinone-2 as acceptors the Km value for choline is also independent of the nature of the acceptor molecule. 5. The mechanism of the Triton X-100-solubilized enzyme is apparently the smae as that for the snake venom solubilized enzyme.

  13. Gastric inhibitory polypeptide analogues

    DEFF Research Database (Denmark)

    Holst, Jens Juul


    of GIP and GLP-1 receptors, the incretin effect is essential for normal glucose tolerance. In patients with type 2 diabetes mellitus it turns out that the incretin effect is severely impaired or abolished. The explanation seems to be that both the secretion of GLP-1 and the effect of GIP are impaired...... (whereas both the secretion of GIP and the effect of GLP-1 are near normal). The impaired GLP-1 secretion is probably a consequence of diabetic metabolic disturbances. The known genetic variations in the GIP receptor sequence are not associated with type 2 diabetes mellitus, but a defective insulinotropic...... and its analogues are attractive as therapeutic agents for type 2 diabetes mellitus, analogues of GIP are unlikely to be effective. On the other hand, GIP seems to play an important role in lipid metabolism, promoting the disposal of ingested lipids, and mice with a targeted deletion of the GIP receptor...

  14. Aspartame and Its Analogues (United States)

    Pavlova, L. A.; Komarova, T. V.; Davidovich, Yurii A.; Rogozhin, S. V.


    The results of studies on the biochemistry of the sweet taste are briefly reviewed. The methods of synthesis of "aspartame" — a sweet dipeptide — are considered, its structural analogues are described, and quantitative estimates are made of the degree of sweetness relative to sucrose. Attention is concentrated mainly on problems of the relation between the structure of the substance and its taste in the series of aspartyl derivatives. The bibliography includes 118 references.

  15. Quantum analogue computing. (United States)

    Kendon, Vivien M; Nemoto, Kae; Munro, William J


    We briefly review what a quantum computer is, what it promises to do for us and why it is so hard to build one. Among the first applications anticipated to bear fruit is the quantum simulation of quantum systems. While most quantum computation is an extension of classical digital computation, quantum simulation differs fundamentally in how the data are encoded in the quantum computer. To perform a quantum simulation, the Hilbert space of the system to be simulated is mapped directly onto the Hilbert space of the (logical) qubits in the quantum computer. This type of direct correspondence is how data are encoded in a classical analogue computer. There is no binary encoding, and increasing precision becomes exponentially costly: an extra bit of precision doubles the size of the computer. This has important consequences for both the precision and error-correction requirements of quantum simulation, and significant open questions remain about its practicality. It also means that the quantum version of analogue computers, continuous-variable quantum computers, becomes an equally efficient architecture for quantum simulation. Lessons from past use of classical analogue computers can help us to build better quantum simulators in future.

  16. Crack path simulation for cylindrical contact under fretting conditions

    Directory of Open Access Journals (Sweden)

    R.A. Cardoso


    Full Text Available In this work different strategies to estimate crack path for cylindrical contacts under fretting conditions are carried out. The main goal is to propose and to evaluate methodologies not only to estimate the direction of crack initiation but also the subsequent propagation in its earlier stages, where the stress field is multiaxial, non-proportional and decays very fast due to the proximity with the contact interface. Such complex conditions pose a substantial challenge to the modelling of crack path. The numerical simulations are provided by a 2D Finite Element Analysis taking into account interactions between the crack faces. The results show that, under fretting conditions, models based on the critical plane method are not effective to estimate the crack initiation orientation, while models based on a so called “critical direction” applied along a critical distance provide better results. Regarding the subsequent crack propagation orientation, it was possible to see that stress intensity factor based models where one considers an infinitesimal virtual crack emerging from an original preexistent crack are powerful mechanisms of crack orientation estimation.

  17. Quantum computing with acceptor spins in silicon. (United States)

    Salfi, Joe; Tong, Mengyang; Rogge, Sven; Culcer, Dimitrie


    The states of a boron acceptor near a Si/SiO2 interface, which bind two low-energy Kramers pairs, have exceptional properties for encoding quantum information and, with the aid of strain, both heavy hole and light hole-based spin qubits can be designed. Whereas a light-hole spin qubit was introduced recently (arXiv:1508.04259), here we present analytical and numerical results proving that a heavy-hole spin qubit can be reliably initialised, rotated and entangled by electrical means alone. This is due to strong Rashba-like spin-orbit interaction terms enabled by the interface inversion asymmetry. Single qubit rotations rely on electric-dipole spin resonance (EDSR), which is strongly enhanced by interface-induced spin-orbit terms. Entanglement can be accomplished by Coulomb exchange, coupling to a resonator, or spin-orbit induced dipole-dipole interactions. By analysing the qubit sensitivity to charge noise, we demonstrate that interface-induced spin-orbit terms are responsible for sweet spots in the dephasing time [Formula: see text] as a function of the top gate electric field, which are close to maxima in the EDSR strength, where the EDSR gate has high fidelity. We show that both qubits can be described using the same starting Hamiltonian, and by comparing their properties we show that the complex interplay of bulk and interface-induced spin-orbit terms allows a high degree of electrical control and makes acceptors potential candidates for scalable quantum computation in Si.

  18. Analogue Methods in Palaeoecology: Using the analogue Package


    Simpson, Gavin L.


    Palaeoecology is an important branch of ecology that uses the subfossil remains of organisms preserved in lake, ocean and bog sediments to inform on changes in ecosystems and the environment through time. The analogue package contains functions to perform modern analogue technique (MAT) transfer functions, which can be used to predict past changes in the environment, such as climate or lake-water pH from species data. A related technique is that of analogue matching, which is concerned with i...

  19. Photoinduced Intramolecular Charge Transfer in Donor-acceptor Dyad and Donor-bridge-acceptor Triad

    Institute of Scientific and Technical Information of China (English)

    Yong Ding; Yuan-zuo Li; Feng-cai Ma


    The ground and excited state properties of the [60]fullerene,diphenylbenzothiadiazole-triphenylamine (PBTDP-TPA) dyad and fullerene-diphenylbenzothiadiazole-triphenylamine (fullerene-PBTDP-TPA) triad were investigated theoretically using density functional theory with B3LYP functional and 3-21G basis set and time-dependent density functional theory with B3LYP functional and STO-3G basis set as well as 2D and 3D real space analysis methods.The 2D site representation reveals the electron-hole coherence on exci- tation.The 3D transition density shows the orientation and strength of the transition dipole moment,and the 3D charge difference density gives the orientation and result of the intramolecular charge transfer.Also, photoinduced intermolecular charge transfer (ICT) in PBTDP-TPA-fullerene triad are identified with 2D and 3D representations,which reveals the mechanisms of ICT in donor-bridge-acceptor triad on excitation. Besides that we also found that the direct superexchange ICT from donor to acceptor (tunneling through the bridge) strongly promotes the ICT in the donor-bridge-acceptor triad.

  20. Partially reduced graphene oxide based FRET on fiber-optic interferometer for biochemical detection (United States)

    Yao, B. C.; Wu, Y.; Yu, C. B.; He, J. R.; Rao, Y. J.; Gong, Y.; Fu, F.; Chen, Y. F.; Li, Y. R.


    Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel ‘FRET on Fiber’ concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based ‘FRET on fiber’ configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated ‘FRET on Fiber’ sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response

  1. Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

    Full Text Available Fluorescence resonance energy transfer (FRET between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

  2. Alkyl Radicals as Hydrogen Bond Acceptors: Computational Evidence

    DEFF Research Database (Denmark)

    Hammerum, Steen


    , and gives rise to pronounced shifts of IR stretching frequencies and to increased absorption intensities. The hydrogen bond acceptor properties of alkyl radicals equal those of many conventional acceptors, e.g., the bond length changes and IR red-shifts suggest that tert-butyl radicals are slightly better...

  3. The role of deep acceptor centers in the oxidation of acceptor-doped wide-band-gap perovskites ABO3 (United States)

    Putilov, L. P.; Tsidilkovski, V. I.


    The impact of deep acceptor centers on defect thermodynamics and oxidation of wide-band-gap acceptor-doped perovskites without mixed-valence cations is studied. These deep centers are formed by the acceptor-bound small hole polarons whose stabilization energy can be high enough (significantly higher than the hole-acceptor Coulomb interaction energy). It is shown that the oxidation enthalpy ΔHox of oxide is determined by the energy εA of acceptor-bound states along with the formation energy EV of oxygen vacancies. The oxidation reaction is demonstrated to be either endothermic or exothermic, and the regions of εA and EV values corresponding to the positive or negative ΔHox are determined. The contribution of acceptor-bound holes to the defect thermodynamics strongly depends on the acceptor states depth εA: it becomes negligible at εA less than a certain value (at which the acceptor levels are still deep). With increasing εA, the concentration of acceptor-bound small hole polarons can reach the values comparable to the dopant content. The results are illustrated with the acceptor-doped BaZrO3 as an example. It is shown that the experimental data on the bulk hole conductivity of barium zirconate can be described both in the band transport model and in the model of hopping small polarons localized on oxygen ions away from the acceptor centers. Depending on the εA magnitude, the oxidation reaction can be either endothermic or exothermic for both mobility mechanisms.

  4. Promising Strategy To Improve Charge Separation in Organic Photovoltaics: Installing Permanent Dipoles in PCBM Analogues. (United States)

    de Gier, Hilde D; Jahani, Fatemeh; Broer, Ria; Hummelen, Jan C; Havenith, Remco W A


    A multidisciplinary approach involving organic synthesis and theoretical chemistry was applied to investigate a promising strategy to improve charge separation in organic photovoltaics: installing permanent dipoles in fullerene derivatives. First, a PCBM analogue with a permanent dipole in the side chain (PCBDN) and its reference analogue without a permanent dipole (PCBBz) were successfully synthesized and characterized. Second, a multiscale modeling approach was applied to investigate if a PCBDN environment around a central donor-acceptor complex indeed facilitates charge separation. Alignment of the embedding dipoles in response to charges present on the central donor-acceptor complex enhances charge separation. The good correspondence between experimentally and theoretically determined electronic and optical properties of PCBDN, PCBBz, and PCBM indicates that the theoretical analysis of the embedding effects of these molecules gives a reliable expectation for their influence on the charge separation process at a microscopic scale in a real device. This work suggests the following strategies to improve charge separation in organic photovoltaics: installing permanent dipoles in PCBM analogues and tuning the concentration of these molecules in an organic donor/acceptor blend.

  5. Fretting Fatigue Improvement of Ti6Al4V by Coating and Shot Peening

    Institute of Scientific and Technical Information of China (English)

    Daoxin LIU; Xiaodong ZHU; Bin TANG; Jiawen HE


    Ion beam enhanced deposition (IBED) was employed to increase the fretting fatigue resistance of Ti6AI4V. CrN and TiN hard coatings were applied on the base material and shot peening was combined with the hard coatings to study the duplex effect on fretting fatigue resistance, The IBED coatings exhibited a good bonding strength. They did not spall off even after shot peening. However, an optimum composition of CrN showed better fretting fatigue resistance than that of TiN with the same processing parameters.

  6. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape


    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.


    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unrav...

  7. The Effect of Elevated Temperature on the Fretting Fatigue Behavior of Nickel Alloy IN-100 (United States)


    Saladin S-N data points for plain fatigue 86 4.16 Comparison of Ownby and Saladin S-N data points for fretting fatigue 87 4.17 Comparison of Ownby...and Saladin S-N data for all tests 88 4.18 Plot comparing ΔQ vs Nf from Ownby, Saladin & Madhi data points 89 4.19 Plot from Kawagoishi et al. [ this study 93 4.3 Madhi fretting and plain fatigue testing data 94 4.4 Saladin fretting and plain fatigue testing data 94 4.5 Comparison of

  8. Preparation of mononucleosomal templates for analysis of transcription with RNA polymerase using spFRET. (United States)

    Kudryashova, Kseniya S; Chertkov, Oleg V; Nikitin, Dmitry V; Pestov, Nikolai A; Kulaeva, Olga I; Efremenko, Anastasija V; Solonin, Alexander S; Kirpichnikov, Mikhail P; Studitsky, Vasily M; Feofanov, Alexey V


    Single positioned nucleosomes have been extensively employed as simple model experimental systems for analysis of various intranuclear processes. Here we describe an experimental system containing positioned mononucleosomes allowing transcription by various RNA polymerases. Each DNA template contains a pair of fluorescent labels (Cy3 and Cy5) allowing measuring relative distances between the neighboring coils of nucleosomal DNA using Forster resonance energy transfer (FRET). The single-particle FRET (spFRET) approach for analysis of DNA uncoiling from the histone octamer during transcription through chromatin is described in detail.

  9. A Short Term Analogue Memory

    DEFF Research Database (Denmark)

    Shah, Peter Jivan


    A short term analogue memory is described. It is based on a well-known sample-hold topology in which leakage currents have been minimized partly by circuit design and partly by layout techniques. Measurements on a test chip implemented in a standard 2.4 micron analogue CMOS process show a droop...

  10. Fretting wear in titanium, Monel-400, and cobalt 25-percent-molybdenum using scanning electron microscopy (United States)

    Bill, R. C.


    Damage scar volume measurements taken from like metal fretting pairs combined with scanning electron microscopy observations showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt - 25-percent molybdenum. Initially, adhesion and plastic deformation of the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism which produced spall-like pits in the damage scar. Finally, a combination of oxidation and abrasion by debris particles became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

  11. Fretting Behavior of SPR Joining Dissimilar Sheets of Titanium and Copper Alloys

    Directory of Open Access Journals (Sweden)

    Xiaocong He


    Full Text Available The fretting performance of self-piercing riveting joining dissimilar sheets in TA1 titanium alloy and H62 copper alloy was studied in this paper. Load-controlled cyclic fatigue tests were carried out using a sine waveform and in tension-tension mode. Scanning electron microscopy and energy-dispersive X-ray techniques were employed to analyze the fretting failure mechanisms of the joints. The experimental results showed that there was extremely severe fretting at the contact interfaces of rivet and sheet materials for the joints at relatively high loads levels. Moreover, the severe fretting in the region on the locked sheet in contact with the rivet was the major cause of the broken locked sheet for the joints at low load level.

  12. Nucleic Acid Base Analog FRET-Pair Facilitating Detailed Structural Measurements in Nucleic Acid Containing Systems

    DEFF Research Database (Denmark)

    Börjesson, Karl; Preus, Søren; El-Sagheer, Afaf;


    toward detailed studies of the inherent dynamics of nucleic acid structures. Moreover, the placement of FRET-pair chromophores inside the base stack will be a great advantage in studies where other (biomacro)molecules interact with the nucleic acid. Lastly, our study gives possibly the first truly solid...... distances covering up to more than one turn of the DNA duplex. Importantly, we show that the rigid stacking of the two base analogs, and consequently excellent control of their exact positions and orientations, results in a high control of the orientation factor and hence very distinct FRET changes...... as the number of bases separating tCO and tC(nitro) is varied. A set of DNA strands containing the FRET-pair at wisely chosen locations will, thus, make it possible to accurately distinguish distance- from orientation-changes using FRET. In combination with the good nucleobase analog properties, this points...

  13. Influence of fretting on the fatigue strength at the vise clamp–specimen interface

    Indian Academy of Sciences (India)

    Abdulhaqq A Hamid; Rafi K Yahya


    Fretting fatigue is one of the most important phenomena for inducing a significant reduction of fatigue strength and consequently, leading to unexpected failure accidents of the engineering structures even at very low stresses. In the present study, both plain and fretting fatigue tests with zero mean stress were carried out on two different types of steel, low-carbon steel and martensitic stainless steel, by means of a reversed bending fatigue testing machine. The drop in the fatigue strengths through fretting at vise clamp–specimen interface were significant for both tested steels. The fretting processes produced a reduction in fatigue strength of about 27% for low-carbon steel and 16% for martensitic stainless steel.

  14. Methodological considerations for global analysis of cellular FLIM/FRET measurements (United States)

    Adbul Rahim, Nur Aida; Pelet, Serge; Kamm, Roger D.; So, Peter T. C.


    Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

  15. Single-molecule three-color FRET with both negligible spectral overlap and long observation time.

    Directory of Open Access Journals (Sweden)

    Sanghwa Lee

    Full Text Available Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF microscopy.

  16. Vorticity in analogue gravity

    CERN Document Server

    Cropp, Bethan; Turcati, Rodrigo


    In the analogue gravity framework, the acoustic disturbances in a moving fluid can be described by an equation of motion identical to a relativistic scalar massless field propagating in a curved spacetime. This description is possible only when the fluid under consideration is barotropic, inviscid and irrotational. In this case, the propagation of the perturbations is governed by an acoustic metric which depends algebrically on the local speed of sound, density and the background flow velocity, the latter assumed to be vorticity free. In this work we provide an straightforward extension in order to go beyond the irrotational constraint. Using a charged --- relativistic and non-relativistic --- Bose--Einstein condensate as a physical system, we show that in the low momentum limit and performing the eikonal approximation we can derive a d'Alembertian equation of motion for the charged phonons where the emergent acoustic metric depends on a flow velocity in the presence of vorticity.

  17. Donor–Acceptor Oligorotaxanes Made to Order

    Energy Technology Data Exchange (ETDEWEB)

    Basu, Subhadeep [Northwestern Univ., Evanston, IL (United States); Coskun, Ali [Northwestern Univ., Evanston, IL (United States); Friedman, Douglas C. [Northwestern Univ., Evanston, IL (United States); Olson, Mark A. [Northwestern Univ., Evanston, IL (United States); Benitez, Diego [California Institute of Technology (Caltech), Pasadena, CA (United States); Tkatchouk, Ekaterina [California Institute of Technology (Caltech), Pasadena, CA (United States); Barin, Gokhan [Northwestern Univ., Evanston, IL (United States); Yang, Jeffrey [Northwestern Univ., Evanston, IL (United States); Fahrenbach, Albert C. [Northwestern Univ., Evanston, IL (United States); Goddard, William A. [California Institute of Technology (Caltech), Pasadena, CA (United States); Stoddart, J. Fraser [Northwestern Univ., Evanston, IL (United States)


    Five donor–acceptor oligorotaxanes made up of dumbbells composed of tetraethylene glycol chains, interspersed with three and five 1,5-dioxynaphthalene units, and terminated by 2,6-diisopropylphenoxy stoppers, have been prepared by the threading of discrete numbers of cyclobis(paraquat-p-phenylene) rings, followed by a kinetically controlled stoppering protocol that relies on click chemistry. The well-known copper(I)-catalyzed alkyne–azide cycloaddition between azide functions placed at the ends of the polyether chains and alkyne-bearing stopper precursors was employed during the final kinetically controlled template-directed synthesis of the five oligorotaxanes, which were characterized subsequently by ¹H NMR spectroscopy at low temperature (233 K) in deuterated acetonitrile. The secondary structures, as well as the conformations, of the five oligorotaxanes were unraveled by spectroscopic comparison with the dumbbell and ring components. By focusing attention on the changes in chemical shifts of some key probe protons, obtained from a wide range of low-temperature spectra, a picture emerges of a high degree of folding within the thread protons of the dumbbells of four of the five oligorotaxanes—the fifth oligorotaxane represents a control compound in effect—brought about by a combination of C[BOND]H···O and π–π stacking interactions between the π-electron-deficient bipyridinium units in the rings and the π-electron-rich 1,5-dioxynaphthalene units and polyether chains in the dumbbells. The secondary structures of a foldamer-like nature have received further support from a solid-state superstructure of a related [3]pseudorotaxane and density functional calculations performed thereon.


    Directory of Open Access Journals (Sweden)

    Ştefan GHIMISI


    Full Text Available The present paper argues that adhesion forces and elastic deformation in the contact zone may contribute significantly to the relative displacement during fretting of metals. A simultaneously applied tangential force and normal into contact appears a adhesion force. A tangential force whose magnitude is less equal on greater than the force of limiting friction will not give rise on give rise to a sliding motion.It is determined the energy loss dissipated per fretting cycle.

  19. Effects of Contact Load on the Fretting Fatigue Behavior of IN-100 at Elevated Temperature (United States)


    experimental setup which did not accurately model the turbine engine environment. Kawagoishi et al. [23] studied the nickel-based alloy Inconel 718 ...In their study with Inconel 718 [48, 49], it was found that the beneficial glaze oxide had formed and did affect the fretting fatigue life. Their...Waterhouse [52] investigated the fretting fatigue behaviors of austenitic steel type 321 and nickel-based alloy Inconel 718 at elevated temperatures. He

  20. Alteration of cartilage glycosaminoglycan protein acceptor by somatomedin and cortisol. (United States)

    Kilgore, B S; McNatt, M L; Meador, S; Lee, J A; Hughes, E R; Elders, M J


    The effect of somatomedin and cortisol on embryonic chick cartilage in vitro indicates that somatomedin stimulates 35SO4 uptake while cortisol decreases it with no effect on glycosaminoglycan turnover. Xylosyltransferase activity is increased in crude fractions of somatomedin-treated cartilage but decreased in cortisol-treated cartilage. By using a Smith-degraded proteoglycan as an exogenous acceptor, xylosyltransferase activities from both treatments were equivalent, suggesting that the enzyme was not rate limiting. The results of xylosyltransferase assays conducted by mixing enzyme and endogenous acceptor from control, cortisol-treated and somatomedin-treated cartilage, suggest both effects to be at the level of the acceptor protein.

  1. Asymmetric Synthesis and Binding Study of New Long-Chain HPA-12 Analogues as Potent Ligands of the Ceramide Transfer Protein CERT. (United States)

    Ďuriš, Andrej; Daïch, Adam; Santos, Cécile; Fleury, Laurence; Ausseil, Frédéric; Rodriguez, Frédéric; Ballereau, Stéphanie; Génisson, Yves; Berkeš, Dušan


    A series of 12 analogues of the Cer transfer protein (CERT) antagonist HPA-12 with long aliphatic chains were prepared as their (1R,3S)-syn and (1R,3R)-anti stereoisomers from pivotal chiral oxoamino acids. The enantioselective access to these intermediates as well as their ensuing transformation relied on a practical crystallization-induced asymmetric transformation (CIAT) process. Sonogashira coupling followed by triple bond reduction and thiophene ring hydrodesulfurization (HDS) into the corresponding alkane moieties was then implemented to complete the synthetic routes delivering the targeted HPA-12 analogues in concise 4- to 6-step reaction sequences. Ten compounds were evaluated regarding their ability to bind to the CERT START domain by using the recently developed time-resolved FRET-based homogeneous (HTR-FRET) binding assay. The introduction of a lipophilic appendage on the phenyl moiety led to an overall 10- to 1000-fold enhancement of the protein binding, with the highest effect being observed for a n-hexyl residue in the meta position. The importance of the phenyl ring for the activity was indicated by the reduced potency of the 3-deoxyphytoceramide aliphatic analogues. The 1,3-syn stereoisomers were systematically more potent than their 1,3-anti analogues. In silico studies were used to rationalized these trends, leading to a model of protein recognition coherent with the stronger binding of (1R,3S)-syn HPAs.

  2. Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. (United States)

    Clifton, Ben E; Whitfield, Jason H; Sanchez-Romero, Inmaculada; Herde, Michel K; Henneberger, Christian; Janovjak, Harald; Jackson, Colin J


    Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here, we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic information to improve the thermostability of proteins, while circular permutation enables the termini of an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and discuss considerations for in situ characterization of the FRET sensors.

  3. Test Methodology of Reproducing Fuel Rod Failure by Debris Fretting Wear

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Oh Joon; Park, Nam Gyu; Kim, Jae Ik [KEPCO NF, Daejeon (Korea, Republic of)


    A test was conducted with simple debris to reproduce debris fretting wear. 68% of fuel rod cladding thickness is worn out by Inconel debris in 75 hours. The test result shows that a simple link system is useful to accommodate debris oscillation, and mid grid mixing vanes could be a source of debris forcing. Additional tests will be conducted with various debris such as wire brush, metal chip, etc which are suspected to generate actual debris fretting wear in future works. Debris fretting is one of the most common cause of the nuclear fuel rod failure. Even the most of the nuclear fuels has debris protection system, debris still cause fuel rod failure. From 1994 to 2006, debris fretting failure is around 11% of the total fuel failure. In 2006-2010, the portion of debris rises to over 13%. The total number of fuel rods failure is decreasing, but the portion of the debris fretting wear is growing with time. Therefore reproducing and identifying the mechanism of fuel rod failure by debris fretting wear is needed to improve reliability of the nuclear fuel.

  4. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening. (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam


    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

  5. Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species.

    Directory of Open Access Journals (Sweden)

    Chongzhao Ran

    Full Text Available BACKGROUND: Systematic differentiation of amyloid (Aβ species could be important for diagnosis of Alzheimer's disease (AD. In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathology, and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher molecular weight species (HrMW would be beneficial for drug screening, diagnosis, and molecular mechanism studies. However, fast and cheap methods for these specific aims are still lacking. PRINCIPAL FINDINGS: We demonstrated the feasibility of a non-conjugated FRET (Förster resonance energy transfer technique that utilized amyloid beta (Aβ species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivatives that served as the small molecule FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer solution. Lastly, this FRET technique enabled us to quantify the concentrations of Aβ monomers and high molecular weight species in solution. SIGNIFICANCE: We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.

  6. NASA/ESMD Analogue Mission Plans (United States)

    Hoffman, Stephen J.


    A viewgraph presentation exploring Earth and its analogues is shown. The topics include: 1) ESMD Goals for the Use of Earth Analogues; 2) Stakeholders Summary; 3) Issues with Current Analogue Situation; 4) Current state of Analogues; 5) External Implementation Plan (Second Step); 6) Recent Progress in Utilizing Analogues; 7) Website Layout Example-Home Page; 8) Website Layout Example-Analogue Site; 9) Website Layout Example-Analogue Mission; 10) Objectives of ARDIG Analog Initiatives; 11) Future Plans; 12) Example: Cold-Trap Sample Return; 13) Example: Site Characterization Matrix; 14) Integrated Analogue Studies-Prerequisites for Human Exploration; and 15) Rating Scale Definitions.

  7. Development of Polymer Acceptors for Organic Photovoltaic Cells

    Directory of Open Access Journals (Sweden)

    Yujeong Kim


    Full Text Available This review provides a current status report of the various n-type polymer acceptors for use as active materials in organic photovoltaic cells (OPVs. The polymer acceptors are divided into four categories. The first section of this review focuses on rylene diimide-based polymers, including perylene diimide, naphthalene diimide, and dithienocoronene diimide-based polymers. The high electron mobility and good stability of rylene diimides make them suitable for use as polymer acceptors in OPVs. The second section deals with fluorene and benzothiadiazole-based polymers such as poly(9,9’-dioctylfluorene-co-benzothiadiazole, and the ensuing section focuses on the cyano-substituted polymer acceptors. Cyano-poly(phenylenevinylene and poly(3-cyano-4-hexylthiophene have been used as acceptors in OPVs and exhibit high electron affinity arising from the electron-withdrawing cyano groups in the vinylene group of poly(phenylenevinylene or the thiophene ring of polythiophene. Lastly, a number of other electron-deficient groups such as thiazole, diketopyrrolopyrrole, and oxadiazole have also been introduced onto polymer backbones to induce n-type characteristics in the polymer. Since the first report on all-polymer solar cells in 1995, the best power conversion efficiency obtained with these devices to date has been 3.45%. The overall trend in the development of n-type polymer acceptors is presented in this review.

  8. Effects of temperature, slip amplitude, contact pressure on fretting fatigue behavior of Ti811 alloys at elevated temperatures

    Institute of Scientific and Technical Information of China (English)

    Xiaohua ZHANG; Daoxin LIU


    Effects of the temperature, slip amplitude, and contact pressure on fretting fatigue (FF) behavior of the Ti811 titanium alloy were investigated using a high frequency fatigue machine and a home-made high temperature apparatus. The fretting fatigue failure mechanism was studied by observing the fretting surface morphology features. The results show that the sensitivity to fretting fatigue is high at both 350 and 500 ℃. The higher the temperature, the more sensitive to the fretting fatigue failure is. Creep is an important factor that influences the fretting fatigue failure process at elevated temperatures. The fretting fatigue life of the Ti811 alloy does not change in a monotonic way as the slip amplitude and contact pressure increase. This is owing to the fact that the slip amplitude affects the action of fatigue and wear in the fretting process, and the nominal contact pressure affects the distribution and concentration of the stress and the amplitude of fretting slip at the contact surface, and thus further influences the crack initiation probability and the driving force for propagation.

  9. Analysis of initial crack path in fretting fatigue

    Directory of Open Access Journals (Sweden)

    J. Vázquez


    Full Text Available The initial crack path is analysed in a fretting fatigue test with cylindrical contact, where there is a stress gradient and a multiaxial and non-proportional stress state. For this, a cylindrical pad is pressed, with a constant normal load, N, against a dog-bone type fatigue test specimen. Then, the test specimen is subjected to a cyclic axial stress, σ. Due to the cyclical axial stress, the assembly used and the friction between the contact pair, a tangential cyclic load Q is generated. In these tests, both components are made of Al7075-T651 alloy. The crack initiation path along the fracture surface is optically measured using a focus variation technique. The contact stress/strain fields obtained analytically, in junction with the Fatemi-Socie (FS and Smith-Watson- Topper (SWT multiaxial fatigue parameters, allow us to determine the controlling parameters of the crack initiation process observed in the tests and to estimate the crack path during the early stage of the crack growth.

  10. Interference Assembly and Fretting Wear Analysis of Hollow Shaft

    Directory of Open Access Journals (Sweden)

    Chuanjun Han


    Full Text Available Fretting damage phenomenon often appears in the interference fit assembly. The finite element model of hollow shaft and shaft sleeve was established, and the equivalent stress and contact stress were computed after interference assembly. The assembly body of hollow shaft and shaft sleeve was in whirling bending load, and the contact status (sticking, sliding, and opening and the distribution of stress along one typical contact line were computed under different loads, interferences, hollow degrees, friction coefficient, and wear quantity. Judgment formula of contact state was fixed by introducing the corrected coefficient k. The computation results showed that the “edge effect” appears in the contact surface after interference fit. The size of slip zone is unchanged along with the increase of bending load. The greater the interference value, the bigger the wear range. The hollow degree does not influence the size of stick zone but controls the position of the junction point of slip-open. Tangential contact stress increases with the friction coefficient, which has a little effect on normal contact stress. The relationship between open size and wear capacity is approximately linear.

  11. Near infrared organic light-emitting diodes based on acceptor-donor-acceptor (ADA) using novel conjugated isatin Schiff bases

    Energy Technology Data Exchange (ETDEWEB)

    Taghi Sharbati, Mohammad, E-mail: [Department of Electrical and Electronics Engineering, Shiraz University of Technology, Shiraz (Iran, Islamic Republic of); Soltani Rad, Mohammad Navid, E-mail: [Department of Chemistry, Shiraz University of Technology, Shiraz 71555-313 (Iran, Islamic Republic of); Behrouz, Somayeh [Department of Chemistry, Shiraz University of Technology, Shiraz 71555-313 (Iran, Islamic Republic of); Gharavi, Alireza [Photonics Lab, Department of Electrical and Computer Engineering, Shiraz University, Shiraz (Iran, Islamic Republic of); Emami, Farzin [Department of Electrical and Electronics Engineering, Shiraz University of Technology, Shiraz (Iran, Islamic Republic of)


    Fabrications of a single layer organic light emitting diodes (OLEDs) based on two conjugated acceptor-donor-acceptor (ADA) isatin Schiff bases are described. The electroluminescent spectra of these materials range from 630 to 700 nm and their band gaps were measured between 1.97 and 1.77 eV. The measured maximum external quantum efficiencies (EQE) for fabricated OLEDs are 0.0515% and 0.054% for two acceptor-donor-acceptor chromophores. The Commission International De L'Eclairage (CIE) (1931) coordinates of these two compounds were attained and found to be (0.4077, 0.4128) and (0.4411, 0.4126) for two used acceptor-donor-acceptor chromophores. The measured I-V curves demonstrated the apparent diode behavior of two ADA chromophores. The turn-on voltages in these OLEDs are directly dependent on the thickness. These results have demonstrated that ADA isatin Schiff bases could be considered as promising electroluminescence-emitting materials for fabrication of OLEDs.

  12. A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology. (United States)

    Li, Chunjie; Imanishi, Ayako; Komatsu, Naoki; Terai, Kenta; Amano, Mutsuki; Kaibuchi, Kozo; Matsuda, Michiyuki


    Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.

  13. Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor. (United States)

    Ahmad, Mohammad; Ameen, Seema; Siddiqi, Tariq Omar; Khan, Parvez; Ahmad, Altaf


    Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Förster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells.

  14. A Study on Surface Modification of Al7075-T6 Alloy against Fretting Fatigue Phenomenon

    Directory of Open Access Journals (Sweden)

    E. Mohseni


    Full Text Available Aircraft engines, fuselage, automobile parts, and energy saving strategies in general have promoted the interest and research in the field of lightweight materials, typically on alloys based on aluminum. Aluminum alloy itself does not have suitable wear resistance; therefore, it is necessary to enhance surface properties for practical applications, particularly when aluminum is in contact with other parts. Fretting fatigue phenomenon occurs when two surfaces are in contact with each other and one or both parts are subjected to cyclic load. Fretting drastically decreases the fatigue life of materials. Therefore, investigating the fretting fatigue life of materials is an important subject. Applying surface modification methods is anticipated to be a supreme solution to gradually decreasing fretting damage. In this paper, the authors would like to review methods employed so far to diminish the effect of fretting on the fatigue life of Al7075-T6 alloy. The methods include deep rolling, shot peening, laser shock peening, and thin film hard coatings. The surface coatings techniques are comprising physical vapor deposition (PVD, hard anodizing, ion-beam-enhanced deposition (IBED, and nitriding.

  15. Designer Metallic Acceptor-Containing Halogen Bonding: General Strategies. (United States)

    Zhang, Xinxing; Bowen, Kit H


    Being electrostatic interactions in nature, hydrogen bonding (HB) and halogen bonding (XB) are considered to be two parallel worlds. In principle, all the applications that HB has could also be applied to XB. However, there has been no report on a metallic XB acceptor but metal anions have been observed to be good HB acceptors. This missing mosaic piece of XB is because common metal anions are reactive for XB donors. In view of this, we propose two strategies for designing metallic acceptor-containing XB using ab initio calculations. The first one is to utilize a metal cluster anion with a high electron detachment energy, such as the superatom, Al13- as the XB acceptor. The second strategy is to design a ligand passivated/protected metal core while it still can maintain the negative charge; several exotic clusters, such as PtH5-, PtZnH5- and PtMgH5-, are utilized as examples. Based on these two strategies, we anticipate that more metallic acceptor-containing XB will be discovered.

  16. Role of surface roughness on corrosion and fretting corrosion behaviour of commercially pure titanium in Ringer's solution for bio-implant application (United States)

    Sivakumar, Bose; Pathak, Lokesh Chandra; Singh, Raghuvir


    Influence of roughness (ra) from 43 to 474 nm on corrosion and fretting corrosion of commercially pure titanium (CpTi) was studied in the Ringer's solution. The anodic polarization and electrochemical impedance spectroscopy (EIS) revealed the highest corrosion resistance of CpTi with ra 43 nm and correlated well with the surface energy (SE). The highest potential drop associated with the fretting corrosion is observed for CpTi with ra 43 nm followed by 474 nm; this is found to correspond with the worn out area. The fretting current density (ifretting) is several order higher than obtained during the potentiodynamic polarization (without fretting) study. Fretting corrosion manifested by the drop in electrochemical potential is simulated with high accuracy using fretting current density and an initial contact area. Fretting corrosion at an applied potential (+250 mV(SCE)) is produced much larger fretting corrosion current density than during the open circuit potential (OCP).

  17. CdS-ZnSe和CdSe-ZnS量子点的合成和F(o)rster能量转移研究%Synthesis and F(o)rster Resonant Energy Transfer (FRET) Study of Quantum Dots Assembly

    Institute of Scientific and Technical Information of China (English)

    弓亚琼; 詹寰; 张贺楠; 卫增岩; 苏伟


    One F(o)rster resonant energy transfer (FRET) donor-acceptor assembly was built from quantum dots (QDs) of type Ⅰ CdSe-ZnS (QD491) and type Ⅲ CdS-ZnSe (QD560) QDs.The assembly was further characterized by steady-state and time-resolved photoluminescence (TRPL) measurements at different donor-to-acceptor ratios.Both photoluminescence quenching and reduction of the exciton lifetime of donor QDs provide concrete evidence of FRET between the type Ⅰ QDs and the type Ⅱ QDs.Because the emission wavelengths of CdS-ZnSe and CdSe-ZnS QDs can be spectrally separated by tuning the size of these QDs,the binary QD assembly of type Ⅰ-type Ⅱ QDs could be an attractive approach for the development of efficient light emitters and bio-sensors.%采用分步合成法成功合成了CdS-ZnSe(Ⅰ型)和CdSe-ZnS(Ⅱ型)核-壳结构量子点.并首次构建了CdS-ZnSe/CdSe-ZnS结构F(o)rster能量转移对.利用吸收光谱(UV-Vis),光致发光谱(PL),时间分辨光致发光谱(TRPL),透射电子显微镜(TEM)对比研究了不同配比的CdS-ZnSe/CdSe-ZnS结构Fö rster能量转移.分析结果表明:在能量转移发生时,CdS-ZnSe荧光强度显著下降,荧光寿命明显缩短,F(o)rster能量转移确实存在于CdS-ZnSe/CdSe-ZnS量子点之间.

  18. An overview of molecular acceptors for organic solar cells

    Directory of Open Access Journals (Sweden)

    Hudhomme Piétrick


    Full Text Available Organic solar cells (OSCs have gained serious attention during the last decade and are now considered as one of the future photovoltaic technologies for low-cost power production. The first dream of attaining 10% of power coefficient efficiency has now become a reality thanks to the development of new materials and an impressive work achieved to understand, control and optimize structure and morphology of the device. But most of the effort devoted to the development of new materials concerned the optimization of the donor material, with less attention for acceptors which to date remain dominated by fullerenes and their derivatives. This short review presents the progress in the use of non-fullerene small molecules and fullerene-based acceptors with the aim of evaluating the challenge for the next generation of acceptors in organic photovoltaics.

  19. Hyperpolarizability studies of some nonconjugated twin donor–acceptor molecules

    Indian Academy of Sciences (India)

    Elizabeth Chirackal Varkey; Krishnapillai Sreekumar


    Extensive theoretical calculation on the effects of spacer length enhancement on the second-order NLO properties of twin donor acceptor molecules having two amide units bridged by the CH2 spacers was performed. The role of such aliphatic bridges on the Donor–Acceptor groups was computed by ZINDO/CV quantum chemical formalism. The odd-even effects were observed in twin donor acceptor systems (with two aliphatic units) linked by an alkyl spacer of varying length from = 1 to = 12. The system considered for the present study was ,'-alkane-(1, ) diyl bis-4-hydroxy hexanamides. For an odd number of CH2 spacers, the value was an order of magnitude higher than that for the even number of CH2 spacers. The origin for such oscillation is attributed to the similar oscillations in the dipole moment difference between the ground state and the dipole allowed state and to some extent on the variation in the oscillator strength.

  20. Fretting fatigue behaviour of Ni-free high-nitrogen stainless steel in a simulated body fluid

    Directory of Open Access Journals (Sweden)

    Norio Maruyama, Sachiko Hiromoto, Eiji Akiyama and Morihiko Nakamura


    Full Text Available Fretting fatigue behaviour of Ni-free high-nitrogen steel (HNS with a yield strength of about 800 MPa, which was prepared by nitrogen gas pressurized electroslag remelting, was studied in air and in phosphate-buffered saline (PBS(-. For comparison, fretting fatigue behaviour of cold-rolled SUS316L steel (SUS316L(CR with similar yield strength was examined. The plain fatigue limit of HNS was slightly lower than that of SUS316L(CR although the former had a higher tensile strength than the latter. The fretting fatigue limit of HNS was higher than that of SUS316L(CR both in air and in PBS(-. A decrease in fatigue limit of HNS by fretting was significantly smaller than that of SUS316L(CR in both environments, indicating that HNS has better fretting fatigue resistance than SUS316L(CR. The decrease in fatigue limit by fretting is discussed taking into account the effect of friction stress due to fretting and the additional influences of wear, tribocorrosion and plastic deformation in the fretted area.

  1. Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene-perylene 2'-α-l-amino-LNA FRET pairs

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Santhosh Kumar, T; Campbell, Meghan A;


    Novel pyrene-perylene α-l-LNA FRET pairs described herein effectively detect assembly of 2- and 3-way branched DNA nanostructures prepared by postsynthetic microwave-assisted CuAAC click chemistry. The fluorescent signalling of assembly by internally positioned FRET pairs is achieved with low to ...

  2. Low-Frequency Reciprocating Fretting Wear Testing System Design and Experiment Research

    Institute of Scientific and Technical Information of China (English)

    Zhongnan Wang∗,Wuyi Wang; Guangyu Zhang


    The fretting wear is resulted from different or same sample’ s surfaces by the small variationand leads to mechanism failures. The main factors consist of the variation of normal load and oscillation frequencies, among which surface topography of different materials are the main factors to the problems of the fretting wear. Therefore, a novel low⁃frequency reciprocating fretting wear test system is designed upon the principle of Friction coefficient measurement. Four metal and non⁃metallic samples are measured under various normal load and oscillation frequencies to obtain the instantaneous friction coefficient in the repeat experiments. In fact, the experimental results show that CoF curves of different samples with the increase of the normal load are the similar exponential decay or parabolic shapes, which are consistent with the literatures to verify the rational design and reliable⁃operation of the system under the conditions of different frequencies.

  3. Analysis of Fretting Fatigue Strength of Integral Shroud Blade for Steam Turbine (United States)

    Kaneko, Yasutomo; Tomii, Masayuki; Ohyama, Hiroharu; Kurimura, Takayuki

    To improve the reliability and the thermal efficiency of LP (Low Pressure) end blades of steam turbine, new standard series of LP end blades have been developed. The new LP end blades are characterized by the ISB (Integral Shroud Blade) structure. In the ISB structure, blades are continuously coupled by blade untwist due to centrifugal force when the blades rotate at high speed. One of the probable failure modes of the ISB structure seems to be fretting fatigue, because the ISB utilizes friction damping between adjacent shrouds and stubs. Therefore, in order to design a blade with high reliability, the design procedure for evaluating the fretting fatigue strength was established by the model test and the nonlinear contact analysis. This paper presents the practical design method for predicting the fretting fatigue strength of the ISB structure, and the some applications are explained.


    Institute of Scientific and Technical Information of China (English)

    CAO Weixiao; ZHANG Peng; FENG Xinde


    Photoinduced charge transfer polymerization of styrene(St) with electron acceptor as initiator was investigated. In case of fumaronitrile (FN) or maleic anhydride (MA) as initiator the polymerization takes place regularly, whereas the tetrachloro-1, 4-benzenequinone (TCQ), 2, 3-dichloro-5, 6-dicyano-1, 4-benzenequinone (DDQ) . or tetracyano ethylene (TCNE) as initiator the polymerization proceeds reluctantly only after the photoaddition reaction. A mechanism was proposed that free radicals would be formed following the charge and proton transfer in the exciplex formed between St and electron acceptors.

  5. Introduction to electronic analogue computers

    CERN Document Server

    Wass, C A A


    Introduction to Electronic Analogue Computers, Second Revised Edition is based on the ideas and experience of a group of workers at the Royal Aircraft Establishment, Farnborough, Hants. This edition is almost entirely the work of Mr. K. C. Garner, of the College of Aeronautics, Cranfield. As various advances have been made in the technology involving electronic analogue computers, this book presents discussions on the said progress, including some acquaintance with the capabilities of electronic circuits and equipment. This text also provides a mathematical background including simple differen

  6. Analogue Methods in Palaeoecology: Using the analogue Package

    Directory of Open Access Journals (Sweden)

    Gavin L. Simpson


    Full Text Available Palaeoecology is an important branch of ecology that uses the subfossil remains of organisms preserved in lake, ocean and bog sediments to inform on changes in ecosystems and the environment through time. The analogue package contains functions to perform modern analogue technique (MAT transfer functions, which can be used to predict past changes in the environment, such as climate or lake-water pH from species data. A related technique is that of analogue matching, which is concerned with identifying modern sites that are floristically and faunistically similar to fossil samples. These techniques, and others, are increasingly being used to inform public policy on environmental pollution and conservation practices. These methods and other functionality in analogue are illustrated using the Surface Waters Acidification Project diatom:pH training set and diatom counts on samples of a sediment core from the Round Loch of Glenhead, Galloway, Scotland. The paper is aimed at palaeoecologists who are familiar with the techniques described but not with R.

  7. Role of bicarbonate at the acceptor side of photosystem II

    NARCIS (Netherlands)

    Rensen, van J.J.S.


    Besides being the substrate for the carboxylation reaction of photosynthesis, CO2 (bicarbonate) is required for the activity of Photosystem II (water plastoquinone oxido-reductase). It plays a role on the electron donor side as well as the electron acceptor side. In this contribution, attention will

  8. Covalent non-fused tetrathiafulvalene-acceptor systems. (United States)

    Pop, Flavia; Avarvari, Narcis


    Covalent donor-acceptor (D-A) systems have significantly contributed to the development of many organic materials and to molecular electronics. Tetrathiafulvalene (TTF) represents one of the most widely studied donor precursors and has been incorporated into the structure of many D-A derivatives with the objective of obtaining redox control and modulation of the intramolecular charge transfer (ICT), in order to address switchable emissive systems and to take advantage of its propensity to form regular stacks in the solid state. In this review, we focus on the main families of non-fused TTF-acceptors, which are classified according to the nature of the acceptor: nitrogen-containing heterocycles, BODIPY, perylenes and electron poor unsaturated hydrocarbons, as well as radical acceptors. We describe herein the most representative members of each family with a brief mention of their synthesis and a special focus on their D-A characteristics. Special attention is given to ICT and its modulation, fluorescence quenching and switching, photoconductivity, bistability and spin distribution by discussing and comparing spectroscopic and electrochemical features, photophysical properties, solid-state properties and theoretical calculations.

  9. Acceptors in II-IV Semiconductors - Incorporation and Complex Formation

    CERN Multimedia


    A strong effort is currently devoted to the investigation of defects and the electrical activation of dopant atoms in II-VI semiconductors. In particular, the knowledge about the behaviour of acceptors, prerequisite for the fabrication of p-type semiconductors, is rather limited. The perturbed $\\,{\\gamma\\gamma}$ -angular correlation technique (PAC) and the photoluminescence spectroscopy (PL) using the radioactive isotopes $^{77}\\!$Br and $^{111}\\!$Ag will be applied for investigating the behaviour of acceptor dopant atoms and their interactions with defects in II-VI semiconductors. The main topic will be the identification of the technical conditions for the incorporation of electrically active acceptors in the II-VI semiconductors ~ZnS, ZnSe, ZnTe, CdS, CdSe, and CdTe with particular emphasis on the compounds~ CdTe, ZnSe, and ZnTe. The investigations will be supplemented by first exploratory PL experiments with the group V acceptors $^{71}\\!$As and $^{121}\\!$Sb. With help of the probe $^{111}\\!$Ag, the pos...

  10. Excitation energy transfer in donor-bridge-acceptor systems. (United States)

    Albinsson, Bo; Mårtensson, Jerker


    This perspective will focus on the mechanistic aspects of singlet and triplet excitation energy transfer. Well defined donor-bridge-acceptor systems specifically designed for investigating the distance and energy gap dependencies of the energy transfer reactions are discussed along with some recent developments in computational modeling of the electronic coupling.

  11. Poly(trifluoromethyl)azulenes: structures and acceptor properties. (United States)

    Clikeman, Tyler T; Bukovsky, Eric V; Kuvychko, Igor V; San, Long K; Deng, Shihu H M; Wang, Xue-Bin; Chen, Yu-Sheng; Strauss, Steven H; Boltalina, Olga V


    Six new poly(trifluoromethyl)azulenes prepared in a single high-temperature reaction exhibit strong electron accepting properties in the gas phase and in solution and demonstrate the propensity to form regular π-stacked columns in donor-acceptor crystals when mixed with pyrene as a donor.

  12. Anaerobic methanotrophy in tidal wetland: Effects of electron acceptors (United States)

    Lin, Li-Hung; Yu, Zih-Huei; Wang, Pei-Ling


    Wetlands have been considered to represent the largest natural source of methane emission, contributing substantially to intensify greenhouse effect. Despite in situ methanogenesis fueled by organic degradation, methanotrophy also plays a vital role in controlling the exact quantity of methane release across the air-sediment interface. As wetlands constantly experience various disturbances of anthropogenic activities, biological burrowing, tidal inundation, and plant development, rapid elemental turnover would enable various electron acceptors available for anaerobic methanotrophy. The effects of electron acceptors on stimulating anaerobic methanotrophy and the population compositions involved in carbon transformation in wetland sediments are poorly explored. In this study, sediments recovered from tidally influenced, mangrove covered wetland in northern Taiwan were incubated under the static conditions to investigate whether anaerobic methanotrophy could be stimulated by the presence of individual electron acceptors. Our results demonstrated that anaerobic methanotrophy was clearly stimulated in incubations amended with no electron acceptor, sulfate, or Fe-oxyhydroxide. No apparent methane consumption was observed in incubations with nitrate, citrate, fumarate or Mn-oxides. Anaerobic methanotrophy in incubations with no exogenous electron acceptor appears to proceed at the greatest rates, being sequentially followed by incubations with sulfate and Fe-oxyhydroxide. The presence of basal salt solution stimulated methane oxidation by a factor of 2 to 3. In addition to the direct impact of electron acceptor and basal salts, incubations with sediments retrieved from low tide period yielded a lower rate of methane oxidation than from high tide period. Overall, this study demonstrates that anaerobic methanotrophy in wetland sediments could proceed under various treatments of electron acceptors. Low sulfate content is not a critical factor in inhibiting methane

  13. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine. (United States)

    Ma, Dong Hee; Kim, Dokyoung; Akisawa, Takuya; Lee, Kyung-Ha; Kim, Kyong-Tai; Ahn, Kyo Han


    A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

  14. Fretting fatigue behavior of high-strength steel monostrands under bending load

    DEFF Research Database (Denmark)

    Winkler, Jan; Georgakis, Christos T.; Fischer, Gregor


    In this paper, the fretting fatigue behavior of pretensioned high-strength steel monostrands is investigated. To measure the local deformations on the strands, a novel method based on the digital image correlation (DIC) technique was used to quantify the relative movement between individual wires...

  15. Time-gated FRET nanoassemblies for rapid and sensitive intra- and extracellular fluorescence imaging

    NARCIS (Netherlands)

    Afsari, Hamid Samareh; Cardoso Dos Santos, Marcelina; Lindén, Stina; Chen, Ting; Qiu, Xue; van Bergen En Henegouwen, Paul M P; Jennings, Travis L; Susumu, Kimihiro; Medintz, Igor L; Hildebrandt, Niko; Miller, Lawrence W


    Time-gated Förster resonance energy transfer (FRET) using the unique material combination of long-lifetime terbium complexes (Tb) and semiconductor quantum dots (QDs) provides many advantages for highly sensitive and multiplexed biosensing. Although time-gated detection can efficiently suppress samp

  16. Metal ion induced FRET OFF-ON in tren/dansyl-appended rhodamine. (United States)

    Lee, Min Hee; Kim, Hyun Jung; Yoon, Sangwoon; Park, Noejung; Kim, Jong Seung


    A series of new fluorescent probes bearing tren-spaced rhodamine B and dansyl groups have been synthesized. Compound 1 exhibits selective changes in the absorption and the emission spectra toward Cu2+ ion over miscellaneous metal cations. Among 1-3, 1 shows the best FRET efficiency through dansyl emission to rhodamine absorption for the Cu2+ ion.

  17. Single-Molecule FRET Measurements in Additive-Enriched Aqueous Solutions. (United States)

    Kempe, Daryan; Cerminara, Michele; Poblete, Simón; Schöne, Antonie; Gabba, Matteo; Fitter, Jörg


    The addition of high amounts of chemical denaturants, salts, viscosity enhancers or macro-molecular crowding agents has an impact on the physical properties of buffer solutions. Among others, the (microscopic) viscosity, the refractive index, the dielectric constant, and the ionic strength can be affected. Here, we systematically evaluate the importance of solvent characteristics with respect to single-molecule FRET (smFRET) data. First, we present a confocal based method for the determination of fluorescence quantum yields to facilitate a fast characterization of smFRET-samples at sub-nM-concentrations. As a case study, we analyze smFRET data of structurally rigid, double-stranded DNA-oligonucleotides in aqueous buffer and in buffers with specific amounts of glycerol, guanidine hydrochloride (GdnHCl), and sodium chloride (NaCl) added. We show that the calculation of interdye distances, without taking into account solvent-induced spectral and photophysical changes of the labels, leads to deviations of up to 4 Å from the real interdye distances. Additionally, we demonstrate that electrostatic dye-dye repulsions are negligible for the interdye distance regime considered here (>50 Å). Finally, we use our approach to validate the further compaction of the already unfolded state of phosphoglycerate kinase (PGK) with decreasing denaturant concentrations, a mechanism known as coil-globule transition.

  18. Imaging real-time HIV-1 virion fusion with FRET-based biosensors (United States)

    Jones, Daniel M.; Padilla-Parra, Sergi


    We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously. PMID:26300212

  19. Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements

    Directory of Open Access Journals (Sweden)

    Pauline Vandame


    Full Text Available Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i fluorophores and (ii linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.

  20. Titanium carbide nanoparticles reinforcing nickel matrix for improving nanohardness and fretting wear properties in wet conditions (United States)

    Dănăilă, Eliza; Benea, Lidia; Caron, Nadège; Raquet, Olivier


    In this study Ni/nano-TiC functional composite coatings were produced by electro-codeposition of TiC nanoparticles (50 nm mean diameter) with nickel on 304L stainless steel support. Coatings were obtained from a Watts classical solution in which TiC nanoparticles were added. The surface morphology, chemical composition, structure, roughness and thickness, were evaluated in relation to the effect of TiC nanoparticles incorporation into Ni matrix. It was found that incorporation of TiC nanoparticles into the nickel matrix produces morphological changes in the deposit and increases the roughness. The fretting wear behavior in wet conditions of the obtained coatings was evaluated on a ball-on-plate configuration. To evaluate the wet fretting wear (tribocorrosion) behavior the open circuit potential was measured before, during and after the fretting tests at room temperature in the solution that simulates the primary water circuit of Pressurized Water Reactors. The results show that Ni/nano-TiC composite coatings exhibited a low friction coefficient, high nanohardness and fretting wear resistance in wet conditions compared with pure Ni coatings.

  1. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

    NARCIS (Netherlands)

    Hohlbein, J.; Kapanidis, A.N.


    Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open

  2. Corrosion and Fretting of a Modular Hip System: A Retrieval Analysis of 60 Rejuvenate Stems. (United States)

    De Martino, Ivan; Assini, Joseph B; Elpers, Marcella E; Wright, Timothy M; Westrich, Geoffrey H


    Femoral stems with dual-taper modularity were introduced to allow independent control of length, offset, and version. Corrosion and fretting related to micromotion at the neck-stem junction are thought to stimulate an adverse local tissue reaction (ALTR). Analysis of 60 consecutively retrieved modular-neck stem implants (Rejuvenate, Stryker) revised primarily for ALTR was done to determine the variables influencing corrosion and fretting patterns at the neck-stem interface. Taper damage evaluation was performed with stereomicrocopic analysis with two observers. Evidence of fretting and corrosion was seen at the neck-stem taper in all implants, including three implants revised for periprosthetic fractures within four weeks of the index surgery indicating that this process starts early. Femoral stems paired with the long overall neck lengths had significantly higher corrosion scores. Correlation of the corrosion severity at particular locations with the length of implantation suggests that the neck-stem junction experiences cyclic cantilever bending in vivo. The positive correlation between the length of implantation and fretting/corrosion scores bodes poorly for patients who still have this implant. Scanning electron microscopy on a subset of specimens was also performed to evaluate the black corrosion material. We strongly urge frequent follow-up exams for every patient with this particular modular hip stem.

  3. A dual-color far-red to near-infrared firefly luciferin analogue designed for multiparametric bioluminescence imaging. (United States)

    Jathoul, Amit P; Grounds, Helen; Anderson, James C; Pule, Martin A


    Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λ(max)=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

  4. A biocatalytic approach to capuramycin analogues by exploiting a substrate permissive N-transacylase CapW. (United States)

    Liu, Xiaodong; Jin, Yuanyuan; Cai, Wenlong; Green, Keith D; Goswami, Anwesha; Garneau-Tsodikova, Sylvie; Nonaka, Koichi; Baba, Satoshi; Funabashi, Masanori; Yang, Zhaoyong; Van Lanen, Steven G


    Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from a methyl ester precursor as an acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed that this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.

  5. Measuring Mental Imagery with Visual Analogue Scales. (United States)

    Quilter, Shawn M.; Band, Jennie P.; Miller, Gary M.


    Investigates some of the psychometric characteristics of the results from visual-analogue scales used to measure mental imagery. Reports that the scores from visual-analogue scales are positively related to scores from longer pencil-and-paper measures of mental imagery. Implications and limitations for the use of visual-analogue scales to measure…

  6. Influence of oil and water media on fretting behaviour of AISI 52100 steel rubbing against AISI 1045 steel

    Institute of Scientific and Technical Information of China (English)

    REN Ping-di; CHEN Guang-xiong; ZHU Min-hao; ZHOU Zhong-rong


    A series of fretting test were carried out using a DELTA PLINT testing system to study the influence of hydraulic oil and water on fretting behaviour of AISI 52100 steel rubbing against AISI 1045 steel. The test result shows that media hydraulic oil and water have a distinct influence on fretting behaviour of the tested materials. Medium water can lead to shifting of the partial slip regime in the fretting map from a larger displacement amplitude toward a smaller one and enlargement of the mixed slip regime, in comparison with that in ambient atmosphere. While medium hydraulic oil can result in shifting of the partial slip regime from a smaller displacement amplitude toward a larger one. In the gross slip regime, hydraulic oil and water play a positive role as lubrication media. They can clearly decrease the fretting friction coefficient between AISI 52100 and AISI 1045. The test result also demonstrates that this lubrication effect will get better with increasing displacement amplitude and that hydraulic oil is better than water for lubrication. SEM observation of the wear scars displays that the fretting wear mainly results from abrasive wear and delamination of the fretted materials when using these two kinds of substances as lubrication media.

  7. 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.

    Directory of Open Access Journals (Sweden)

    Jeffrey Klarenbeek

    Full Text Available 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 μM, 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.

  8. Temporal Data Set Reduction Based on D-Optimality for Quantitative FLIM-FRET Imaging. (United States)

    Omer, Travis; Intes, Xavier; Hahn, Juergen


    Fluorescence lifetime imaging (FLIM) when paired with Förster resonance energy transfer (FLIM-FRET) enables the monitoring of nanoscale interactions in living biological samples. FLIM-FRET model-based estimation methods allow the quantitative retrieval of parameters such as the quenched (interacting) and unquenched (non-interacting) fractional populations of the donor fluorophore and/or the distance of the interactions. The quantitative accuracy of such model-based approaches is dependent on multiple factors such as signal-to-noise ratio and number of temporal points acquired when sampling the fluorescence decays. For high-throughput or in vivo applications of FLIM-FRET, it is desirable to acquire a limited number of temporal points for fast acquisition times. Yet, it is critical to acquire temporal data sets with sufficient information content to allow for accurate FLIM-FRET parameter estimation. Herein, an optimal experimental design approach based upon sensitivity analysis is presented in order to identify the time points that provide the best quantitative estimates of the parameters for a determined number of temporal sampling points. More specifically, the D-optimality criterion is employed to identify, within a sparse temporal data set, the set of time points leading to optimal estimations of the quenched fractional population of the donor fluorophore. Overall, a reduced set of 10 time points (compared to a typical complete set of 90 time points) was identified to have minimal impact on parameter estimation accuracy (≈5%), with in silico and in vivo experiment validations. This reduction of the number of needed time points by almost an order of magnitude allows the use of FLIM-FRET for certain high-throughput applications which would be infeasible if the entire number of time sampling points were used.

  9. Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements.

    Directory of Open Access Journals (Sweden)

    Rosemary A Bamford

    Full Text Available Förster resonance energy transfer (FRET technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5 complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

  10. An organic donor/acceptor lateral superlattice at the nanoscale. (United States)

    Otero, Roberto; Ecija, David; Fernandez, Gustavo; Gallego, José María; Sanchez, Luis; Martín, Nazario; Miranda, Rodolfo


    A precise control of the nanometer-scale morphology in systems containing mixtures of donor/acceptor molecules is a key factor to improve the efficiency of organic photovoltaic devices. Here we report on a scanning tunneling microscopy study of the first stages of growth of 2-[9-(1,3-dithiol-2-ylidene)anthracen-10(9H)-ylidene]-1,3-dithiole, as electron donor, and phenyl-C61-butyric acid methyl ester, as electron acceptor, on a Au(111) substrate under ultrahigh vacuum conditions. Due to differences in bonding strength with the substrate and different interactions with the Au(111) herringbone surface reconstruction, mixed thin films spontaneously segregate into a lateral superlattice of interdigitated nanoscale stripes with a characteristic width of about 10-20 nm, a morphology that has been predicted to optimize the efficiency of organic solar cells.

  11. Lipase-catalyzed biodiesel synthesis with different acyl acceptors

    Directory of Open Access Journals (Sweden)

    Ognjanović Nevena D.


    Full Text Available Biodiesel is an alternative fuel for diesel engine that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The aim of the present work was to investigate novel acyl acceptors for biodiesel production. 2-Propanol and n-butanol have a less negative effect on lipase stability, and they also improve low temperature properties of the fuel. However, excess alcohol leads to inactivation of the enzyme, and glycerol, a major byproduct, can block the immobilized enzyme, resulting in low enzymatic activity. This problem was solved by using methyl acetate as acyl acceptor. Triacetylglycerol is produced instead of glycerol, and it has no negative effect on the activity of the lipase.

  12. Selection of electron acceptors and strategies for in situ bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Norris, R.D. [Eckenfelder, Inc., Nashville, TN (United States)


    The most critical aspect of designing in situ bioremediation systems is, typically, the selection and method of delivery of the electron acceptor. Nitrate, sulfate, and several forms of oxygen can be introduced, depending on the contaminants and the site conditions. Oxygen can be added as air, pure oxygen, hydrogen peroxide, or an oxygen release compound. Simplistic cost calculations can illustrate the advantages of some methods over others, providing technical requirements can be met.

  13. Electron Acceptor Materials Engineering in Colloidal Quantum Dot Solar Cells

    KAUST Repository

    Liu, Huan


    Lead sulfide colloidal quantum dot (CQD) solar cells with a solar power conversion efficiency of 5.6% are reported. The result is achieved through careful optimization of the titanium dioxide electrode that serves as the electron acceptor. Metal-ion-doped sol-gel-derived titanium dioxide electrodes produce a tunable-bandedge, well-passivated materials platform for CQD solar cell optimization. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Neuronal Analogues of Conditioning Paradigms (United States)


    Although the mechanisms of interneuronal communication have been well established, the changes underlying most forms of learning have thus far eluded...stimulating electrodes on one of the connectives was adjusted so as to produce a small excitatory postsynaptic potential ( EPSP ) in the impaled cell...two stimuli would constitute a neuronal analogue of conditioning by producing an increased EPSP in response to the test stimulus alone. If so, then

  15. Income-generating activities for family planning acceptors. (United States)


    The Income Generating Activities program for Family Planning Acceptors was introduced in Indonesia in 1979. Capital input by the Indonesian National Family Planning Coordination Board and the UN Fund for Population Activities was used to set up small businesses by family planning acceptors. In 2 years, when the businesses become self-sufficient, the loans are repaid, and the money is used to set up new family planning acceptors in business. The program strengthens family planning acceptance, improves the status of women, and enhances community self-reliance. The increase in household income generated by the program raises the standards of child nutrition, encourages reliance on the survival of children, and decreases the value of large families. Approximately 18,000 Family Planning-Income Generating Activities groups are now functioning all over Indonesia, with financial assistance from the central and local governments, the World Bank, the US Agency for International Development, the UN Population Fund, the Government of the Netherlands, and the Government of Australia through the Association of South East Asian Nations.

  16. Fullerene derivatives as electron acceptors for organic photovoltaic cells. (United States)

    Mi, Dongbo; Kim, Ji-Hoon; Kim, Hee Un; Xu, Fei; Hwang, Do-Hoon


    Energy is currently one of the most important problems humankind faces. Depletion of traditional energy sources such as coal and oil results in the need to develop new ways to create, transport, and store electricity. In this regard, the sun, which can be considered as a giant nuclear fusion reactor, represents the most powerful source of energy available in our solar system. For photovoltaic cells to gain widespread acceptance as a source of clean and renewable energy, the cost per watt of solar energy must be decreased. Organic photovoltaic cells, developed in the past two decades, have potential as alternatives to traditional inorganic semiconductor photovoltaic cells, which suffer from high environmental pollution and energy consumption during production. Organic photovoltaic cells are composed of a blended film of a conjugated-polymer donor and a soluble fullerene-derivative acceptor sandwiched between a poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)-coated indium tin oxide positive electrode and a low-work-function metal negative electrode. Considerable research efforts aim at designing and synthesizing novel fullerene derivatives as electron acceptors with up-raised lowest unoccupied molecular orbital energy, better light-harvesting properties, higher electron mobility, and better miscibility with the polymer donor for improving the power conversion efficiency of the organic photovoltaic cells. In this paper, we systematically review novel fullerene acceptors synthesized through chemical modification for enhancing the photovoltaic performance by increasing open-circuit voltage, short-circuit current, and fill factor, which determine the performance of organic photovoltaic cells.

  17. Donor-Acceptor Block Copolymers: Synthesis and Solar Cell Applications

    Directory of Open Access Journals (Sweden)

    Kazuhiro Nakabayashi


    Full Text Available Fullerene derivatives have been widely used for conventional acceptor materials in organic photovoltaics (OPVs because of their high electron mobility. However, there are also considerable drawbacks for use in OPVs, such as negligible light absorption in the visible-near-IR regions, less compatibility with donor polymeric materials and high cost for synthesis and purification. Therefore, the investigation of non-fullerene acceptor materials that can potentially replace fullerene derivatives in OPVs is increasingly necessary, which gives rise to the possibility of fabricating all-polymer (polymer/polymer solar cells that can deliver higher performance and that are potentially cheaper than fullerene-based OPVs. Recently, considerable attention has been paid to donor-acceptor (D-A block copolymers, because of their promising applications as fullerene alternative materials in all-polymer solar cells. However, the synthesis of D-A block copolymers is still a challenge, and therefore, the establishment of an efficient synthetic method is now essential. This review highlights the recent advances in D-A block copolymers synthesis and their applications in all-polymer solar cells.

  18. Virtual screening of electron acceptor materials for organic photovoltaic applications (United States)

    Halls, Mathew D.; Djurovich, Peter J.; Giesen, David J.; Goldberg, Alexander; Sommer, Jonathan; McAnally, Eric; Thompson, Mark E.


    Virtual screening involves the generation of structure libraries, automated analysis to predict properties related to application performance and subsequent screening to identify lead systems and estimate critical structure-property limits across a targeted chemical design space. This approach holds great promise for informing experimental discovery and development efforts for next-generation materials, such as organic semiconductors. In this work, the virtual screening approach is illustrated for nitrogen-substituted pentacene molecules to identify systems for development as electron acceptor materials for use in organic photovoltaic (OPV) devices. A structure library of tetra-azapentacenes (TAPs) was generated by substituting four nitrogens for CH at 12 sites on the pentacene molecular framework. Molecular properties (e.g. ELUMO, Eg and μ) were computed for each candidate structure using hybrid DFT at the B3LYP/6-311G** level of theory. The resulting TAPs library was then analyzed with respect to intrinsic properties associated with OPV acceptor performance. Marcus reorganization energies for charge transport for the most favorable TAP candidates were then calculated to further determine suitability as OPV electron acceptors. The synthesis, characterization and OPV device testing of TAP materials is underway, guided by these results.

  19. Design directed self-assembly of donor-acceptor polymers. (United States)

    Marszalek, Tomasz; Li, Mengmeng; Pisula, Wojciech


    Donor-acceptor polymers with an alternating array of donor and acceptor moieties have gained particular attention during recent years as active components of organic electronics. By implementation of suitable subunits within the conjugated backbone, these polymers can be made either electron-deficient or -rich. Additionally, their band gap and light absorption can be precisely tuned for improved light-harvesting in solar cells. On the other hand, the polymer design can also be modified to encode the desired supramolecular self-assembly in the solid-state that is essential for an unhindered transport of charge carriers. This review focuses on three major factors playing a role in the assembly of donor-acceptor polymers on surfaces which are (1) nature, geometry and substitution position of solubilizing alkyl side chains, (2) shape of the conjugated polymer defined by the backbone curvature, and (3) molecular weight which determines the conjugation length of the polymer. These factors adjust the fine balance between attractive and repulsive forces and ensure a close polymer packing important for an efficient charge hopping between neighboring chains. On the microscopic scale, an appropriate domain formation with a low density of structural defects in the solution deposited thin film is crucial for the charge transport. The charge carrier transport through such thin films is characterized by field-effect transistors as basic electronic elements.


    Energy Technology Data Exchange (ETDEWEB)

    McCusker, James


    The upcoming incarnation of the Gordon Research Conference on Electron Donor Acceptor Interactions will feature sessions on classic topics including proton-coupled electron transfer, dye-sensitized solar cells, and biological electron transfer, as well as emerging areas such as quantum coherence effects in donor-acceptor interactions, spintronics, and the application of donor-acceptor interactions in chemical synthesis.

  1. Novel acetylcholine and carbamoylcholine analogues

    DEFF Research Database (Denmark)

    Hansen, Camilla Petrycer; Jensen, Anders Asbjørn; Christensen, Jeppe K.;


    A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha 4beta 2 nAChR and pronounced selectivity for this ......A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha 4beta 2 nAChR and pronounced selectivity...... for this subtype over alpha 3beta 4, alpha 4beta 4, and alpha 7 nAChRs. The high nAChR activity of carbamoylcholine analogue 5d was found to reside in its R-enantiomer, a characteristic most likely true for all other compounds in the series. Interestingly, the pronounced alpha 4beta 2 selectivities exhibited......AChR agonists published to date. Ligand-protein docking experiments using homology models of the amino-terminal domains of alpha 4beta 2 and alpha 3beta 4 nAChRs identified residues Val111(beta 2)/Ile113(beta 4), Phe119(beta 2)/Gln121(beta 4), and Thr155(alpha 4)/Ser150(alpha 3) as possible key determinants...

  2. Substrate analogues for isoprenoid enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Stremler, K.E.


    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  3. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method (United States)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju


    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  4. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design (United States)

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten


    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (ɛΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

  5. Spectral, thermal and kinetic studies of charge-transfer complexes formed between the highly effective antibiotic drug metronidazole and two types of acceptors: σ- and π-acceptors. (United States)

    Refat, Moamen S; Saad, Hosam A; Adam, Abdel Majid A


    Understanding the interaction between drugs and small inorganic or organic molecules is critical in being able to interpret the drug-receptor interactions and acting mechanism of these drugs. A combined solution and solid state study was performed to describe the complexation chemistry of drug metronidazole (MZ) which has a broad-spectrum antibacterial activity with two types of acceptors. The acceptors include, σ-acceptor (i.e., iodine) and π-acceptors (i.e., dichlorodicyanobenzoquinone (DDQ), chloranil (CHL) and picric acid (PA)). The molecular structure, spectroscopic characteristics, the binding modes as well as the thermal stability were deduced from IR, UV-vis, (1)H NMR and thermal studies. The binding ratio of complexation (MZ: acceptor) was determined to be 1:2 for the iodine acceptor and 1:1 for the DDQ, CHL or PA acceptor, according to the CHN elemental analyses and spectrophotometric titrations. It has been found that the complexation with CHL and PA acceptors increases the values of enthalpy and entropy, while the complexation with DDQ and iodine acceptors decreases the values of these parameters compared with the free MZ donor.

  6. Ultrafast Photoinduced Electron Transfer in Bimolecular Donor-Acceptor Systems

    KAUST Repository

    Alsulami, Qana A.


    The efficiency of photoconversion systems, such as organic photovoltaic (OPV) cells, is largely controlled by a series of fundamental photophysical processes occurring at the interface before carrier collection. A profound understanding of ultrafast interfacial charge transfer (CT), charge separation (CS), and charge recombination (CR) is the key determinant to improving the overall performances of photovoltaic devices. The discussion in this dissertation primarily focuses on the relevant parameters that are involved in photon absorption, exciton separation, carrier transport, carrier recombination and carrier collection in organic photovoltaic devices. A combination of steady-state and femtosecond broadband transient spectroscopies was used to investigate the photoinduced charge carrier dynamics in various donor-acceptor systems. Furthermore, this study was extended to investigate some important factors that influence charge transfer in donor-acceptor systems, such as the morphology, energy band alignment, electronic properties and chemical structure. Interestingly, clear correlations among the steady-state measurements, time-resolved spectroscopy results, grain alignment of the electron transporting layer (ETL), carrier mobility, and device performance are found. In this thesis, we explored the significant impacts of ultrafast charge separation and charge recombination at donor/acceptor (D/A) interfaces on the performance of a conjugated polymer PTB7-Th device with three fullerene acceptors: PC71BM, PC61BM and IC60BA. Time-resolved laser spectroscopy and high-resolution electron microscopy can illustrate the basis for fabricating solar cell devices with improved performances. In addition, we studied the effects of the incorporation of heavy metals into π-conjugated chromophores on electron transfer by monitoring the triplet state lifetime of the oligomer using transient absorption spectroscopy, as understanding the mechanisms controlling intersystem crossing and

  7. Study of fretting wear in titanium, Monel-400, and cobalt-25 percent molybdenum using scanning electron microscopy. (United States)

    Bill, R. C.


    Damage scar volume measurements taken from like metal fretting pairs, combined with scanning electron microscopy observations, showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt-25% molybdenum. Initially, adhesion and plastic deformation on the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism, producing spall-like pits in the damage scar. Finally, an oxidation-related mechanism became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

  8. Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells (United States)

    Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.


    Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

  9. Fretting corrosion behavior of nitinol spinal rods in conjunction with titanium pedicle screws. (United States)

    Lukina, Elena; Kollerov, Mikhail; Meswania, Jay; Khon, Alla; Panin, Pavel; Blunn, Gordon W


    Untypical corrosion damage including erosions combined with the build-up of titanium oxide as a corrosion product on the surface of explanted Nitinol spinal rods in the areas where it was in contact with titanium pedicle screw head is reported. It was suggested that Nitinol rods might have inferior fretting corrosion resistance compared with that made of titanium or CoCr. Fretting corrosion of Nitinol spinal rods with titanium (Ti6Al4V) pedicle screws were tested in-vitro by conducting a series of potentiostatic measurements of the peak-to-peak values of fretting corrosion current under bending in a 10% solution of calf serum in PBS. The test included Nitinol rods locked in titanium pedicle screws of different designs. Performance of commercially available titanium (Ti6Al4V) and CoCr spinal rods was also investigated for a comparison. Corrosion damage observed after the in-vitro tests was studied using SEM and EDAX analysis and was compared with patterns on Nitinol rods retrieved 12months after initial surgery. Metal ions level was measured in the test media after in-vitro experiments and in the blood and tissues of the patients who had the rods explanted. The results of this study revealed that Nitinol spinal rods locked in Ti pedicle screws are susceptible to fretting corrosion demonstrating higher fretting corrosion current compared with commercially used Ti6Al4V and CoCr rods. On the surface of Nitinol rods after in-vitro tests and on those retrieved from the patients similar corrosion patterns were observed. Improved resistance to fretting corrosion was observed with Nitinol rods in the in-vitro tests where pedicle screws were used with a stiffer locking mechanism. Since the development of the localized corrosion damage might increase the risk of premature fatigue failure of the rods and result in leaching of Ni ions, it is concluded that Nitinol rods should not be used in conjunction with Ti pedicle screws without special protection especially where the

  10. The effect of intramolecular donor–acceptor moieties with donor–π-bridge–acceptor structure on the solar photovoltaic performance

    Directory of Open Access Journals (Sweden)

    T. L. Wang


    Full Text Available A series of intramolecular donor–acceptor polymers containing different contents of (E-1-(2-ethylhexyl-6,9-dioctyl-2-(2-(thiophen-3-ylvinyl-1H-phenanthro[9,10-d]imidazole (thiophene-DOPI moiety and 4,4-diethylhexylcyclopenta[ 2,1-b:3,4-b']dithiophene (CPDT unit was synthesized via Grignard metathesis (GRIM polymerization. The synthesized random copolymers and homopolymer of thiophene-DOPI contain the donor–π-bridge–acceptor conjugated structure to tune the absorption spectra and energy levels of the resultant polymers. UV-vis spectra of the three polymer films exhibit panchromatic absorptions ranging from 300 to 1100 nm and low band gaps from 1.38 to 1.51 eV. It is found that more thiophene-DOPI moieties result in the decrease of band gap and lower the highest occupied molecular orbital (HOMO and lowest unoccupied molecular orbital (LUMO values of polymers. Photovoltaic performance results indicate that if the content of the intramolecular donor–acceptor moiety is high enough, the copolymer structure may be better than homopolymer due to more light-harvesting afforded by both monomer units.

  11. Dynamics of iron-acceptor-pair formation in co-doped silicon

    Energy Technology Data Exchange (ETDEWEB)

    Bartel, T.; Gibaja, F.; Graf, O.; Gross, D.; Kaes, M.; Heuer, M.; Kirscht, F. [Calisolar GmbH, Magnusstrasse 11, 12489 Berlin (Germany); Möller, C. [CiS Forschungsinstitut für Mikrosensorik und Photovoltaik GmbH, Konrad-Zuse-Str. 14, 99099 Erfurt (Germany); TU Ilmenau, Institut für Physik, Weimarer Str. 32, 98693 Ilmenau (Germany); Lauer, K. [CiS Forschungsinstitut für Mikrosensorik und Photovoltaik GmbH, Konrad-Zuse-Str. 14, 99099 Erfurt (Germany)


    The pairing dynamics of interstitial iron and dopants in silicon co-doped with phosphorous and several acceptor types are presented. The classical picture of iron-acceptor pairing dynamics is expanded to include the thermalization of iron between different dopants. The thermalization is quantitatively described using Boltzmann statistics and different iron-acceptor binding energies. The proper understanding of the pairing dynamics of iron in co-doped silicon will provide additional information on the electronic properties of iron-acceptor pairs and may become an analytical method to quantify and differentiate acceptors in co-doped silicon.

  12. Recognition of Gene Acceptor Site Based on Multi-objective Optimization

    Institute of Scientific and Technical Information of China (English)

    Jing ZHAO; Yue-Min ZHU; Pei-Ming SONG; Qing FANG; Jian-Hua LUO


    A new method for predicting the gene acceptor site based on multi-objective optimization is introduced in this paper. The models for the acceptor, branch and distance between acceptor site and branch site were constructed according to the characteristics of the sequences from the exon-intron database and using common biological knowledge. The acceptor function, branch function and distance function were defined respectively, and the multi-objective optimization model was constructed to recognize the splice site. The test results show that the algorithm used in this study performs better than the SplicePredictor,which is one of the leading acceptor site detectors.

  13. Experimental and Numerical Investigations of Fretting Fatigue Behavior for Steel Q235 Single-Lap Bolted Joints

    Directory of Open Access Journals (Sweden)

    Yazhou Xu


    Full Text Available This work aims to investigate the fretting fatigue life and failure mode of steel Q235B plates in single-lap bolted joints. Ten specimens were prepared and tested to fit the S-N curve. SEM (scanning electron microscope was then employed to observe fatigue crack surfaces and identify crack initiation, crack propagation, and transient fracture zones. Moreover, a FEM model was established to simulate the stress and displacement fields. The normal contact stress, tangential contact stress, and relative slipping displacement at the critical fretting zone were used to calculate FFD values and assess fretting fatigue crack initiation sites, which were in good agreement with SEM observations. Experimental results confirmed the fretting fatigue failure mode for these specimens. It was found that the crack initiation resulted from wear regions at the contact surfaces between plates, and fretting fatigue cracks occurred at a certain distance away from hole edges. The proposed FFD-N relationship is an alternative approach to evaluate fretting fatigue life of steel plates in bolted joints.

  14. FRET-based nanoscale point-to-point and broadcast communications with multi-exciton transmission and channel routing. (United States)

    Kuscu, Murat; Akan, Ozgur B


    Nanoscale communication based on Förster Resonance Energy Transfer (FRET) enables nanoscale single molecular devices to communicate with each other utilizing excitons generated on fluorescent molecules as information carriers. Based on the point-to-point single-exciton FRET-based nanocommunication model, we investigate the multiple-exciton case for point-to-point and broadcast communications following an information theoretical approach and conducting simulations through Monte Carlo approach. We demonstrate that the multi-exciton transmission significantly improves the channel reliability and the range of the communication up to tens of nanometers for immobile nanonodes providing high data transmission rates. Furthermore, our analyses indicate that multi-exciton transmission enables broadcasting of information from a transmitter nanonode to many receiver nanonodes pointing out the potential of FRET-based communication to extend over nanonetworks. In this study, we also propose electrically and chemically controllable routing mechanisms exploiting the strong dependence of FRET rate on spectral and spatial characteristics of fluorescent molecules. We show that the proposed routing mechanisms enable the remote control of information flow in FRET-based nanonetworks. The high transmission rates obtained by multi-exciton scheme for point-to-point and broadcast communications, as well as the routing opportunities make FRET-based communication promising for future molecular computers.

  15. Evaluation of quantum dot-based concentric FRET configurations with a fluorescent dye and dark quencher for multiplexed bioanalyses (United States)

    Conroy, Erin M.; Algar, W. Russ


    Semiconductor quantum dots (QDs) continue to emerge as a highly advantageous platform for bioanalysis. Their unique physical and optical properties are especially well suited for Förster resonance energy transfer (FRET)-based bioprobes. Concentric FRET configurations are a recent development in this area of research and are best described as QD bioconjugates where multiple energy transfer pathways have been assembled around the central QD. Concentric FRET configurations permit multiplexed bioanalysis using one type of QD vector, but require more sophisticated analyses than conventional FRET pairs. In this paper, we describe the design and characterization of a new concentric FRET configuration that assembles both a fluorescent dye, Alexa Fluor 555 or Alexa Fluor 647, and a dark quencher, QSY9, at different ratios around a central CdSeS/ZnS QD. It was found that the magnitudes of the total photoluminescence (PL) intensity and either the A555/QD or A647/QD PL ratio can be related to the number of QSY9 and A555 or A647 per QD. The trends in these parameters with changes in the number of each dye molecule per QD have both similarities and differences between configurations with A555 and A647. In each case, a system of equations can be defined to permit calculation of the number of each dye molecule per QD from PL measurements. Both of these dark quencher-based concentric FRET configurations are therefore good candidates for quantitative, multiplexed bioanalysis.

  16. Fretting properties of biodegradable Mg-Nd-Zn-Zr alloy in air and in Hank’s solution (United States)

    Li, Wenting; Li, Nan; Zheng, Yufeng; Yuan, Guangyin


    Fretting is a significant cause for the failure of orthopedic implants. Currently, since magnesium and its alloys have been developed as promising biodegradable implant materials, the fretting behavior of the Mg alloys is of great research significance. In this study, a Mg-Nd-Zn-Zr alloy (hereafter, denoted as JDBM alloy) was selected as experimental material, and its fretting behaviors were evaluated under 5 N, 10 N and 20 N normal loads with a displacement of 200 μm under the frequency of 10 Hz at 37 °C in air and in Hank’s solution, respectively. The results indicated that while the friction coefficient decreased with the increment of the normal load, the wear volume of the alloy increased with the increment of the normal load both in air and in Hank’s solution. Both the friction coefficients and the wear volume of the fretting in Hank’s solution were much lower than those in air environment. The evolution trend of friction coefficients with time had different performance in air environment and the Hank’s solution group. Although oxidation occurred during the fretting tests in Hank’s solution, the damage of JDBM alloy was still reduced due to the lubrication effects of Hank’s solution. Moreover, the addition of Fetal bovine serum (FBS) could act as lubrication and result in the reduction of the fretting damage.

  17. Kinetic Studies of Lysine Riboswitch Folding Using Single-Molecule FRET (United States)

    Fiegland, Larry; Garst, Andrew; Fiore, J.; Batey, Robert; Nesbitt, David


    Riboswitches regulate gene expression through conformational changes induced by metabolite binding. This regulation of gene expression depends on the kinetics of metabolite binding and structural changes. Therefore, an understanding of these dynamics is crucial to developing a compete knowledge of riboswitch functionality. To probe the binding of a metabolite and subsequent folding, a metabolite-binding domain of the Bacillus subtilis lysine riboswitch was transcribed and hybridized to a fluorescent-labeled RNA strand, which allows FRET monitoring of ligand-induced conformational changes. The RNA construct was studied using single-molecule FRET methods that allowed for characterization of the folding dynamics. In the presence of lysine, we observed two states, of which the relative populations are perturbed by lysine concentration. We measured the folding and unfolding rates of the inter-conversion between these states. We also observe that [Mg^2+] affects the lysine-free conformation and the lysine sensitivity of the riboswitch.

  18. High-efficiency FRET-enhanced photoacoustic probes for in vivo tumor imaging (United States)

    Qin, Huan; Liu, Liming


    Photoacoustic imaging can provide high-resolution and high-contrast image under unprecedented depth compared with pure optical imaging techniques by making use of laser-induced ultrasound waves. Although a series of absorption-enhanced optical contrast agents for photoacoustic imaging were developed, the probe with fully conversion from absorbed light energy to acoustic energy has not been achieved so far. Here we develop a high-efficiency photoacoustic probes with fluorescence resonance energy transfer (FRET) effect for enhancement of nonradiative energy. Graphene oxide (GO) binding optical dyes (GO-dyes) were achieved to show highly fluorescence quenching and violently increased photoacoustic signal intensity. GO-dyes were constructed and testified for multi-spectral photoacoustic imaging. As a representative probe, GO-Cy7 nanoparticles were used to validate the feasibility of photoacoustic tumor molecular imaging in vivo. Our work demonstrated a new approach to construct high-efficiency FRET-enhanced multi-spectrum probes for photoacoustic molecular imaging.

  19. Cold-Sprayed Cu-MoS2 and Its Fretting Wear Behavior (United States)

    Zhang, Yinyin; Descartes, Sylvie; Vo, Phuong; Chromik, Richard R.


    Cu and Cu-MoS2 coatings were fabricated by cold spray, and the fretting wear performance of the two coatings was compared. A mixture (95 wt.% Cu + 5 wt.% MoS2) was used as feedstock for the composite coating. Coatings were sprayed with identical gas flow conditions on the substrates pre-heated to approximately 170 °C. The morphology of coating top surface and polished cross sections was analyzed by scanning electron microscopy (SEM) and light optical microscopy (LOM). The influence of MoS2 on Cu deposition was examined. The local MoS2 concentration within the coating was found to affect the hardness. Fretting tests were carried out at two different normal loads, and the influence of MoS2 on friction and wear was studied. The morphology and elemental compositions of the wear scars and wear debris were observed by SEM and energy dispersive x-ray spectroscopy (EDS), respectively.

  20. Noise-Assisted Quantum Electron Transfer in Multi-Level Donor-Acceptor System

    CERN Document Server

    Gurvitz, Shmuel; Berman, Gennady P


    We analytically and numerically study noise-assisted quantum electron transfer (ET) in bio-complexes consisting of a single-level electron donor and an acceptor which is modeled by many electron energy levels. Interactions are included between the donor and the acceptor energy levels and with the protein environment, which is modeled by a diagonal classical noise acting on all donor and acceptor energy levels. Different regions of parameters characterizing (i) the number of the acceptor levels, (ii) the acceptor "band-width", and (iii) the amplitude of noise and its correlation time are considered. Under some conditions, we derive analytical expressions for the ET rate and efficiency, which reveal the coarse-grain features. We obtain equal occupation of all levels at large times, independently of the structure of the acceptor band. We discuss the multi-scale regime of the acceptor population, and the accompanying effect of quantum coherent oscillations, which are analogous to those observed in experiments on ...

  1. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1


    Raina, Shweta A.; Jeffrey Tsai; Montserrat Samsó; Fessenden, James D.


    Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) inse...

  2. Handheld Fluorescence Resonance Energy Transfer (FRET)-Aptamer Sensor for Bone Markers (United States)

    Bruno, John G.


    Astronauts lose significant bone mass during lengthy space flights. NASA wishes to monitor this bone loss in order to develop nutritional and exercise countermeasures. Operational Technologies Corporation (OpTech) has developed a handheld device that quantifies bone loss in a spacecraft environment. The innovation works by adding fluorescent dyes and quenchers to aptamers to enable pushbutton, one-step bind-and-detect FRET assays that can be freeze-dried, rehydrated with body fluids, and used to quantify bone loss.

  3. Ecstasy analogues found in cacti. (United States)

    Bruhn, Jan G; El-Seedi, Hesham R; Stephanson, Nikolai; Beck, Olof; Shulgin, Alexander T


    Human interest in psychoactive phenethylamines is known from the use of mescaline-containing cacti and designer drugs such as Ecstasy. From the alkaloid composition of cacti we hypothesized that substances resembling Ecstasy might occur naturally. In this article we show that lophophine, homopiperonylamine and lobivine are new minor constituents of two cactus species, Lophophora williamsii (peyote) and Trichocereus pachanoi (San Pedro). This is the first report of putatively psychoactive phenethylamines besides mescaline in these cacti. A search for further biosynthetic analogues may provide new insights into the structure-activity relationships of mescaline. An intriguing question is whether the new natural compounds can be called "designer drugs."

  4. Antimicrobial evaluation of mangiferin analogues

    Directory of Open Access Journals (Sweden)

    Singh S


    Full Text Available The naturally occurring xanthone glycoside mangiferin has been isolated by column chromatography from the ethanol extract of stem bark of Mangifera indica. Mangiferin was further converted to 5-(N-phenylaminomethylenomangiferin, 5-(N-p-chlorophenylaminomethyleno mangiferin, 5-(N-2-methylphenylaminomethyleno mangiferin, 5-(N-p-methoxyphenylaminomethyleno mangiferin, 5-(N,N-diphenylaminomethyleno mangiferin, 5-(N--napthylaminomethyleno mangiferin and 5-(N-4-methylphenylaminomethyleno mangiferin. Mangiferin and its analogues were characterized by melting point and R f value determination and through spectral technique like UV, IR, and NMR spectral analysis. The synthesized compounds were screened for antimicrobial activity.

  5. Antimicrobial Evaluation of Mangiferin Analogues (United States)

    Singh, S. K.; Kumar, Y.; Kumar, S. Sadish; Sharma, V. K.; Dua, K.; Samad, A.


    The naturally occurring xanthone glycoside mangiferin has been isolated by column chromatography from the ethanol extract of stem bark of Mangifera indica. Mangiferin was further converted to 5-(N-phenylaminomethyleno)mangiferin, 5-(N-p-chlorophenylaminomethyleno) mangiferin, 5-(N-2-methylphenylaminomethyleno) mangiferin, 5-(N-p-methoxyphenylaminomethyleno) mangiferin, 5-(N, N-diphenylaminomethyleno) mangiferin, 5-(N--napthylaminomethyleno) mangiferin and 5-(N-4-methylphenylaminomethyleno) mangiferin. Mangiferin and its analogues were characterized by melting point and Rf value determination and through spectral technique like UV, IR, and NMR spectral analysis. The synthesized compounds were screened for antimicrobial activity. PMID:20490307

  6. A graphical approach to analogue behavioural modelling


    Moser, Vincent; Nussbaum, Pascal; Amann, Hans-Peter; Astier, Luc; Pellandini, Fausto


    In order to master the growing complexity of analogue electronic systems, modelling and simulation of analogue hardware at various levels is absolutely necessary. This paper presents an original modelling method based on the graphical description of analogue electronic functional blocks. This method is intended to be automated and integrated into a design framework: specialists create behavioural models of existing functional blocks, that can then be used through high-level selection and spec...

  7. Effects of Plasma ZrN Metallurgy and Shot Peening Duplex Treatment on Fretting Wear and Fretting Fatigue Behavior of Ti6Al4V Alloy

    Directory of Open Access Journals (Sweden)

    Jingang Tang


    Full Text Available A metallurgical zirconium nitride (ZrN layer was fabricated using glow metallurgy using nitriding with zirconiuming prior treatment of the Ti6Al4V alloy. The microstructure, composition and microhardness of the corresponding layer were studied. The influence of this treatment on fretting wear (FW and fretting fatigue (FF behavior of the Ti6Al4V alloy was studied. The composite layer consisted of an 8-μm-thick ZrN compound layer and a 50-μm-thick nitrogen-rich Zr–Ti solid solution layer. The surface microhardness of the composite layer is 1775 HK0.1. A gradient in cross-sectional microhardness distribution exists in the layer. The plasma ZrN metallurgical layer improves the FW resistance of the Ti6Al4V alloy, but reduces the base FF resistance. This occurs because the improvement in surface hardness results in lowering of the toughness and increasing in the notch sensitivity. Compared with shot peening treatment, plasma ZrN metallurgy and shot peening composite treatment improves the FW resistance and enhances the FF resistance of the Ti6Al4V alloy. This is attributed to the introduction of a compressive stress field. The combination of toughness, strength, FW resistance and fatigue resistance enhance the FF resistance for titanium alloy.

  8. FRET Fluctuation Spectroscopy of Diffusing Biopolymers: Contributions of Conformational Dynamics and Translational Diffusion (United States)

    Gurunathan, Kaushik; Levitus, Marcia


    The use of Fluorescence Correlation Spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in Fluorescence Resonance Energy Transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments. Results show that diffusion does not contribute to the measured fluctuations in FRET efficiency in conditions where the relaxation time of the kinetic process is much shorter than the mean transit time of the molecules in the optical observation volume. However, in contrast to what has been suggested in previous work, the contributions of diffusion are otherwise significant. Neglecting the contributions of diffusion can potentially lead to an erroneous interpretation of the kinetic mechanisms. As an example, we demonstrate that the analysis of FRET fluctuations in terms of a purely kinetic model would generally lead to the conclusion that the system presents complex kinetic behavior even for an idealized two-state system PMID:20030305

  9. Single-molecule FRET reveals hidden complexity in a protein energy landscape. (United States)

    Tsytlonok, Maksym; Ibrahim, Shehu M; Rowling, Pamela J E; Xu, Wenshu; Ruedas-Rama, Maria J; Orte, Angel; Klenerman, David; Itzhaki, Laura S


    Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

  10. Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors. (United States)

    Kamioka, Yuji; Sumiyama, Kenta; Mizuno, Rei; Sakai, Yoshiharu; Hirata, Eishu; Kiyokawa, Etsuko; Matsuda, Michiyuki


    Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.

  11. FRET based biosensor for detection of active NF-kB (United States)

    Baldini, Francesco; Citti, Lorenzo; Domenici, Claudio; Giannetti, Ambra; Tedeschi, Lorena; Vo-Dinh, Tuan; Wabuyele, Musundi B.


    The Nuclear Factor kB is a transcription factor, ubiquitously expressed, involved in the regulation of a large number of genes and in a variety of human disease including inflammation, asthma, atherosclerosis, AIDS, septic shock, arthritis and cancer. The critical need for a simple and direct method to evaluate the quantity of active NF-kB in a biological sample can be addressed using a suitable and reusable biosensor. For this purpose, a novel method, using fluorescence resonance energy transfer (FRET), to detect the active form of NF-kB binding a specific DNA sequence has been developed. A single-stranded DNA (ssDNA) with auto-complementary sequence has been properly designed and synthesized. In order to evaluate FRET due to the DNA/protein binding interaction taking place between double-stranded DNA (dsDNA) immobilized in a capillary wall and NF-kB proteins, a highly sensitive FRET-based biosensor system developed in our laboratory was used. Preliminary results show that our system was capable of detecting the active form of NF-kB protein with a detection efficiency of about 90% and that the system has a good regenerability.

  12. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy. (United States)

    Day, Richard N; Tao, Wen; Dunn, Kenneth W


    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

  13. Influence of surface coating on Ti811 alloy resistance to fretting fatigue at elevated temperature

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiaohua; LIU Daoxin


    An extensive study of the composition distribution, bonding strength, hardness, and wear resistance of a 0Cr18Ni9 film deposited on a Ti811 titanium alloy surface by ion beam enhanced deposition (IBED) is presented. Shot peening was introduced to post-treat the modified surface to synergistically improve the fretting fatigue resistance of the Ti811 alloy at 350~C. The results indicate that the 0Cr18Ni9 film with high density, small grain size, low void radio, and high bonding strength can be prepared using IBED. As a result, the hardness, wear resistance, and fretting fatigue resistance of the Ti811 alloy are increased to a remarkable extent. Compared with shot peening treatment or IBED 0Cr18Ni9 film alone, the Ti811 titanium alloy with an IBED 0Cr18Ni9 film combined with shot peening shows a higher fretting fatigue resistance at 350℃. This is due to the synergistic effect of the high wear resistance of the film surface and the residual compressive stress induced by shot peening.

  14. What Can We Learn From Analogue Experiments?

    CERN Document Server

    Thebault, Karim P Y


    In 1981 Unruh proposed that fluid mechanical experiments could be used to probe key aspects of the quantum phenomenology of black holes. In particular, he claimed that an analogue to Hawking radiation could be created within a fluid mechanical `dumb hole', with the event horizon replaced by a sonic horizon. Since then an entire sub-field of `analogue gravity' has been created. In 2016 Steinhauer reported the experimental observation of quantum Hawking radiation and its entanglement in a Bose-Einstein condensate analogue black hole. What can we learn from such analogue experiments? In particular, in what sense can they provide evidence of novel phenomena such as black hole Hawking radiation?

  15. Acceptor and donor impurities in GaN nanocrystals


    Echeverría-Arrondo, C.; Pérez-Conde, J.; Bhattacharjee, A. K.


    We investigate acceptor and donor states in GaN nanocrystals doped with a single substitutional impurity. Quantum dots (QD's) of zinc-blende structure and spherical shape are considered with the radius ranging from 4.5 to 67.7 A. The size-dependent energy spectra are calculated within the sp3d5s* tight-binding model, which yields a good agreement with the confinement-induced blue shifts observed in undoped QD's. The computed binding energy is strongly enhanced with respect to the experimental...

  16. The Valles natural analogue project

    Energy Technology Data Exchange (ETDEWEB)

    Stockman, H.; Krumhansl, J.; Ho, C. [Sandia National Labs., Albuquerque, NM (United States); McConnell, V. [Alaska Univ., Fairbanks, AK (United States). Geophysical Inst.


    The contact between an obsidian flow and a steep-walled tuff canyon was examined as an analogue for a highlevel waste repository. The analogue site is located in the Valles Caldera in New Mexico, where a massive obsidian flow filled a paleocanyon in the Battleship Rock tuff. The obsidian flow provided a heat source, analogous to waste panels or an igneous intrusion in a repository, and caused evaporation and migration of water. The tuff and obsidian samples were analyzed for major and trace elements and mineralogy by INAA, XRF, X-ray diffraction; and scanning electron microscopy and electron microprobe. Samples were also analyzed for D/H and {sup 39}Ar/{sup 4O} isotopic composition. Overall,the effects of the heating event seem to have been slight and limited to the tuff nearest the contact. There is some evidence of devitrification and migration of volatiles in the tuff within 10 meters of the contact, but variations in major and trace element chemistry are small and difficult to distinguish from the natural (pre-heating) variability of the rocks.

  17. FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D (United States)

    Bruno, John G.


    Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is

  18. Classic maximum entropy recovery of the average joint distribution of apparent FRET efficiency and fluorescence photons for single-molecule burst measurements. (United States)

    DeVore, Matthew S; Gull, Stephen F; Johnson, Carey K


    We describe a method for analysis of single-molecule Förster resonance energy transfer (FRET) burst measurements using classic maximum entropy. Classic maximum entropy determines the Bayesian inference for the joint probability describing the total fluorescence photons and the apparent FRET efficiency. The method was tested with simulated data and then with DNA labeled with fluorescent dyes. The most probable joint distribution can be marginalized to obtain both the overall distribution of fluorescence photons and the apparent FRET efficiency distribution. This method proves to be ideal for determining the distance distribution of FRET-labeled biomolecules, and it successfully predicts the shape of the recovered distributions.

  19. Glutathione Adduct Patterns of Michael-Acceptor Carbonyls. (United States)

    Slawik, Christian; Rickmeyer, Christiane; Brehm, Martin; Böhme, Alexander; Schüürmann, Gerrit


    Glutathione (GSH) has so far been considered to facilitate detoxification of soft organic electrophiles through covalent binding at its cysteine (Cys) thiol group, followed by stepwise catalyzed degradation and eventual elimination along the mercapturic acid pathway. Here we show that in contrast to expectation from HSAB theory, Michael-acceptor ketones, aldehydes and esters may form also single, double and triple adducts with GSH involving β-carbon attack at the much harder N-terminus of the γ-glutamyl (Glu) unit of GSH. In particular, formation of the GSH-N single adduct contradicts the traditional view that S alkylation always forms the initial reaction of GSH with Michael-acceptor carbonyls. To this end, chemoassay analyses of the adduct formation of GSH with nine α,β-unsaturated carbonyls employing high performance liquid chromatography and tandem mass spectrometry have been performed. Besides enriching the GSH adductome and potential biomarker applications, electrophilic N-terminus functio-nalization is likely to impair GSH homeostasis substantially through blocking the γ-glutamyl transferase catalysis of the first breakdown step of modified GSH, and thus its timely reconstitution. The discussion includes a comparison with cyclic adducts of GSH and furan metabolites as reported in literature, and quantum chemically calculated thermodynamics of hard-hard, hard-soft and soft-soft adducts.

  20. ETP-0474: Evaluation of Electroless Nickel Coatings to Achieve Interference Fit in the RSRM Without Fretting (United States)

    Schaffnit, William O.


    Part of the redesign of the SRMs for the Space Shuttle involved the substitution of three new capture cylinders for three of the previously used cylinders. These new cylinders mate with the old standard case segments in each of the three field joints. The new capture cylinders contain an integral capture latch on the tang end which mates with a case clevis during stackup at KSC. The capture cylinders also contain a groove in the capture latch to provide for a third 0-ring in the joint and are designed to achieve a metal-to- metal interference fit between the capture latch and the mating clevis. An unexpected fretting problem has occurred on the tang capture feature and the inner clevis leg interference fit surfaces on flight hardware since STS-26. Varying degrees of fretting damage have been found on the case segments from different flight motors. Fretting is a wear phenomena that occurs when two tightly fitting metal surfaces are subject to cyclic relative motion of extremely small amplitudes (generally less than 0.010-inch) in the absence of adequate lubrication. It is adhesive ("cold" - welding) in nature and vibration is its essential causative factor. This problem has manifested itself on the flight motors as a series of pits and axial gouges on the inside diameter (ID) surfaces of the inner clevis legs and the outside diameter (OD) surfaces of the tang capture features. The problem occurs in varying degrees of severity in all of the field joints. It is not believed that fretting is a flight safety issue. However, it could become a reusability issue if left unattended. Fretting has been encountered in other industries for many years and measures that will prevent or reduce it have been devised. These include: elimination or reduction of vibration (amplitudes and/or frequencies), elimination of slip, improved lubrication between parts, increased surface separation, increased interference, inducing residual compressive stresses in the surfaces of the mating

  1. Probing riboswitch-ligand interactions using thiamine pyrophosphate analogues. (United States)

    Chen, Liuhong; Cressina, Elena; Dixon, Neil; Erixon, Karl; Agyei-Owusu, Kwasi; Micklefield, Jason; Smith, Alison G; Abell, Chris; Leeper, Finian J


    The Escherichia coli thiM riboswitch forms specific contacts with its natural ligand, thiamine pyrophosphate (TPP or thiamine diphosphate), allowing it to generate not only nanomolar binding affinity, but also a high degree of discrimination against similar small molecules. A range of synthetic TPP analogues have been used to probe each of the riboswitch-ligand interactions. The results show that the pyrimidine-sensing helix of thiM is exquisitely tuned to select for TPP by recognising the H-bonding donor and acceptors around its aminopyrimidine ring and also by forming π-stacking interactions that may be sensitive to the electronics of the ring. The central thiazolium ring of TPP appears to be more important for ligand recognition than previously thought. It may contribute to binding via long-range electrostatic interactions and/or by exerting an electron withdrawing effect on the pyrimidine ring, allowing its presence to be sensed indirectly and thereby allowing discrimination between thiamine (and its phosphate esters) and other aminopyrimidines found in vivo. The pyrophosphate moiety is essential for submicromolar binding affinity, but unexpectedly, it does not appear to be strictly necessary for modulation of gene expression.

  2. Binding characteristics of homogeneous molecularly imprinted polymers for acyclovir using an (acceptor-donor-donor)-(donor-acceptor-acceptor) hydrogen-bond strategy, and analytical applications for serum samples. (United States)

    Wu, Suqin; Tan, Lei; Wang, Ganquan; Peng, Guiming; Kang, Chengcheng; Tang, Youwen


    This paper demonstrates a novel approach to assembling homogeneous molecularly imprinted polymers (MIPs) based on mimicking multiple hydrogen bonds between nucleotide bases by preparing acyclovir (ACV) as a template and using coatings grafted on silica supports. (1)H NMR studies confirmed the AAD-DDA (A for acceptor, D for donor) hydrogen-bond array between template and functional monomer, while the resultant monodisperse molecularly imprinted microspheres (MIMs) were evaluated using a binding experiment, high performance liquid chromatography (HPLC), and solid phase extraction. The Langmuir isothermal model and the Langmuir-Freundlich isothermal model suggest that ACV-MIMs have more homogeneous binding sites than MIPs prepared through normal imprinting. In contrast to previous MIP-HPLC columns, there were no apparent tailings for the ACV peaks, and ACV-MIMs had excellent specific binding properties with a Ka peak of 3.44 × 10(5)M(-1). A complete baseline separation is obtained for ACV and structurally similar compounds. This work also successfully used MIMs as a specific sorbent for capturing ACV from serum samples. The detection limit and mean recovery of ACV was 1.8 ng/mL(-1) and 95.6%, respectively, for molecularly imprinted solid phase extraction coupled with HPLC. To our knowledge, this was the first example of MIPs using AAD-DDA hydrogen bonds.

  3. Strategy and methodology of dynamical analogue prediction

    Institute of Scientific and Technical Information of China (English)


    In order to effectively improve numerical prediction level by using current models and data, the strategy and methodology of dynamical analogue prediction (DAP) is deeply studied in the present paper. A new idea to predict the prediction errors of dynamical model on the basis of historical analogue information is put forward so as to transform the dynamical prediction problem into the estimation problem of prediction errors. In terms of such an idea, a new prediction method of final analogue correction of errors (FACE) is developed. Furthermore, the FACE is applied to extra-seasonal prediction experiments on an operational atmosphere-ocean coupled general circulation model. Prediction results of summer mean circulation and total precipitation show that the FACE can to some extent reduce prediction errors, recover prediction variances, and improve prediction skills. Besides, sensitive experiments also show that predictions based on the FACE are evidently influenced by the number of analogues, analogue-selected variables and analogy metric.

  4. The future of somatostatin analogue therapy. (United States)

    Stewart, P M; James, R A


    Since its discovery almost 30 years ago, the mode of action and therapeutic applications of somatostatin have been defined. In particular the cloning and characterization of somatostatin receptor subtypes has facilitated the development of high affinity analogues. In the context of pituitary disease, long-acting somatostatin analogues (octreotide, lanreotide) have been used to treat a variety of pituitary tumours but are most efficacious for the treatment of GH and TSH-secreting adenomas. In patients with acromegaly, depot preparations of these analogues are administered intramuscularly every 10-28 days and provide consistent suppression of GH levels to < 5 mU/l in approximately 50-65% of all cases. Even more specific somatostatin receptor analogues are under development. Finally, radiolabelled somatostatin analogue scintigraphy and, in larger doses, therapy, are now established tools in the evaluation and treatment of neuroendocrine tumours.

  5. Progress in ZnO Acceptor Doping: What Is the Best Strategy?

    Directory of Open Access Journals (Sweden)

    Judith G. Reynolds


    Full Text Available This paper reviews the recent progress in acceptor doping of ZnO that has been achieved with a focus toward the optimum strategy. There are three main approaches for generating p-type ZnO: substitutional group IA elements on a zinc site, codoping of donors and acceptors, and substitution of group VA elements on an oxygen site. The relevant issues are whether there is sufficient incorporation of the appropriate dopant impurity species, does it reside on the appropriate lattice site, and lastly whether the acceptor ionization energy is sufficiently small to enable significant p-type conduction at room temperature. The potential of nitrogen doping and formation of the appropriate acceptor complexes is highlighted although theoretical calculations predict that nitrogen on an oxygen site is a deep acceptor. We show that an understanding of the growth and annealing steps to achieve the relevant acceptor defect complexes is crucial to meet requirements.

  6. The structure and bonding of iron-acceptor pairs in silicon

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, S.; Assali, L.V.C.; Kimerling, L.C. [Massachusetts Inst. of Technology, Cambridge, MA (United States)


    The highly mobile interstitial iron and Group III impurities (B, Al, Ga, In) form iron-acceptor pairs in silicon. Based on the migration kinetics and taking host silicon as a dielectric medium, we have simulated the pairing process in a static silicon lattice. Different from the conventional point charge ionic model, our phenomenological calculations include (1) a correction that takes into account valence electron cloud polarization which adds a short range, attractive interaction in the iron-acceptor pair bonding; and (2) silicon lattice relaxation due to the atomic size difference which causes a local strain field. Our model explains qualitatively (1) trends among the iron-acceptor pairs revealing an increase of the electronic state hole emission energy with increasing principal quantum number of acceptor and decreasing pair separation distance; and (2) the stable and metastable sites and configurational symmetries of the iron-acceptor pairs. The iron-acceptor pairing and bonding mechanism is also discussed.

  7. Donor-Acceptor-Type Semiconducting Polymers Consisting of Benzothiadiazole Derivatives as Electron-Acceptor Units for Organic Photovoltaic Cells. (United States)

    Kim, Hee Su; Park, Jong Baek; Kim, Ji-Hoon; Hwang, Do-Hoon


    We synthesized two fused pentacyclic donor-acceptor structures, where the two different outer electron rich thiophene (DTPBT) and electron poor benzene (ICTh) moieties are covalently bonded to the central electron-deficient benzothiadiazole core by two nitrogen bridges. These new electron-acceptor DTPBT and ICTh building blocks were copolymerized with fluorene, as the electron donor group, via Suzuki coupling polymerization, to produce two new alternating copolymers, PFDTPBT and PFICTh, respectively. The average molecular weights of the synthesized polymers were determined by GPC. The number-average molecular weights of PFDTPBT and PFICTh were 19,000 (PDI = 2.5) and 20,000 (PDI = 4.0), respectively. The optical bandgap energies of the polymers were measured from their absorption onsets to be 2.15 and 2.55 eV, depending on the polymer structure. The HOMO energy levels of the polymers were determined, by measuring the oxidation onsets of the polymer films by cyclic voltammetry. The measured HOMO energy levels of PFDTPBT and PFICTh were -5.10 and -5.57 eV, respectively. When the polymers were blended with PC71BM, as the active layer for bulk-heterojunction photovoltaic devices, power conversion efficiencies were 2.08% and 0.34%, respectively, under AM 1.5 G (100 mW cm(-2)) conditions.

  8. Magnetic anisotropy of single Mn acceptors in GaAs in an external magnetic field


    Bozkurt, M Murat; Mahani, MR; Studer, P; Tang, J-M; Schofield, SR; Curson, NJ; Flatté, ME Michael; Silov, AY Andrei; Hirjibehedin, CF; Canali, CM; Koenraad, PM Paul


    We investigate the effect of an external magnetic field on the physical properties of the acceptor hole states associated with single Mn acceptors placed near the (110) surface of GaAs. Crosssectional scanning tunneling microscopy images of the acceptor local density of states (LDOS) show that the strongly anisotropic hole wavefunction is not significantly affected by a magnetic field up to 6 T. These experimental results are supported by theoretical calculations based on a tightbinding model...

  9. Development of a Fluorescence Resonance Energy Transfer (FRET-Based DNA Biosensor for Detection of Synthetic Oligonucleotide of Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Noremylia Mohd Bakhori


    Full Text Available An optical DNA biosensor based on fluorescence resonance energy transfer (FRET utilizing synthesized quantum dot (QD has been developed for the detection of specific-sequence of DNA for Ganoderma boninense, an oil palm pathogen. Modified QD that contained carboxylic groups was conjugated with a single-stranded DNA probe (ssDNA via amide-linkage. Hybridization of the target DNA with conjugated QD-ssDNA and reporter probe labeled with Cy5 allows for the detection of related synthetic DNA sequence of Ganoderma boninense gene based on FRET signals. Detection of FRET emission before and after hybridization was confirmed through the capability of the system to produce FRET at 680 nm for hybridized sandwich with complementary target DNA. No FRET emission was observed for non-complementary system. Hybridization time, temperature and effect of different concentration of target DNA were studied in order to optimize the developed system. The developed biosensor has shown high sensitivity with detection limit of 3.55 × 10−9 M. TEM results show that the particle size of QD varies in the range between 5 to 8 nm after ligand modification and conjugation with ssDNA. This approach is capable of providing a simple, rapid and sensitive method for detection of related synthetic DNA sequence of Ganoderma boninense.

  10. A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement-Förster-Type Resonance Energy Transfer (PIFE-FRET). (United States)

    Lerner, Eitan; Ploetz, Evelyn; Hohlbein, Johannes; Cordes, Thorben; Weiss, Shimon


    Single-molecule, protein-induced fluorescence enhancement (PIFE) serves as a molecular ruler at molecular distances inaccessible to other spectroscopic rulers such as Förster-type resonance energy transfer (FRET) or photoinduced electron transfer. In order to provide two simultaneous measurements of two distances on different molecular length scales for the analysis of macromolecular complexes, we and others recently combined measurements of PIFE and FRET (PIFE-FRET) on the single molecule level. PIFE relies on steric hindrance of the fluorophore Cy3, which is covalently attached to a biomolecule of interest, to rotate out of an excited-state trans isomer to the cis isomer through a 90° intermediate. In this work, we provide a theoretical framework that accounts for relevant photophysical and kinetic parameters of PIFE-FRET, show how this framework allows the extraction of the fold-decrease in isomerization mobility from experimental data, and show how these results provide information on changes in the accessible volume of Cy3. The utility of this model is then demonstrated for experimental results on PIFE-FRET measurement of different protein-DNA interactions. The proposed model and extracted parameters could serve as a benchmark to allow quantitative comparison of PIFE effects in different biological systems.

  11. Establishment of Plant Acceptor System for Hyperosmosis Transformation.

    Institute of Scientific and Technical Information of China (English)


    Calli and adventitious buds induced from different explants such as young stems and leaves of tomato variety Moneymaker, and mature embryos and young spikelets of rice variety TP309 were used to establish hyperosmotic transformation system at various osmotica treatments.The results revealed that the calli induced from tomato young leaves and rice young spikelets were the ideal transfomation acceptor. The cells of calli were still vigorous when treated wity 0.75 mol/L hyperosmotic solution for 4 hours. The differentiation rates of calli varied from 7.5% to 93.3% in different media. The bud differentiation was apparently inhibited by hyperosmotic treatments. O.75mol/L sucrose hypertonic solution and O.2mol/L CaCl2 solution were favorable hyperosmoticum and hypoosmoticum respectively.

  12. Donor-acceptor pair recombination in gallium sulfide (United States)

    Aydinli, A.; Gasanly, N. M.; Gökşen, K.


    Low temperature photoluminescence of GaS single crystals shows three broad emission bands below 2.4 eV. Temperature and excitation light intensity dependencies of these bands reveal that all of them originate from close donor-acceptor pair recombination processes. Temperature dependence of the peak energies of two of these bands in the visible range follow, as expected, the band gap energy shift of GaS. However, the temperature dependence of the peak energy of the third band in the near infrared shows complex behavior by blueshifting at low temperatures followed by a redshift at intermediate temperatures and a second blueshift close to room temperature, which could only be explained via a configuration coordinate model. A simple model calculation indicates that the recombination centers are most likely located at the nearest neighbor lattice or interstitial sites.

  13. Advanced non-linear flow-induced vibration and fretting-wear analysis capabilities

    Energy Technology Data Exchange (ETDEWEB)

    Toorani, M.; Pan, L.; Li, R.; Idvorian, N. [Babcock and Wilcox Canada Ltd., Cambridge, Ontario (Canada); Vincent, B.


    Fretting wear is a potentially significant degradation mechanism in nuclear steam generators and other shell and tube heat transfer equipment as well. This paper presents an overview of the recently developed code FIVDYNA which is used for the non-linear flow-induced vibration and fretting wear analysis for operating steam generators (OTSG and RSG) and shell-and-tube heat exchangers. FIVDYNA is a non-linear time-history Flow-Induced Vibration (FIV) analysis computer program that has been developed by Babcock and Wilcox Canada to advance the understanding of tube vibration and tube to tube-support interaction. In addition to the dynamic fluid induced forces the program takes into account other tube static forces due to axial and lateral tube preload and thermal interaction loads. The program is capable of predicting the location where the fretting wear is most likely to occur and its magnitude taking into account the support geometry including gaps. FIVDYNA uses the general purpose finite element computer code ABAQUS as its solver. Using ABAQUS gives the user the flexibility to add additional forces to the tube ranging from tube preloads and the support offsets to thermal loads. The forces currently being modeled in FIVDYNA are the random turbulence, steady drag force, fluid-elastic forces, support offset and pre-strain force (axial loads). This program models the vibration of tubes and calculates the structural dynamic characteristics, and interaction forces between the tube and the tube supports. These interaction forces are then used to calculate the work rate at the support and eventually the predicted depth of wear scar on the tube. A very good agreement is found with experiments and also other computer codes. (author)

  14. PEG-Phospholipids Coated Quantum Rods as Amplifiers of the Photosensitization Process by FRET. (United States)

    Timor, Reut; Weitman, Hana; Waiskopf, Nir; Banin, Uri; Ehrenberg, Benjamin


    Singlet oxygen ((1)O2) generated upon photostimulation of photosensitizer molecules is a highly reactive specie which is utilized in photodynamic therapy. Recent studies have shown that semiconductor nanoparticles can be used as donors in fluorescence resonance energy transfer (FRET) process to excite attached photosensitizer molecules. In these studies, their unique properties, such as low nanoscale size, long-term photostability, wide broad absorbance band, large absorption cross section, and narrow and tunable emission bands were used to provide advantages over the traditional methods to produce singlet oxygen. Previous studies that achieved this goal, however, showed some limitations, such as low FRET efficiency, poor colloidal stability, nonspecific interactions, and/or complex preparation procedure. In this work, we developed and characterized a novel system of semiconductor quantum rods (QRs) and the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC), as a model system that produces singlet oxygen without these limitations. A simple two-step preparation method is shown; Hydrophobic CdSe/CdS QRs are solubilized in aqueous solutions by encapsulation with lecithin and PEGylated phospholipid (PEG-PL) of two lipid lengths: PEG350 or PEG2000. Then, the hydrophobic photosensitizer mTHPC, was intercalated into the new amphiphilic PEG-PL coating of the QR, providing a strong attachment to the nanoparticle without covalent linkage. These PEGylated QR (eQR)-mTHPC nanocomposites show efficient FRET processes upon light stimulation of the QR component which results in efficient production of singlet oxygen. The results demonstrate the potential for future use of this concept in photodynamic therapy schemes.

  15. The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

    Directory of Open Access Journals (Sweden)

    Shaoying Lu

    Full Text Available Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP experiments, we have developed a finite element (FE method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2/sec than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2/sec and outside: 0.18+/-0.02 microm(2/sec. The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

  16. Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane. (United States)

    Gordon, Sharona E; Senning, Eric N; Aman, Teresa K; Zagotta, William N


    Biological membranes are complex assemblies of lipids and proteins that serve as platforms for cell signaling. We have developed a novel method for measuring the structure and dynamics of the membrane based on fluorescence resonance energy transfer (FRET). The method marries four technologies: (1) unroofing cells to isolate and access the cytoplasmic leaflet of the plasma membrane; (2) patch-clamp fluorometry (PCF) to measure currents and fluorescence simultaneously from a membrane patch; (3) a synthetic lipid with a metal-chelating head group to decorate the membrane with metal-binding sites; and (4) transition metal ion FRET (tmFRET) to measure short distances between a fluorescent probe and a transition metal ion on the membrane. We applied this method to measure the density and affinity of native and introduced metal-binding sites in the membrane. These experiments pave the way for measuring structural rearrangements of membrane proteins relative to the membrane.

  17. Modeling of Complex Wear Behavior Associated with Grid-to-Rod Fretting in Light Water Nuclear Reactors (United States)

    Blau, P. J.; Qu, J.; Lu, R.


    Fretting wear damage to fuel cladding from flow-induced vibrations can be a significant concern in the operation of light water nuclear reactors. For years, research on the grid-to-rod fretting (GTRF) phenomena has been underway in countries where nuclear power production is a significant industry. Under the auspices of the U.S. Department of Energy Consortium for Advanced Simulation of Light Water Reactors, an effort has been underway to develop and test an engineering wear model for zirconium alloy fuel rod cladding against a supporting grid. The multi-stage model accounts for oxide layers and wear rate transitions. This paper describes the basis for a GTRF engineering wear model, the physical significance of the wear factor it contains, and recent progress toward model validation based on a fretting wear testing apparatus that accounts for coolant temperature, pressure, and the presence of periodic impacts (gaps) in grid/rod contact.

  18. Evaluation of fretting failures on PWR fuel by post-irradiation examinations and modeling in the DEGRAD-1 code

    Energy Technology Data Exchange (ETDEWEB)

    Castanheira, Myrthes; Silva, Jose Eduardo Rosa da; Lucki, Georgi; Terremoto, Luis A.A.; Silva, Antonio Teixeira e; Teodoro, Celso A.; Damy, Margaret de A. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)]. E-mail:


    One of the major recognized causes of fuel rod failures is fretting of the clad due to the entrapment of debris in a fuel rod spacer. Such debris, inadvertently dropped into the primary system during maintenance operations, includes various sizes of particles. Intermediate size particles, such as metal cuttings, electrical connectors, metal fittings, pieces of wire, and small nuts and bolts can become trapped between fuel rods in a spacer where hydraulically induced vibrations can cause fretting failure of the fuel rod. An evaluation of debris fretting failure on PWR fuel is presented. The inquiries on fuel rods failures are based on results of analysis using post-irradiation non-destructive examination. The complementary analysis includes a modeling approach by code DEGRAD-1 to characterize the degradation phenomenon after primary failure integrated in the reactor operational history. (author)

  19. Modeling of Complex Wear Behavior Associated with Grid-to-Rod Fretting in Light Water Nuclear Reactors (United States)

    Blau, P. J.; Qu, J.; Lu, R.


    Fretting wear damage to fuel cladding from flow-induced vibrations can be a significant concern in the operation of light water nuclear reactors. For years, research on the grid-to-rod fretting (GTRF) phenomena has been underway in countries where nuclear power production is a significant industry. Under the auspices of the U.S. Department of Energy Consortium for Advanced Simulation of Light Water Reactors, an effort has been underway to develop and test an engineering wear model for zirconium alloy fuel rod cladding against a supporting grid. The multi-stage model accounts for oxide layers and wear rate transitions. This paper describes the basis for a GTRF engineering wear model, the physical significance of the wear factor it contains, and recent progress toward model validation based on a fretting wear testing apparatus that accounts for coolant temperature, pressure, and the presence of periodic impacts (gaps) in grid/rod contact.

  20. Experimental evaluation of the fretting fatigue behavior of high-strength steel monostrands

    DEFF Research Database (Denmark)

    Winkler, Jan; Fischer, Gregor; Georgakis, Christos T.


    . Moreover, the paper provides relevant information about the monostrand bending stiffness and the extent of relative displacement between core and outer wires of the monostrand undergoing flexural deformations. The results presented herein are of special interest for the fatigue analysis of modern stay......In this paper, the fretting fatigue behavior of pretensioned high-strength steel monostrands is investigated. A method based on the digital image correlation (DIC) technique was used to quantify the relative movement between individual wires along the length of the monostrand. The experimental data...

  1. Compound parabolic concentrator optical fiber tip for FRET-based fluorescent sensors

    DEFF Research Database (Denmark)

    Hassan, Hafeez Ul; Nielsen, Kristian; Aasmul, Soren;


    polymer optical fiber for an excitation and emission wavelength of 550 nm and 650nm, respectively. The model suggests an increase of a factor of 1.6 to 4 in the collected fluorescent power for an ideal CPC tip, as compared to the plane-cut fiber tip for fiber lengths between 5 and 45mm......The Compound Parabolic Concentrator (CPC) optical fiber tip shape has been proposed for intensity based fluorescent sensors working on the principle of FRET (Förster Resonance Energy Transfer). A simple numerical Zemax model has been used to optimize the CPC tip geometry for a step-index multimode...

  2. Analysis of temporal and spatial contact voltage fluctuation during fretting in automotive connectors


    El Mossouess, Sofiane; Benjemâa, N; Carvou, E; El Abdi, R; Obame, H; Doublet, L; Rodari, T


    International audience; Our study is focused on contact voltage fluctuations during fretting with small amplitudes of a few tens microns which generate damage of the contact of connectors. A contact composed by a pin and a curve female part are submitted to vibration cyclic of 25µm at 100Hz and supplied with current ramp from 0.1mA to 3A in two directions. With the help of fast devices, the voltage and position data acquisition are conjointly made with the common DC contact voltage dur-ing fr...

  3. Contrasting performance of donor-acceptor copolymer pairs in ternary blend solar cells and two-acceptor copolymers in binary blend solar cells. (United States)

    Khlyabich, Petr P; Rudenko, Andrey E; Burkhart, Beate; Thompson, Barry C


    Here two contrasting approaches to polymer-fullerene solar cells are compared. In the first approach, two distinct semi-random donor-acceptor copolymers are blended with phenyl-C61-butyric acid methyl ester (PC61BM) to form ternary blend solar cells. The two poly(3-hexylthiophene)-based polymers contain either the acceptor thienopyrroledione (TPD) or diketopyrrolopyrrole (DPP). In the second approach, semi-random donor-acceptor copolymers containing both TPD and DPP acceptors in the same polymer backbone, termed two-acceptor polymers, are blended with PC61BM to give binary blend solar cells. The two approaches result in bulk heterojunction solar cells that have the same molecular active-layer components but differ in the manner in which these molecular components are mixed, either by physical mixing (ternary blend) or chemical "mixing" in the two-acceptor (binary blend) case. Optical properties and photon-to-electron conversion efficiencies of the binary and ternary blends were found to have similar features and were described as a linear combination of the individual components. At the same time, significant differences were observed in the open-circuit voltage (Voc) behaviors of binary and ternary blend solar cells. While in case of two-acceptor polymers, the Voc was found to be in the range of 0.495-0.552 V, ternary blend solar cells showed behavior inherent to organic alloy formation, displaying an intermediate, composition-dependent and tunable Voc in the range from 0.582 to 0.684 V, significantly exceeding the values achieved in the two-acceptor containing binary blend solar cells. Despite the differences between the physical and chemical mixing approaches, both pathways provided solar cells with similar power conversion efficiencies, highlighting the advantages of both pathways toward highly efficient organic solar cells.

  4. Single-molecule FRET reveals the native-state dynamics of the IκBα ankyrin repeat domain. (United States)

    Lamboy, Jorge A; Kim, Hajin; Dembinski, Holly; Ha, Taekjip; Komives, Elizabeth A


    Previous single-molecule fluorescence resonance energy transfer (smFRET) studies in which the second and sixth ankyrin repeats (ARs) of IκBα were labeled with FRET pairs showed slow fluctuations as if the IκBα AR domain was unfolding in its native state. To systematically probe where these slow dynamic fluctuations occur, we now present data from smFRET studies wherein FRET labels were placed at ARs 1 and 4 (mutant named AR 1-4), at ARs 2 and 5 (AR 2-5), and at ARs 3 and 6 (AR 3-6). The results presented here reveal that AR 6 most readily detaches/unfolds from the AR domain, undergoing substantial fluctuations at room temperature. AR 6 has fewer stabilizing consensus residues than the other IκBα ARs, probably contributing to the ease with which AR 6 "loses grip". AR 5 shows almost no fluctuations at room temperature, but a significant fraction of molecules shows fluctuations at 37 °C. Introduction of stabilizing mutations that are known to fold AR 6 dampen the fluctuations of AR 5, indicating that the AR 5 fluctuations are likely due to weakened inter-repeat stabilization from AR 6. AR 1 also fluctuates somewhat at room temperature, suggesting that fluctuations are a general behavior of ARs at ends of AR domains. Remarkably, AR 1 still fluctuates in the bound state, but mainly between 0.6 and 0.9 FRET efficiency, whereas in the free IκBα, the fluctuations extend to <0.5 FRET efficiency. Overall, our results provide a more complete picture of the energy landscape of the native state dynamics of an AR domain.

  5. Fretting Wear Behavior of Medium Carbon Steel Modified by Low Temperature Gas Multi-component Thermo-chemical Treatment

    Institute of Scientific and Technical Information of China (English)

    LUO Jun; ZHENG Jianfeng; PENG Jinfang; HE Liping; ZHU Minhao


    The introduction of surface engineering is expected to be an effective strategy against fretting damage. A large number of studies show that the low gas multi-component (such as carbon, nitrogen, sulphur and oxygen, etc) thermo-chemical treatment(LTGMTT) can overcome the brittleness of nitriding process, and upgrade the surface hardness and improve the wear resistance and fatigue properties of the work-pieces significantly. However, there are few reports on the anti-fretting properties of the LTGMTT modified layer up to now, which limits the applications of fretting. So this paper discusses the fretting wear behavior of modified layer on the surface of LZ50 (0.48%C) steel prepared by low temperature gas multi-component thermo-chemical treatment (LTGMTT) technology. The fretting wear tests of the modified layer flat specimens and its substrate (LZ50 steel) against 52100 steel balls with diameter of 40 mm are carried out under normal load of 150 N and displacement amplitudes varied from 2 μm to 40 μm. Characterization of the modified layer and dynamic analyses in combination with microscopic examinations were performed through the means of scanning electron microscope(SEM), optical microscope(OM), X-ray diffraction(XRD) and surface profilometer. The experimental results showed that the modified layer with a total thickness of 60 μm was consisted of three parts, i.e., loose layer, compound layer and diffusion layer. Compared with the substrate, the range of the mixed fretting regime(MFR) of the LTGMTT modified layer diminished, and the slip regime(SR) of the modified layer shifted to the direction of smaller displacement amplitude. The coefficient of friction(COF) of the modified layer was lower than that of the substrate in the initial stage. For the modified layer, the damage in partial slip regime(PSR) was very slight. The fretting wear mechanism of the modified layer both in MFR and SR was abrasive wear and delamination. The modified layer presented better wear

  6. Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system. (United States)

    Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q


    To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection

  7. Space analogue studies in Antarctica (United States)

    Lugg, D.; Shepanek, M.


    Medical research has been carried out on the Australian National Antarctic Research Expeditions (ANARE) for 50 years. As an extension of this program collaborative Australian/United States research on immunology, microbiology, psychology and remote medicine has produced important data and insight on how humans adapt to the stress of extreme isolation, confinement and the harsh environment of Antarctica. An outstanding analogue for the isolation and confinement of space missions (especially planetary outposts), ANARE has been used as an international research platform by Australia and the United States since 1993. Collaborative research has demonstrated a lowered responsiveness of the immune system under the isolation and confinement of Antarctic winter-over; a reduction of almost 50% in T cell proliferation to mitogen phytohaemogglutinin, as well as changes in latent herpesvirus states and the expansion of the polyclonal latent Epstein-Barr virus infected B cell populations. Although no clinically significant disease has been found to result from these immune changes, research is currently assessing the effects of psychological factors on the immune system. This and associated research performed to date and its relevance to both organisations is discussed, and comment made on possible extensions to the program in both medical and other fields.

  8. Condensed matter analogues of cosmology (United States)

    Kibble, Tom; Srivastava, Ajit


    It is always exciting when developments in one branch of physics turn out to have relevance in a quite different branch. It would be hard to find two branches farther apart in terms of energy scales than early-universe cosmology and low-temperature condensed matter physics. Nevertheless ideas about the formation of topological defects during rapid phase transitions that originated in the context of the very early universe have proved remarkably fruitful when applied to a variety of condensed matter systems. The mathematical frameworks for describing these systems can be very similar. This interconnection has led to a deeper understanding of the phenomena in condensed matter systems utilizing ideas from cosmology. At the same time, one can view these condensed matter analogues as providing, at least in a limited sense, experimental access to the phenomena of the early universe for which no direct probe is possible. As this special issue well illustrates, this remains a dynamic and exciting field. The basic idea is that when a system goes through a rapid symmetry-breaking phase transition from a symmetric phase into one with spontaneously broken symmetry, the order parameter may make different choices in different regions, creating domains that when they meet can trap defects. The scale of those domains, and hence the density of defects, is constrained by the rate at which the system goes through the transition and the speed with which order parameter information propagates. This is what has come to be known as the Kibble-Zurek mechanism. The resultant scaling laws have now been tested in a considerable variety of different systems. The earliest experiments illustrating the analogy between cosmology and condensed matter were in liquid crystals, in particular on the isotropic-to-nematic transition, primarily because it is very easy to induce the phase transition (typically at room temperature) and to image precisely what is going on. This field remains one of the

  9. Antimicrobial Activity of Resveratrol Analogues

    Directory of Open Access Journals (Sweden)

    Malik Chalal


    Full Text Available Stilbenes, especially resveratrol and its derivatives, have become famous for their positive effects on a wide range of medical disorders, as indicated by a huge number of published studies. A less investigated area of research is their antimicrobial properties. A series of 13 trans-resveratrol analogues was synthesized via Wittig or Heck reactions, and their antimicrobial activity assessed on two different grapevine pathogens responsible for severe diseases in the vineyard. The entire series, together with resveratrol, was first evaluated on the zoospore mobility and sporulation level of Plasmopara viticola (the oomycete responsible for downy mildew. Stilbenes displayed a spectrum of activity ranging from low to high. Six of them, including the most active ones, were subsequently tested on the development of Botrytis cinerea (fungus responsible for grey mold. The results obtained allowed us to identify the most active stilbenes against both grapevine pathogens, to compare the antimicrobial activity of the evaluated series of stilbenes, and to discuss the relationship between their chemical structure (number and position of methoxy and hydroxy groups and antimicrobial activity.

  10. A combined study of mesomorphism, optical, and electronic properties of donor-acceptor columnar liquid crystals

    NARCIS (Netherlands)

    Eichhorn, S.H.; Shuai, C.; Ahmida, M.; Demenev, A.; Kayal, H.; Raad, F.S.; Kaafarani, B.R.; Patwardhan, S.; Grozema, F.C.; Siebbeles, L.D.A.; Taerum, T.; Perepichka, D.F.; Klenkler, R.


    Donor-acceptor structures have recently gained great popularity for the design of low band gap polymeric organic semiconductors. Presented here is a first systematic study of organic semiconductors based on columnar liquid crystals that consist of discotic and board-shaped donor-acceptor structures.

  11. Phthalimides as exceptionally efficient single electron transfer acceptors in reductive coupling reactions promoted by samarium diiodide. (United States)

    Vacas, Tatiana; Alvarez, Eleuterio; Chiara, Jose Luis


    Experimental and theoretical evidence shows that phthalimides are highly efficient single electron transfer acceptors in reactions promoted by samarium diiodide, affording ketyl radical anion intermediates, which participate in high-yielding inter- and intramolecular reductive coupling processes with different radicophiles including imides, oxime ethers, nitrones, and Michael acceptors.

  12. Dichotomous Role of Exciting the Donor or the Acceptor on Charge Generation in Organic Solar Cells. (United States)

    Hendriks, Koen H; Wijpkema, Alexandra S G; van Franeker, Jacobus J; Wienk, Martijn M; Janssen, René A J


    In organic solar cells, photoexcitation of the donor or acceptor phase can result in different efficiencies for charge generation. We investigate this difference for four different 2-pyridyl diketopyrrolopyrrole (DPP) polymer-fullerene solar cells. By comparing the external quantum efficiency spectra of the polymer solar cells fabricated with either [60]PCBM or [70]PCBM fullerene derivatives as acceptor, the efficiency of charge generation via donor excitation and acceptor excitation can both be quantified. Surprisingly, we find that to make charge transfer efficient, the offset in energy between the HOMO levels of donor and acceptor that govern charge transfer after excitation of the acceptor must be larger by ∼0.3 eV than the offset between the corresponding two LUMO levels when the donor is excited. As a consequence, the driving force required for efficient charge generation is significantly higher for excitation of the acceptor than for excitation of the donor. By comparing charge generation for a total of 16 different DPP polymers, we confirm that the minimal driving force, expressed as the photon energy loss, differs by about 0.3 eV for exciting the donor and exciting the acceptor. Marcus theory may explain the dichotomous role of exciting the donor or the acceptor on charge generation in these solar cells.

  13. Life in the absence of oxygen: alterative electron acceptors for anaerobic microorganisms in a petroleum environment

    NARCIS (Netherlands)

    Balk, M.


    Anaerobic microorganisms derive energy by transferring electrons from an external source or donor to an external electron sink or terminal acceptor and often have the capacity to reduce 2 or more terminal electron acceptors. The well-known type of microbial respiration, in which oxygen serves as an

  14. Electric-field-induced ionization of acceptors in p-GaAs

    CERN Document Server

    Dargys, A; Cesna, A; Zurauskiene, N


    Hole de-trapping dynamics out of shallow acceptors subjected to high pulsed electric fields is investigated in pure p-GaAs used in radiation detectors. The characteristic de-trapping times are found from current transients due to impact and tunnel ionization of the acceptors. The de-trapping times are presented as a function of electric field strength. (author)

  15. Sulfur analogues of psychotomimetic agents. Monothio analogues of mescaline and isomescaline. (United States)

    Jacob, P; Shulgin, A T


    Two monothio analogues of mescaline and three monothio analogues of 2,3,4-trimethoxyphenethylamine (isomescaline) have been synthesized and characterized. Only the two mescaline analogues (3-and 4-thiomescaline) were found to be psychotomimetics in man, being 6 and 12 times more potent than mescaline, respectively. All five compounds can serve as substrates for bovine plasma monoamine oxidase in vitro, but no positive correlation is apparent between the extent of enzymatic degradation and human psychotomimetic potency.

  16. Heterocyclic chalcone analogues as potential anticancer agents. (United States)

    Sharma, Vikas; Kumar, Vipin; Kumar, Pradeep


    Chalcones, aromatic ketones and enones acting as the precursor for flavonoids such as Quercetin, are known for their anticancer effects. Although, parent chalcones consist of two aromatic rings joined by a three-carbon α,β-unsaturated carbonyl system, various synthetic compounds possessing heterocyclic rings like pyrazole, indole etc. are well known and proved to be effective anticancer agents. In addition to their use as anticancer agents in cancer cell lines, heterocyclic analogues are reported to be effective even against resistant cell lines. In this connection, we hereby highlight the potential of various heterocyclic chalcone analogues as anticancer agents with a brief summary about therapeutic potential of chalcones, mechanism of anticancer action of various chalcone analogues, and current and future prospects related to the chalcones-derived anticancer research. Furthermore, some key points regarding chalcone analogues have been reviewed by analyzing their medicinal properties.

  17. Total Synthesis of the Analogue of Icogenin

    Institute of Scientific and Technical Information of China (English)

    Shu Jie HOU; Peng XU; Liang ZHOU; De Quan YU; Ping Sheng LEI; Chuan Chun ZOU


    One of the analogues of icogenin, a natural furostanol saponin showing strong cytotoxic effect on cancer cell, was first synthesized via convergent strategy by using diosgenin and available monosaccharides as starting materials,

  18. Second-Generation Fluorescent Quadracyclic Adenine Analogues

    DEFF Research Database (Denmark)

    Dumat, Blaise; Bood, Mattias; Wranne, Moa S.;


    Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putativ...

  19. The structure activity relationship of discodermolide analogues. (United States)

    Shaw, Simon J


    The marine polyketide discodermolide is a member of a class of natural products that stabilize microtubules. Many analogues have been synthesized suggesting that few changes can be made to the internal carbon backbone. Both ends of the molecule, however, can be modified. The majority of analogues have been generated via modification of the lactone region. This suggests that significant simplifications can be made in this region provided that the lactone moiety is maintained.

  20. The activation energy for Mg acceptor in the Ga-rich InGaN alloys (United States)

    Zhao, Chuan-Zhen; Wei, Tong; Chen, Li-Ying; Wang, Sha-Sha; Wang, Jun


    The activation energy for Mg acceptor in InxGa1-xN alloys is investigated. It is found that there are three factors to influence the activation energy for Mg acceptor. One is the stronger dependence of the VBM of InxGa1-xN depending on In content than that of the Mg acceptor energy level. The other is the concentration of Mg acceptors. Another is the extending of the valence band-tail states into the band gap. In addition, a model based on modifying the effective mass model is developed. It is found that the model can describe the activation energy for Mg acceptor in the Ga-rich InxGa1-xN alloys well after considering the influence of the valence band-tail states.

  1. Excitation energy transfer in partly ordered polymer films differing in donor and acceptor transition moments orientation (United States)

    Synak, A.; Bojarski, P.; Sadownik, M.; Kułak, L.; Gryczynski, I.; Grobelna, B.; Rangełowa-Jankowska, S.; Jankowski, D.; Kubicki, A.


    Based on spectroscopic measurements selected properties of nonradiative Förster energy transport are studied in uniaxially stretched polyvinyl alcohol thin films for three systems differing in donor and acceptor transition moments orientation relative to the axis of stretching. In particular, donor - acceptor emission anisotropy spectra yield completely different regularities for these systems in uniaxially stretched films, whereas they are similar in unstretched films. In particular it is shown that acceptor fluorescence can be either strongly polarized after nonradiative energy transfer in stretched films or depolarized depending on the angular distribution of acceptor transition moments in the matrix. Donor and acceptor emission anisotropy decays exhibit similar regularities to those of steady-state measurements. The obtained results are analyzed with the help of Monte Carlo simulations.

  2. Hexa-peri-hexabenzocoronene with Different Acceptor Units for Tuning Optoelectronic Properties. (United States)

    Keerthi, Ashok; Hou, Ian Cheng-Yi; Marszalek, Tomasz; Pisula, Wojciech; Baumgarten, Martin; Narita, Akimitsu


    Hexa-peri-hexabenzocoronene (HBC)-based donor-acceptor dyads were synthesized with three different acceptor units, through two pathways: 1) "pre-functionalization" of monobromo-substituted hexaphenylbenzene prior to the cyclodehydrogenation; and 2) "post-functionalization" of monobromo-substituted HBC after the cyclodehydrogenation. The HBC-acceptor dyads demonstrated varying degrees of intramolecular charge-transfer interactions, depending on the attached acceptor units, which allowed tuning of their photophysical and optoelectronic properties, including the energy gaps. The two synthetic pathways described here can be complementary and potentially be applied for the synthesis of nanographene-acceptor dyads with larger aromatic cores, including one-dimensionally extended graphene nanoribbons.

  3. FRET-based genetically-encoded sensors for quantitative monitoring of metabolites. (United States)

    Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad


    Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

  4. Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter (United States)

    Billesbølle, Christian B.; Mortensen, Jonas S.; Sohail, Azmat; Schmidt, Solveig G.; Shi, Lei; Sitte, Harald H.; Gether, Ulrik; Loland, Claus J.


    Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle. PMID:27678200

  5. Transition metal ion FRET uncovers K(+) regulation of a neurotransmitter/sodium symporter. (United States)

    Billesbølle, Christian B; Mortensen, Jonas S; Sohail, Azmat; Schmidt, Solveig G; Shi, Lei; Sitte, Harald H; Gether, Ulrik; Loland, Claus J


    Neurotransmitter/sodium symporters (NSSs) are responsible for Na(+)-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K(+) inhibits Na(+)-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K(+)-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni(2+) bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K(+)-induced closure of the transporter to the outside, which was counteracted by Na(+) and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K(+)-effect. The K(+)-effect depended on an intact Na1 site and mutating the Na2 site potentiated K(+) binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K(+) to regulate the LeuT transport cycle.

  6. FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Hanna


    The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

  7. Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits. (United States)

    Quan, Ke; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Ying, Le; Wang, He; Xie, Nuli; Ou, Min; Wang, Kemin


    Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

  8. The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, Jennifer L.; Kim, Hanseong [Arizona State University, Tempe, AZ 85287-1604 (United States); Markwardt, Michele L. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Chen, Liqing; Fromme, Raimund [Arizona State University, Tempe, AZ 85287-1604 (United States); Rizzo, Mark A. [University of Maryland School of Medicine, Baltimore, MD 21201-1559 (United States); Wachter, Rebekka M., E-mail: [Arizona State University, Tempe, AZ 85287-1604 (United States)


    The high resolution X-ray structure of the cyan fluorescent protein mCerulean3 demonstrates that different combinations of correlated residue substitutions can provide near optimum quantum yield values for fluorescence. Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

  9. Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Wendy E Kaman

    Full Text Available Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM. In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility.

  10. Intravital imaging of fluorescent markers and FRET probes by DNA tattooing

    Directory of Open Access Journals (Sweden)

    Spencer David M


    Full Text Available Abstract Background Advances in fluorescence microscopy and mouse transgenesis have made it possible to image molecular events in living animals. However, the generation of transgenic mice is a lengthy process and intravital imaging requires specialized knowledge and equipment. Here, we report a rapid and undemanding intravital imaging method using generally available equipment. Results By DNA tattooing we transfect keratinocytes of living mice with DNA encoding fluorescent biosensors. Subsequently, the behavior of individual cells expressing these biosensors can be visualized within hours and using conventional microscopy equipment. Using this "instant transgenic" model in combination with a corrected coordinate system, we followed the in vivo behavior of individual cells expressing either FRET- or location-based biosensors for several days. The utility of this approach was demonstrated by assessment of in vivo caspase-3 activation upon induction of apoptosis. Conclusion This "instant skin transgenic" model can be used to follow the in vivo behavior of individual cells expressing either FRET- or location-based probes for several days after tattooing and provides a rapid and inexpensive method for intravital imaging in murine skin.

  11. Nanoblinker: Brownian Motion Powered Bio-Nanomachine for FRET Detection of Phagocytic Phase of Apoptosis (United States)

    Minchew, Candace L.; Didenko, Vladimir V.


    We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5′OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells’ DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis. PMID:25268504

  12. An all-in-one numerical methodology for fretting wear and fatigue life assessment

    Directory of Open Access Journals (Sweden)

    I. Llavori


    Full Text Available Many mechanical components such as, bearing housings, flexible couplings and spines or orthopedic devices are simultaneously subjected to a fretting wear and fatigue damage. For this reason, the combined study on a single model of wear, crack initiation and propagation is of great interest. This paper presents an all-in-one 2D cylinder on flat numerical model for life assessment on coupled fretting wear and fatigue phenomena. In the literature, two stages are usually distinguished: crack nucleation and its subsequent growth. The method combines the Archard wear model, a critical-plane implementation of the Smith-Watson- Topper (SWT multiaxial fatigue criterion coupled with the Miner-Palmgren accumulation damage rule for crack initiation prediction. Then, the Linear Elastic Fracture Mechanics (LEFM via eXtended Finite Element Method (X-FEM embedded into the commercial finite element code Abaqus FEA has been employed to determine the crack propagation stage. Therefore, the sum of the two stages gives a total life prediction. Finally, the numerical model was validated with experimental data reported in the literature and a good agreement was obtained.

  13. Effects of Hydrogen on Acceptor Activation in Ternary Nitride Semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Fioretti, Angela N. [National Renewable Energy Laboratory, Golden CO 80401 USA; Colorado School of Mines, Golden CO 80401 USA; Stokes, Adam [National Renewable Energy Laboratory, Golden CO 80401 USA; Colorado School of Mines, Golden CO 80401 USA; Young, Matthew R. [National Renewable Energy Laboratory, Golden CO 80401 USA; Gorman, Brian [Colorado School of Mines, Golden CO 80401 USA; Toberer, Eric S. [National Renewable Energy Laboratory, Golden CO 80401 USA; Colorado School of Mines, Golden CO 80401 USA; Tamboli, Adele C. [National Renewable Energy Laboratory, Golden CO 80401 USA; Colorado School of Mines, Golden CO 80401 USA; Zakutayev, Andriy [National Renewable Energy Laboratory, Golden CO 80401 USA


    Doping control is necessary to unlock the scientific and technological potential of many materials, including ternary II-IV-nitride semiconductors, which are closely related to binary GaN. In particular, ZnSnN2 has been reported to have degenerate doping density, despite bandgap energies that are well suited for solar energy conversion. Here, we show that annealing Zn-rich Zn1+xSn1-xN2 grown with added hydrogen reduces its free electron density by orders of magnitude, down to 4 x 1016 cm-3. This experimental observation can be explained by hydrogen passivation of acceptors in Zn1+xSn1-xN2 during growth, lowering the driving force for unintentional donor formation. These results indicate that the doping control principles used in GaN can be translated to ZnSnN2, suggesting that other strategies used in binary III-Vs can be applied to accelerate the technological development of ternary II-IV-N2 materials.


    Institute of Scientific and Technical Information of China (English)

    Jian-fei Qu; Jian Liu; Si-da Li; Zhi-yuan Xie; Yan-hou Geng


    Five novel donor-acceptor (D-A) conjugated cooligomers (F4B-hP,F5B-hP,F5B2[1,2]-hP,F5B2[1,3]-hP and F7B2[1,2]-hP) were synthesized.The absorption spectra of the cooligomers cover a wide range from 300 nm to 630 nm.The cooligomers could form films featured by alternating D-A lamellar nanostructures with the periods relative to the molecular lengths after thermal annealing or solvent vapor annealing.Single molecule solar cells were fabricated,and F5B-hP exhibited the best device performance.When the film of F5B-hP was thermally annealed,a power conversion efficiency (PCE) of 1.56% was realized.With solvent vapor annealing,the PCE could be further improved to 1.72% with a short-circuit current (Jsc) of 5.76 mA/cm2,an open-circuit voltage (VoC) of 0.87 V and a fill factor (FF) of 0.34.

  15. Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET. (United States)

    Zagotta, William N; Gordon, Moshe T; Senning, Eric N; Munari, Mika A; Gordon, Sharona E


    Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent, noncanonical amino acid, L-ANAP. A metal chelating lipid was used to decorate the plasma membrane with high-density/high-affinity metal-binding sites. The fluorescence resonance energy transfer (FRET) efficiencies between L-ANAP in TRPV1 and Co(2+) bound to the plasma membrane were consistent with the arrangement of the ARDs in recent cryoelectron microscopy structures of TRPV1. No change in tmFRET was observed with the TRPV1 agonist capsaicin. These results demonstrate the power of tmFRET for measuring structure and rearrangements of membrane proteins relative to the cell membrane.

  16. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda


    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  17. The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues

    Directory of Open Access Journals (Sweden)

    Anna-Karin E. Johnsson


    Full Text Available The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

  18. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda


    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  19. Monitoring apoptosis of TK-GFP-expressing ACC-M cells induced by ACV using FRET technique (United States)

    Xiong, Tao; Zhang, Zhihong; Lin, Juqiang; Yang, Jie; Zeng, Shaoqun; Luo, Qingming


    Apoptosis is an evolutionary conserved cellular process that plays an important role during development, but it is also involved in tissue homeostasis and in many diseases. To study the characteristics of suicide gene system of the herpes simplex virus thymidine kinase (HSV-tk) gene in tumor cells and explore the apoptosis phenomena in this system and its effect on the human adenoid cystic carcinoma line ACC-M cell, we detected apoptosis of CD3- (ECFP-CRS-DsRed) and TK-GFP-expressing ACC-M (ACC-M-TK-GFP-CD3) cells induced by acyclovir (ACV) using fluorescence resonance energy transfer (FRET) technique. CD3 is a FRET-based indicator for activity of caspase-3, which is composed of an enhanced cyan fluorescent protein, a caspase-3 sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property. FRET from ECFP to DsRed could be detected in normal ACC-M-TK-GFP-CD3 cells, and the FRET efficient was remarkably decreased and then disappeared during the cells apoptosis induced by ACV. It was due to the activated caspase-3 cleaved the CD3 fusion protein. In this study, the results suggested that the AVC-induced apoptosis of ACC-M-TK-GFP-CD3 cells was through caspase-3 pathway.

  20. Influence of thermal oxidation duration on the microstructure and fretting wear behavior of Ti6Al4V alloy

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Song [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Liao, Zhenhua [Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Biomechanics and Biotechnology Lab, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China); Liu, Yuhong [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Department of Mechanical Engineering, Tsinghua University, Beijing 100084 (China); Liu, Weiqiang, E-mail: [State Key Laboratory of Tribology, Tsinghua University, Beijing 100084 (China); Biomechanics and Biotechnology Lab, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China)


    Thermal oxidation under water oxidizing atmosphere was performed on Ti6Al4V alloy under different durations from 2 h to 8 h. Surface characterizations were performed using X-ray diffractometery (XRD), scanning electron microscopy (SEM), Raman spectroscopy, nanoindentation and nano scratch testing. Fretting wear behaviors of untreated and oxidized samples were also examined. The formed oxide coating mainly included rutile TiO{sub 2} as well as a little alumina. The weight gain with respect to the oxidation duration obeyed the linear oxidation kinetics law. The growth of oxide grains was in inadequate growth state of incomplete scale coverage from 2nd to 4th hour duration, in normal growth state from 4th to 6th hour duration while in excessive growth state of oxide particle agglomeration and surface roughening from 6th to 8th (or more than 8th) hour duration. The coating thickness increased from 5 μm to 12 μm as oxidation duration increased from 2 h to 8 h. The increase in duration also increased surface roughness and nano hardness as well as adhesion strength of the film/substrate for oxidized samples. The nano hardness value was 10.06 ± 2.15 GPa and the critical load of failure during nano scratch testing was 554.3 ± 6.44 mN for 4 h treated sample. The untreated and oxidized samples showed a same fretting running status and fretting regime with a displacement amplitude of 200 μm while revealing different fretting failure mechanisms. It was mainly abrasive and adhesive wear under ploughing force for untreated sample, while a mix of 3-body abrasion by rolling oxide particles and severe plastic deformation under high contact stress between two ceramic materials for the oxidized samples. The oxide coating was not worn out and improved the fretting wear resistance of titanium alloy. - Highlights: • A thickness of 5–12 μm rutile TiO{sub 2} coating formed under different oxidation durations. • Weight gain with respect to oxidation duration obeyed linear

  1. Between Analogue and Digital Diagrams

    Directory of Open Access Journals (Sweden)

    Zoltan Bun


    Full Text Available This essay is about the interstitial. About how the diagram, as a method of design, has lead fromthe analogue deconstruction of the eighties to the digital processes of the turn of the millennium.Specifically, the main topic of the text is the interpretation and the critique of folding (as a diagramin the beginning of the nineties. It is necessary then to unfold its relationship with immediatelypreceding and following architectural trends, that is to say we have to look both backwards andforwards by about a decade. The question is the context of folding, the exchange of the analogueworld for the digital. To understand the process it is easier to investigate from the fields of artand culture, rather than from the intentionally perplicated1 thoughts of Gilles Deleuze. Both fieldsare relevant here because they can similarly be used as the yardstick against which the era itselfit measured. The cultural scene of the eighties and nineties, including performing arts, movies,literature and philosophy, is a wide milieu of architecture. Architecture responds parallel to itsera; it reacts to it, and changes with it and within it. Architecture is a medium, it has always beena medium, yet the relations are transformed. That’s not to say that technical progress, for exampleusing CAD-software and CNC-s, has led to the digital thinking of certain movements ofarchitecture, (it is at most an indirect tool. But the ‘up-to-dateness’ of the discipline, however,a kind of non-servile reading of an ‘applied culture’ or ‘used philosophy’2 could be the key.(We might recall here, parenthetically, the fortunes of the artistic in contemporary mass society.The proliferation of museums, the magnification of the figure of the artist, the existence of amassive consumption of printed and televised artistic images, the widespread appetite for informationabout the arts, all reflect, of course, an increasingly leisured society, but also relateprecisely to the fact

  2. Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability

    Energy Technology Data Exchange (ETDEWEB)

    Giometti, Carol S.; Khare, Tripti; Verberkmoes, Nathan; O' Loughlin, Ed; Lindberg, Carl; Thompson, Melissa; Hettich, Robert


    Shewanella oneidensis MR-1, a gram negative metal-reducing bacterium, can utilize a large number of electron acceptors. In the natural environment, S. oneidensis utilizes insoluble metal oxides as well as soluble terminal electron acceptors. The purpose of this ERSP project is to identify differentially expressed proteins associated with the membranes of S. oneidensis MR-1 cells grown with different electron acceptors, including insoluble metal oxides. We hypothesize that through the use of surface labeling, subcellular fractionation, and a combination of proteome analysis tools, proteins involved in the reduction of different terminal electron acceptors will be elucidated. We are comparing the protein profiles from cells grown with the soluble electron acceptors oxygen and fumarate and with those from cells grown with the insoluble iron oxides goethite, ferrihydrite and lepidocrocite. Comparison of the cell surface proteins isolated from cells grown with oxygen or anaerobically with fumarate revealed an increase in the abundance of over 25 proteins in anaerobic cells, including agglutination protein and flagellin proteins along with the several hypothetical proteins. In addition, the surface protein composition of cells grown with the insoluble iron oxides varies considerably from the protein composition observed with either soluble electron acceptor as well as between the different insoluble acceptors.

  3. Synthesis and electrochemical studies of charge-transfer complexes of thiazolidine-2,4-dione with σ and π acceptors (United States)

    Singh, Prashant; Kumar, Pradeep; Katyal, Anju; Kalra, Rashmi; Dass, Sujata K.; Prakash, Satya; Chandra, Ramesh


    In the present work, we report the synthesis and characterization of novel charge-transfer complexes of thiazolidine-2,4-dione (TZD) with sigma acceptor (iodine) and pi acceptors (chloranil, dichlorodicyanoquinone, picric acid and duraquinone). We also evaluated their thermal and electrochemical properties and we conclude that these complexes are frequency dependent. Charge-transfer complex between thiazolidine-2,4-dione and iodine give best conductivity. In conclusion, complex with sigma acceptors are more conducting than with pi acceptors.

  4. Wide-field lifetime-based FRET imaging for the assessment of early functional distribution of transferrin-based delivery in breast tumor-bearing small animals (United States)

    Sinsuebphon, Nattawut; Rudkouskaya, Alena; Barroso, Margarida; Intes, Xavier


    Targeted drug delivery is a critical aspect of successful cancer therapy. Assessment of dynamic distribution of the drug provides relative concentration and bioavailability at the target tissue. The most common approach of the assessment is intensity-based imaging, which only provides information about anatomical distribution. Observation of biomolecular interactions can be performed using Förster resonance energy transfer (FRET). Thus, FRET-based imaging can assess functional distribution and provide potential therapeutic outcomes. In this study, we used wide-field lifetime-based FRET imaging for the study of early functional distribution of transferrin delivery in breast cancer tumor models in small animals. Transferrin is a carrier for cancer drug delivery. Its interaction with its receptor is within a few nanometers, which is suitable for FRET. Alexa Fluor® 700 and Alexa Fluor® 750 were conjugated to holo-transferrin which were then administered via tail vein injection to the mice implanted with T47D breast cancer xenografts. Images were continuously acquired for 60 minutes post-injection. The results showed that transferrin was primarily distributed to the liver, the urinary bladder, and the tumor. The cellular uptake of transferrin, which was indicated by the level of FRET, was high in the liver but very low in the urinary bladder. The results also suggested that the fluorescence intensity and FRET signals were independent. The liver showed increasing intensity and increasing FRET during the observation period, while the urinary bladder showed increasing intensity but minimal FRET. Tumors gave varied results corresponding to their FRET progression. These results were relevant to the biomolecular events that occurred in the animals.

  5. Sliding Wear and Fretting Wear of DLC-Based, Functionally Graded Nanocomposite Coatings (United States)

    Miyoshi, K.; Pohlchuck, B.; Street, Kenneth W.; Zabinski, J. S.; Sanders, J. H.; Voevodin, A. a.; Wu, R. L. C.


    Improving the tribological functionality of diamondlike carbon (DLC) films--developing, good wear resistance, low friction, and high load-carrying capacity-was the aim of this investigation. Nanocomposite coatings consisting of an amorphous DLC (a-DLC) top layer and a functionally graded titanium-titanium carbon-diamondlike carbon (Ti-Ti(sub x) C(sub y)-DLC) underlayer were produced on AISI 440C stainless steel substrates by the hybrid technique of magnetron sputtering and pulsed-laser deposition. The resultant DLC films were characterized by Raman spectroscopy, scanning electron microscopy, and surface profilometry. Two types of wear experiment were conducted in this investioation: sliding friction experiments and fretting wear experiments. Unidirectional ball-on-disk sliding friction experiments were conducted to examine the wear behavior of an a-DLC/Ti-Ti(sub x) C(sub y)-DLC-coated AISI 440C stainless steel disk in sliding contact with a 6-mm-diameter AISI 440C stainless steel ball in ultrahigh vacuum, dry nitrogen, and humid air. Although the wear rates for both the coating and ball were low in all three environments, the humid air and dry nitrogen caused mild wear with burnishing, in the a-DLC top layer, and the ultrahigh vacuum caused relatively severe wear with brittle fracture in both the a-DLC top layer and the Ti-Ti(sub x) C(sub y)-DLC underlayer. For reference, amorphous hydrogenated carbon (H-DLC) films produced on a-DLC/Ti-Ti(sub x) C(sub y)-DLC nanocomposite coatings by using an ion beam were also examined in the same manner. The H-DLC films markedly reduced friction even in ultrahigh vacuum without sacrificing wear resistance. The H-DLC films behaved much like the a-DLC/Ti-Ti(sub x) C(sub y)-DLC nanocomposite coating in dry nitrogen and humid air, presenting low friction and low wear. Fretting wear experiments were conducted in humid air (approximately 50% relative humidity) at a frequency of 80 Hz and an amplitude of 75 micron on an a

  6. Friction and wear properties of high-velocity oxygen fuel sprayed WC-17Co coating under rotational fretting conditions (United States)

    Luo, Jun; Cai, Zhenbing; Mo, Jiliang; Peng, Jinfang; Zhu, Minhao


    Rotational fretting which exist in many engineering applications has incurred enormous economic loss. Thus, accessible methods are urgently needed to alleviate or eliminate damage by rotational fretting. Surface engineering is an effective approach that is successfully adopted to enhance the ability of components to resist the fretting damage. In this paper, using a high-velocity oxygen fuel sprayed (HVOF) technique WC-17Co coating is deposited on an LZ50 steel surface to study its properties through Vickers hardness testing, scanning electric microscope (SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffractrometry (XRD). Rotational fretting wear tests are conducted under normal load varied from 10 N to 50 N, and angular displacement amplitudes vary from 0.125° to 1°. Wear scars are examined using SEM, EDX, optical microscopy (OM), and surface topography. The experimental results reveal that the WC-17Co coating adjusted the boundary between the partial slip regime (PSR) and the slip regime (SR) to the direction of smaller amplitude displacement. As a result, the coefficients of friction are consistently lower than the substrate's coefficients of friction both in the PSR and SR. The damage to the coating in the PSR is very slight. In the SR, the coating exhibits higher debris removal efficiency and load-carrying capacity. The bulge is not found for the coating due to the coating's higher hardness to restrain plastic flow. This research could provide experimental bases for promoting industrial application of WC-17Co coating in prevention of rotational fretting wear.

  7. New Type of Donor-Acceptor Through-Space Conjugated Polymer

    Directory of Open Access Journals (Sweden)

    Lin Lin


    Full Text Available We report the synthesis and properties of a novel through-space conjugated polymer with a [2.2]paracyclophane skeleton. The obtained polymer possessed donor (fluorene and acceptor (2,1,3-benzothiadiazole segments that were alternately π-stacked in proximity via the [2.2]paracyclophane moieties. The good overlap between the emission peak of the donor unit (fluorene and the CT band of the acceptor unit (2,1,3-benzothiadiazole caused fluorescence resonance energy transfer, and the visible green light emission from the acceptor unit was observed.

  8. Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif. (United States)

    Filipski, Kevin J; Guzman-Perez, Angel; Bian, Jianwei; Perreault, Christian; Aspnes, Gary E; Didiuk, Mary T; Dow, Robert L; Hank, Richard F; Jones, Christopher S; Maguire, Robert J; Tu, Meihua; Zeng, Dongxiang; Liu, Shenping; Knafels, John D; Litchfield, John; Atkinson, Karen; Derksen, David R; Bourbonais, Francis; Gajiwala, Ketan S; Hickey, Michael; Johnson, Theodore O; Humphries, Paul S; Pfefferkorn, Jeffrey A


    Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

  9. Optical spectroscopy of single beryllium acceptors in GaAs/AlGaAs quantum well (United States)

    Petrov, P. V.; Kokurin, I. A.; Klimko, G. V.; Ivanov, S. V.; Ivánov, Yu. L.; Koenraad, P. M.; Silov, A. Yu.; Averkiev, N. S.


    We carry out microphotoluminescence measurements of an acceptor-bound exciton (A0X ) recombination in the applied magnetic field with a single impurity resolution. In order to describe the obtained spectra we develop a theoretical model taking into account a quantum well (QW) confinement, an electron-hole and hole-hole exchange interaction. By means of fitting the measured data with the model we are able to study the fine structure of individual acceptors inside the QW. The good agreement between our experiments and the model indicates that we observe single acceptors in a pure two-dimensional environment whose states are unstrained in the QW plain.

  10. Photochemistry of free and bound Zn-chlorophyll analogues to synthetic peptides depend on the quinone and pH. (United States)

    Razeghifard, Reza


    A synthetic peptide was used as a scaffold to bind Zn-Chlorophyll (ZnChl) analogues through histidine ligation to study their photochemistry in the presence of different type of quinones. The Chl analogues were chlorin e6 (Ce6), chlorin e6 trimethyl ester, pyropheophorbide a, and pheophorbide a while the quinones were PPBQ, DMBQ, NPHQ, DBTQ, DCBQ and PBQ. The binding of each ZnChl analogue to the peptide was verified by native gel electrophoresis. First the photo-stability of the ZnChl analogues were tested under continuous light. The ZnCe6 and ZnCe6TM analogues showed the least stability judged by the loss of optical signal intensity at their Qy band. The photoactivity of each ZnChl analogue was measured in the presence of each of the six quinones using time-resolved EPR spectroscopy. DMBQ was found to be the most efficient electron acceptor when all four ZnChl analogues were compared. The light-induced electron transfer between the ZnChl analogues complexed with the peptide and DMBQ were also measured using time-resolved EPR spectroscopy. The ZnCe6-peptide complex exhibited the highest photoactivity. The electron transfer in the complex was faster and the photoactivity yield was higher than those values obtained for free ZnCe6 and DMBQ. The fast phase of kinetics can be attributed to intra-protein electron transfer in the complex since it was not observed in the presence of DMBQ-glutathione adduct. Unlike free ZnCe6, the ZnCe6-peptide complex was robust and demonstrated very similar photoactivity efficiency in pH values 10, 8.0 and 5.0. The electron transfer kinetics were pH dependent and appeared to be modulated by the peptide charge and possibly fold. The charge recombination rate was slowed by an order of magnitude when the pH value was changed from 10.0 to 5.0. The implications of constructing the photoactive peptide complexes in terms of artificial photosynthesis are discussed.

  11. CASL Structural Mechanics Modeling of Grid-to-Rod Fretting (GTRF) (United States)

    Lu, Wei; Thouless, M. D.; Hu, Zupan; Wang, Hai; Ghelichi, Ramin; Wu, Chen-Hung; Kamrin, Ken; Parks, David


    Fluid-induced grid-to-rod fretting (GTRF) wear is responsible for over 70% of fuel leaks in pressurized water reactors (PWRs) in the US. The Consortium for Advanced Simulation of Light Water Reactors (CASL) has identified GTRF as a challenge problem that is very important to nuclear plants. GTRF is a complex problem that involves multiple physical phenomena. This paper summarizes several GTRF-related problems being addressed by the University of Michigan and the Massachusetts Institute of Technology, CASL partners. These include analyses of cladding creep, wear, structural mechanics, and the effects of the rod-to-grid gap. Also outlined are additional aspects of material science and computational modeling that will be needed to realize the ultimate goal of high-fidelity predictive modeling and design tools to address GTRF.

  12. A Study on Fretting Wear Property of CVD SiC and Sintered SiC

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Hyun-Jin; Jang, Ki-Nam; An, Ji-Hyeong; Kim, Kyu-Tae [Dongguk University, Gyeongju (Korea, Republic of)


    Silicon Carbide is broadly used as high temperature structure material because of its high temperature tolerance and superior mechanical properties. After the Fukushima nuclear power plant accident, SiC proposed as one of the alternative materials for LWR fuel cladding to provide enhanced safety margin. Grid-to-rod fretting wear-induced fuel failure is known to occur due to flow-induced vibration of the reactor core and grid to- rod gap. In this paper, wear tests for CVD SiC plate and sintered SiC tube were performed with two types of spacer grids. Wear test of corroded and non-corroded CVD SiC plates indicate that wear resistance of corroded specimen is lower than one of non-corroded specimen in contrast with zirconium alloy cladding tube. It may be affected by rough surface of corroded specimen caused by grain boundary attack.

  13. A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

    DEFF Research Database (Denmark)

    Schneider, Uffe V.; Severinsen, Jette K.; Géci, Imrich;


    BACKGROUND: Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic....... The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm...... and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes. RESULTS: A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection...

  14. spFRET reveals changes in nucleosome breathing by neighboring nucleosomes. (United States)

    Buning, Ruth; Kropff, Wietske; Martens, Kirsten; van Noort, John


    Chromatin, the structure in which DNA is compacted in eukaryotic cells, plays a key role in regulating DNA accessibility. FRET experiments on single nucleosomes, the basic units in chromatin, have revealed a dynamic nucleosome where spontaneous DNA unwrapping from the ends provides access to the nucleosomal DNA. Here we investigated how this DNA breathing is affected by extension of the linker DNA and by the presence of a neighboring nucleosome. We found that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Interactions between neighboring nucleosomes are more likely in dinucleosomes spaced by 55 bp of linker DNA than in dinucleosomes spaced by 50 bp of linker DNA. Such increased unwrapping may not only increase the accessibility of nucleosomal DNA in chromatin fibers, it may also be key to folding of nucleosomes into higher order structures.

  15. Multirate Simulations of String Vibrations Including Nonlinear Fret-String Interactions Using the Functional Transformation Method (United States)

    Trautmann, L.; Rabenstein, R.


    The functional transformation method (FTM) is a well-established mathematical method for accurate simulations of multidimensional physical systems from various fields of science, including optics, heat and mass transfer, electrical engineering, and acoustics. This paper applies the FTM to real-time simulations of transversal vibrating strings. First, a physical model of a transversal vibrating lossy and dispersive string is derived. Afterwards, this model is solved with the FTM for two cases: the ideally linearly vibrating string and the string interacting nonlinearly with the frets. It is shown that accurate and stable simulations can be achieved with the discretization of the continuous solution at audio rate. Both simulations can also be performed with a multirate approach with only minor degradations of the simulation accuracy but with preservation of stability. This saves almost 80% of the computational cost for the simulation of a six-string guitar and therefore it is in the range of the computational cost for digital waveguide simulations.

  16. Geomagnetic properties of Proxima Centauri b analogues

    CERN Document Server

    Zuluaga, Jorge I


    The recent discovery of a planet around the closest star, Proxima Centauri, could represent a quantum leap on the testability of models in exoplanet sciences. Unlike any other discovered exoplanet, models of planetary processes in Proxima b could be contrasted against near future telescopic observations and far future in-situ measurements. In this paper we study the geomagnetic properties of Proxima b analogues, namely, solid planets with masses close but larger than Earth's mass, periods of rotation of several days and habitable surface conditions. Assuming different planetary masses, bulk compositions and periods of rotations, we calculate for each planetary analogue its radius, heat flux, time of inner core formation, dynamo lifetime and minimum dipole magnetic moment. We find that most ($\\gtrsim$70\\%) Proxima b analogues develop intrinsic dynamos that last at least 3 Gyr, although only half of them are older than the present age of the host star ($4-6$ Gyr). Relying in our planetary evolution models, we p...

  17. Synthesis and anticancer evaluation of spermatinamine analogues

    KAUST Repository

    Moosa, Basem


    Spermatinamine was isolated from an Australian marine sponge, Pseudoceratina sp. as an inhibitor of isoprenylcystiene carboxyl methyltransferase (Icmt), an attractive and novel anticancer target. Herein, we report the synthesis of spermatinamine analogues and their cytotoxic evaluation against three human cancer cell lines i.e. cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF-7), and prostate carcinoma (DU145). Analogues 12, 14 and 15 were found to be the most potent against one or more cell lines with the IC50 values in the range of 5 - 10 μM. The obtained results suggested that longer polyamine linker along with aromatic oxime substitution provided the most potent analogue compounds against cancer cell lines.

  18. Molecular dynamics simulations of the cardiac troponin complex performed with FRET distances as restraints.

    Directory of Open Access Journals (Sweden)

    Jayant James Jayasundar

    Full Text Available Cardiac troponin (cTn is the Ca(2+-sensitive molecular switch that controls cardiac muscle activation and relaxation. However, the molecular detail of the switching mechanism and how the Ca(2+ signal received at cardiac troponin C (cTnC is communicated to cardiac troponin I (cTnI are still elusive. To unravel the structural details of troponin switching, we performed ensemble Förster resonance energy transfer (FRET measurements and molecular dynamic (MD simulations of the cardiac troponin core domain complex. The distance distributions of forty five inter-residue pairs were obtained under Ca(2+-free and saturating Ca(2+ conditions from time-resolved FRET measurements. These distances were incorporated as restraints during the MD simulations of the cardiac troponin core domain. Compared to the Ca(2+-saturated structure, the absence of regulatory Ca(2+ perturbed the cTnC N-domain hydrophobic pocket which assumed a closed conformation. This event partially unfolded the cTnI regulatory region/switch. The absence of Ca(2+, induced flexibility to the D/E linker and the cTnI inhibitory region, and rotated the cTnC N-domain with respect to rest of the troponin core domain. In the presence of saturating Ca(2+ the above said phenomenon were absent. We postulate that the secondary structure perturbations experienced by the cTnI regulatory region held within the cTnC N-domain hydrophobic pocket, coupled with the rotation of the cTnC N-domain would control the cTnI mobile domain interaction with actin. Concomitantly the rotation of the cTnC N-domain and perturbation of the D/E linker rigidity would control the cTnI inhibitory region interaction with actin to effect muscle relaxation.

  19. Olive secoiridoids and semisynthetic bioisostere analogues for the control of metastatic breast cancer. (United States)

    Busnena, Belnaser A; Foudah, Ahmed I; Melancon, Tina; El Sayed, Khalid A


    (-)-Oleocanthal (1) and ligstroside aglycone (2) are common bioactive olive oil secoiridoids. Secoiridoid 1 has been previously reported as a c-MET inhibitor. Chemically, (-)-oleocanthal is the elenolic acid ester of the common olive phenolic alcohol tyrosol. Therefore, several analogues (4-13) were synthesized by esterification and carbamoylation of tyrosol using diverse phenolic naturally occurring in olive and heterocyclic acids as elenolic acid bioisosteres to assess the effect of replacing the acid moiety of (-)-oleocanthal. Their c-MET inhibitory activity as well as their antiproliferative, antimigratory, and anti-invasive activities against the highly metastatic human breast cancer cell line MDA-MB231 has been assessed. Ligstroside aglycone (2) showed the best antimigratory activity. Generally, tyrosol esters showed better activities versus carbamate analogues. Tyrosol sinapate (5) showed the best c-MET phosphorylation inhibitory activity in Z'-LYTE kinase assay. Both 1 and 5 competitively inhibited the ATP binding into its pocket in the c-MET catalytic domain. Compound 5 showed selective activities against tumor cells without toxicity to the non-tumorigenic human breast MCF10A epithelial cell line. Tyrosol esters with a phenolic acid containing hydrogen bond donor and/or acceptor groups at the para-position have better anticancer and c-MET inhibitory activities. Olive oil secoiridoids are excellent scaffolds for the design of novel c-MET inhibitors.

  20. Benzoylphenylurea sulfur analogues with potent antitumor activity. (United States)

    Hallur, Gurulingappa; Jimeno, Antonio; Dalrymple, Susan; Zhu, Tao; Jung, M Katherine; Hidalgo, Manuel; Isaacs, John T; Sukumar, Saraswati; Hamel, Ernest; Khan, Saeed R


    A novel series of BPU analogues were synthesized and evaluated for antitumor activity. In particular, BPU sulfur analogues 6n and 7d were shown to possess up to 10-fold increased potency, when compared to 1 (NSC-639829), against cancer cell lines. 6n was more effective than 1 in causing apoptosis of MCF-7 cells. When compared to other drugs with a similar mechanism of action, 6n retained significant ability to inhibit tubulin assembly, with an IC(50) of 2.1 microM.

  1. Time-resolved spectroscopy of the fluorescence quenching of a donor — acceptor pair by halothane (United States)

    Sharma, A.; Draxler, S.; Lippitsch, M. E.


    Donor (anthracene) sensitized acceptor (perylene) fluorescence is quenched more efficiently by halothane than is intrinsic perylene fluorescence. The underlying process of dynamic fluorescence quenching is investigated by time-resolved fluorescence spectroscopy.

  2. Computational design of donor-bridge-acceptor systems exhibiting pronounced quantum interference effects. (United States)

    Gorczak, Natalie; Renaud, Nicolas; Galan, Elena; Eelkema, Rienk; Siebbeles, Laurens D A; Grozema, Ferdinand C


    Quantum interference is a well-known phenomenon that dictates charge transport properties of single molecule junctions. However, reports on quantum interference in donor-bridge-acceptor molecules are scarce. This might be due to the difficulties in meeting the conditions for the presence of quantum interference in a donor-bridge-acceptor system. The electronic coupling between the donor, bridge, and acceptor moieties must be weak in order to ensure localised initial and final states for charge transfer. Yet, it must be strong enough to allow all bridge orbitals to mediate charge transfer. We present the computational route to the design of a donor-bridge-acceptor molecule that features the right balance between these contradicting requirements and exhibits pronounced interference effects.

  3. Interface-induced heavy-hole/light-hole splitting of acceptors in silicon

    Energy Technology Data Exchange (ETDEWEB)

    Mol, J. A. [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); Department of Materials, University of Oxford, 16 Parks Road, Oxford OX1 3PH (United Kingdom); Salfi, J.; Simmons, M. Y.; Rogge, S., E-mail: [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); Rahman, R.; Hsueh, Y.; Klimeck, G. [Purdue University, West Lafayette, Indiana 47906 (United States); Miwa, J. A. [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); Department of Physics and Astronomy, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, 8000 Aarhus C (Denmark)


    The energy spectrum of spin-orbit coupled states of individual sub-surface boron acceptor dopants in silicon have been investigated using scanning tunneling spectroscopy at cryogenic temperatures. The spatially resolved tunnel spectra show two resonances, which we ascribe to the heavy- and light-hole Kramers doublets. This type of broken degeneracy has recently been argued to be advantageous for the lifetime of acceptor-based qubits [R. Ruskov and C. Tahan, Phys. Rev. B 88, 064308 (2013)]. The depth dependent energy splitting between the heavy- and light-hole Kramers doublets is consistent with tight binding calculations, and is in excess of 1 meV for all acceptors within the experimentally accessible depth range (<2 nm from the surface). These results will aid the development of tunable acceptor-based qubits in silicon with long coherence times and the possibility for electrical manipulation.

  4. A Novel Donor Acceptor Substituted Chiroptical Molecular Switch : Physical Properties and Photoisomerization Behavior

    NARCIS (Netherlands)

    Delden, R.A. van; Schoevaars, A.M.; Feringa, B.L.


    A novel donor acceptor substituted sterically overcrowded alkene was synthesized and characterized. Photoisomerization experiments it showed that this system could be converted with high efficiency towards a cis photostationary state and although the reverse isomerization towards the trans state was

  5. Grain boundary defect chemistry of acceptor-doped titanates: Space charge layer width

    Energy Technology Data Exchange (ETDEWEB)

    Vollman, M.; Waser, R. [Inst. fuer Werkstoffe der Elektrotechnik, Aachen (Germany)


    The grain boundary space charge depletion layers in acceptor-doped SrTiO{sub 3} ceramics were investigated by impedance spectroscopy in the time and frequency domain. Based on the layer and its dependence on the acceptor concentration, the temperature, and the oxygen partial pressure during annealing, a suggestion for a refined Schottky model is proposed. The local distribution of the donor type grain boundary states causing the depletion layer and the resulting band bending are discussed.

  6. Electron Donor-Acceptor Quenching and Photoinduced Electron Transfer for Coumarin Dyes. (United States)


    Mechanism of cousarin photodegradation . Ithe behavior of eoiuma dyes is water ad In aqueous detergent media,. and the effsects of medism aud, additives on...D-i36 345 ELECTRON DONOR-ACCEPTOR UENCHING AND PHOTOINDUCED i/i Ai ELECTRON TRANSFER FOR COUMARIN DYES (U) BOSTON UNIY MR DEPT OF CHEMISTRY G JONES...TYPE OF REPORT & PEIOD COVERED Electron Donor-acceptor Quenching and Photo- Technical, 1/1/82-10/31/82 induced Electron Transfer for Coumarin Dyes S

  7. Ternary Organic Solar Cells Based on Two Compatible Nonfullerene Acceptors with Power Conversion Efficiency >10. (United States)

    Liu, Tao; Guo, Yuan; Yi, Yuanping; Huo, Lijun; Xue, Xiaonan; Sun, Xiaobo; Fu, Huiting; Xiong, Wentao; Meng, Dong; Wang, Zhaohui; Liu, Feng; Russell, Thomas P; Sun, Yanming


    Two different nonfullerene acceptors and one copolymer are used to fabricate ternary organic solar cells (OSCs). The two acceptors show unique interactions that reduce crystallinity and form a homogeneous mixed phase in the blend film, leading to a high efficiency of ≈10.3%, the highest performance reported for nonfullerene ternary blends. This work provides a new approach to fabricate high-performance OSCs.

  8. Analogue alternative the electronic analogue computer in Britain and the USA, 1930-1975

    CERN Document Server

    Small, James S


    We are in the midst of a digital revolution - until recently, the majority of appliances used in everyday life have been developed with analogue technology. Now, either at home or out and about, we are surrounded by digital technology such as digital 'film', audio systems, computers and telephones. From the late 1940s until the 1970s, analogue technology was a genuine alternative to digital, and the two competing technologies ran parallel with each other. During this period, a community of engineers, scientists, academics and businessmen continued to develop and promote the analogue computer.

  9. Beyond Fullerenes: Designing Alternative Molecular Electron Acceptors for Solution-Processable Bulk Heterojunction Organic Photovoltaics. (United States)

    Sauvé, Geneviève; Fernando, Roshan


    Organic photovoltaics (OPVs) are promising candidates for providing a low cost, widespread energy source by converting sunlight into electricity. Solution-processable active layers have predominantly consisted of a conjugated polymer donor blended with a fullerene derivative as the acceptor. Although fullerene derivatives have been the acceptor of choice, they have drawbacks such as weak visible light absorption and poor energy tuning that limit overall efficiencies. This has recently fueled new research to explore alternative acceptors that would overcome those limitations. During this exploration, one question arises: what are the important design principles for developing nonfullerene acceptors? It is generally accepted that acceptors should have high electron affinity, electron mobility, and absorption coefficient in the visible and near-IR region of the spectra. In this Perspective, we argue that alternative molecular acceptors, when blended with a conjugated polymer donor, should also have large nonplanar structures to promote nanoscale phase separation, charge separation and charge transport in blend films. Additionally, new material design should address the low dielectric constant of organic semiconductors that have so far limited their widespread application.

  10. Molecular helices as electron acceptors in high-performance bulk heterojunction solar cells. (United States)

    Zhong, Yu; Trinh, M Tuan; Chen, Rongsheng; Purdum, Geoffrey E; Khlyabich, Petr P; Sezen, Melda; Oh, Seokjoon; Zhu, Haiming; Fowler, Brandon; Zhang, Boyuan; Wang, Wei; Nam, Chang-Yong; Sfeir, Matthew Y; Black, Charles T; Steigerwald, Michael L; Loo, Yueh-Lin; Ng, Fay; Zhu, X-Y; Nuckolls, Colin


    Despite numerous organic semiconducting materials synthesized for organic photovoltaics in the past decade, fullerenes are widely used as electron acceptors in highly efficient bulk-heterojunction solar cells. None of the non-fullerene bulk heterojunction solar cells have achieved efficiencies as high as fullerene-based solar cells. Design principles for fullerene-free acceptors remain unclear in the field. Here we report examples of helical molecular semiconductors as electron acceptors that are on par with fullerene derivatives in efficient solar cells. We achieved an 8.3% power conversion efficiency in a solar cell, which is a record high for non-fullerene bulk heterojunctions. Femtosecond transient absorption spectroscopy revealed both electron and hole transfer processes at the donor-acceptor interfaces. Atomic force microscopy reveals a mesh-like network of acceptors with pores that are tens of nanometres in diameter for efficient exciton separation and charge transport. This study describes a new motif for designing highly efficient acceptors for organic solar cells.

  11. An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions. (United States)

    Salonikidis, Petrus S; Niebert, Marcus; Ullrich, Tim; Bao, Guobin; Zeug, Andre; Richter, Diethelm W


    Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.

  12. Synthesis and antimicrobial activity of squalamine analogue. (United States)

    Kim, H S; Choi, B S; Kwon, K C; Lee, S O; Kwak, H J; Lee, C H


    Synthesis and antimicrobial activity of squalamine analogue 2 are reported. The synthesis of 2 was accomplished from bisnoralcohol 3. The spermidine moiety was introduced via reductive amination of an appropriately functionalized 3beta-aminosterol with spermidinyl aldehyde 17 utilizing sodium triacetoxyborohydride as the reducing agent. Compound 2 shows weaker antimicrobial activity than squalamine.

  13. Dumb holes: analogues for black holes. (United States)

    Unruh, W G


    The use of sonic analogues to black and white holes, called dumb or deaf holes, to understand the particle production by black holes is reviewed. The results suggest that the black hole particle production is a low-frequency and low-wavenumber process.

  14. Solanapyrone analogues from a Hawaiian fungicolous fungus (United States)

    Four new solanayrone analogues (solanapyrones J-M; 1-4) have been isolated from an unidentified fungicolous fungus collected in Hawaii. The structures and relative configurations of these compounds were determined by analysis of ID NMR, 2D NMR, and MS data. Solanapyrone J(1) showed antifungal acti...

  15. Prussian Blue Analogues of Reduced Dimensionality

    NARCIS (Netherlands)

    Gengler, Regis Y. N.; Toma, Luminita M.; Pardo, Emilio; Lloret, Francesc; Ke, Xiaoxing; Van Tendeloo, Gustaaf; Gournis, Dimitrios; Rudolf, Petra


    Mixed-valence polycyanides (Prussian Blue analogues) possess a rich palette of properties spanning from room-temperature ferromagnetism to zero thermal expansion, which can be tuned by chemical modifications or the application of external stimuli (temperature, pressure, light irradiation). While mol

  16. [Dmt(1)]DALDA analogues modified with tyrosine analogues at position 1. (United States)

    Cai, Yunxin; Lu, Dandan; Chen, Zhen; Ding, Yi; Chung, Nga N; Li, Tingyou; Schiller, Peter W


    Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity were prepared by replacing Dmt with various 2',6'-dialkylated Tyr analogues, including 2',4',6'-trimethyltyrosine (Tmt), 2'-ethyl-6'-methyltyrosine (Emt), 2'-isopropyl-6'-methyltyrosine (Imt) and 2',6'-diethyltyrosine (Det). All compounds were selective μ opioid agonists and the Tmt(1)-, Emt(1) and Det(1)-analogues showed subnanomolar μ opioid receptor binding affinities. The Tmt(1)- and Emt(1)-analogues showed improved antioxidant activity compared to the Dmt(1)-parent peptide in the DPPH radical-scavenging capacity assay, and thus are of interest as drug candidates for neuropathic pain treatment.

  17. Surface Characterizations of Fretting Fatigue Damage in Aluminum Alloy 7075-T6 Clamped Joints: The Beneficial Role of Ni–P Coatings

    Directory of Open Access Journals (Sweden)

    Reza H. Oskouei


    Full Text Available This paper aims to characterize the surface damage as a consequence of fretting fatigue in aluminum alloy 7075-T6 plates in double-lap bolted joints through XRD, surface profilometry, and SEM analyses. The main focus was on the surface roughness and chemical phase composition of the damaged zone along with the identification of fretting fatigue crack initiations over the surface of the material. The surface roughness of the fretted zone was found to increase when the joint was clamped with a higher tightening torque and tested under the same cyclic loading. Additionally, MgZn2 (η/ή precipitates and ZnO phase were found to form onto the surface of uncoated aluminum plate in the fretted and worn zones. The formation of the ZnO phase was understood to be a result of frictional heat induced between the surface of contacting uncoated Al 7075-T6 plates during cyclic loading and exposure to the air. The beneficial role of electroless nickel-phosphorous (Ni–P coatings in minimizing the fretting damage and thus improving the fretting fatigue life of the aluminum plates was also studied. The results showed that the surface roughness decreased by approximately 40% after applying Ni–P coatings to the Al 7075-T6 plates.

  18. Förster Resonance Energy Transfer (FRET as a Tool for Dissecting the Molecular Mechanisms for Maturation of the Shigella Type III Secretion Needle Tip Complex

    Directory of Open Access Journals (Sweden)

    William D. Picking


    Full Text Available Förster resonance energy transfer (FRET provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying Å-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system.

  19. Dynamique en fretting : influence du type d'asservissement et apport de la technique d'émission acoustique


    Benitez, Alberto


    La plupart des études de fretting analyse la réponse des matériaux en contact à partir de critères issus directement des paramètres caractéristiques des cycles de fretting (ouverture du cycle, énergie dissipée, raideur de contact...). La première partie de cette étude s’intéresse à l’influence du dispositif, premier élément du triplet tribologique, sur la réponse en glissement total d’un contact sphère/plan. Le rôle du mode de commande (déplacement imposé ou force actionneur imposée), de la r...

  20. A q-analogue of the four functions theorem


    Christofides, Demetres


    In this article we give a proof of a q-analogue of the celebrated four functions theorem. This analogue was conjectured by Bjorner and includes as special cases both the four functions theorem and also Bjorner's q-analogue of the FKG inequality.

  1. Tryptophan analogues. 1. Synthesis and antihypertensive activity of positional isomers. (United States)

    Safdy, M E; Kurchacova, E; Schut, R N; Vidrio, H; Hong, E


    A series of tryptophan analogues having the carboxyl function at the beta-position was synthesized and tested for antihypertensive activity. The 5-methoxy analogue 46 exhibited antihypertensive activity in the rat via the oral route and was much more potent than the normal tryptophan analogue. The methyl ester was found to be a critical structural feature for activity.

  2. Analogue VLSI for probabilistic networks and spike-time computation. (United States)

    Murray, A


    The history and some of the methods of analogue neural VLSI are described. The strengths of analogue techniques are described, along with residual problems to be solved. The nature of hardware-friendly and hardware-appropriate algorithms is reviewed and suggestions are offered as to where analogue neural VLSI's future lies.

  3. Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay. (United States)

    Karpenko, Iuliia A; Margathe, Jean-François; Rodriguez, Thiéric; Pflimlin, Elsa; Dupuis, Elodie; Hibert, Marcel; Durroux, Thierry; Bonnet, Dominique


    The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

  4. Fretting wear behavior of Cr-coated fuel rod for accident-tolerant fuel in flowing fluid

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Ho; Kim, Hyung Kyu; Kim, Hyun Gil; Koo, Yang Hyun [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    Fretting wear test of the Cr-coated fuel clading candidate have been performed in the flowing fluid condition in order to verify the reliability of Cr-coated layer on zirconium-based fuel cladding. Rod wear volume at each grid spring and dimple is dramaically increased with GTR gap even though each wear scar is not evenly distributed within a 1x1 grid cell.

  5. A FRET-based ratiometric fluorescent and colorimetric probe for the facile detection of organophosphonate nerve agent mimic DCP. (United States)

    Xuan, Weimin; Cao, Yanting; Zhou, Jiahong; Wang, Wei


    A FRET ratiometric fluorescent probe enabling a fast and highly sensitive response to OP nerve agent mimic DCP within 1 min and with as low as 0.17 ppm concentration detection limit has been developed. Moreover, the probe exhibits noticeable color changes under UV light and even with the naked eye. It is also demonstrated that it can detect both liquid and gas nerve agents.

  6. Measuring distances between TRPV1 and the plasma membrane using a noncanonical amino acid and transition metal ion FRET


    Zagotta, William N.; Gordon, Moshe T.; Senning, Eric N.; Munari, Mika A.; Gordon, Sharona E.


    Despite recent advances, the structure and dynamics of membrane proteins in cell membranes remain elusive. We implemented transition metal ion fluorescence resonance energy transfer (tmFRET) to measure distances between sites on the N-terminal ankyrin repeat domains (ARDs) of the pain-transducing ion channel TRPV1 and the intracellular surface of the plasma membrane. To preserve the native context, we used unroofed cells, and to specifically label sites in TRPV1, we incorporated a fluorescent...

  7. Metabolic response of Geobacter sulfurreducens towards electron donor/acceptor variation

    Directory of Open Access Journals (Sweden)

    Lovley Derek R


    Full Text Available Abstract Background Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen and acceptors (Fe(III, fumarate was investigated via 13C-based metabolic flux analysis. We examined the 13C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with 13C-acetate. Results Using 13C-based metabolic flux analysis, we observed that donor and acceptor variations gave rise to differences in gluconeogenetic initiation, tricarboxylic acid cycle activity, and amino acid biosynthesis pathways. Culturing G. sulfurreducens cells with Fe(III as the electron acceptor and acetate as the electron donor resulted in pyruvate as the primary carbon source for gluconeogenesis. When fumarate was provided as the electron acceptor and acetate as the electron donor, the flux analysis suggested that fumarate served as both an electron acceptor and, in conjunction with acetate, a carbon source. Growth on fumarate and acetate resulted in the initiation of gluconeogenesis by phosphoenolpyruvate carboxykinase and a slightly elevated flux through the oxidative tricarboxylic acid cycle as compared to growth with Fe(III as the electron acceptor. In addition, the direction of net flux between acetyl-CoA and pyruvate was reversed during growth on fumarate relative to Fe(III, while growth in the presence of Fe(III and acetate which provided hydrogen as an electron donor, resulted in decreased flux through the tricarboxylic acid cycle. Conclusions We gained detailed insight into the metabolism of G. sulfurreducens cells under various electron donor/acceptor conditions using 13C-based metabolic flux analysis. Our results can be used for the development of G. sulfurreducens as a chassis for a variety of applications including bioremediation and renewable biofuel production.

  8. Predicting the steady state thickness of passive films with the Point Defect Model in fretting corrosion experiments

    CERN Document Server

    Geringer, Jean; Taylor, Mathew L


    Some implants have approximately a lifetime of 15 years. The femoral stem, for example, should be made of 316L/316LN stainless steel. Fretting corrosion, friction under small displacements, should occur during human gait, due to repeated loadings and un-loadings, between stainless steel and bone for instance. Some experimental investigations of fretting corrosion have been practiced. As well known, metallic alloys and especially stainless steels are covered with a passive film that prevents from the corrosion and degradation. This passive layer of few nanometers, at ambient temperature, is the key of our civilization according to some authors. This work is dedicated to predict the passive layer thicknesses of stainless steel under fretting corrosion with a specific emphasis on the role of proteins. The model is based on the Point Defect Model (micro scale) and an update of the model on the friction process (micro-macro scale). Genetic algorithm was used for finding solution of the problem. The major results a...

  9. Evaluation and Description of Friction between an Electro-Deposited Coating and a Ceramic Ball under Fretting Condition

    Directory of Open Access Journals (Sweden)

    Kyungmok Kim


    Full Text Available This article describes fretting behavior of zirconia and silicon nitride balls on an electro-deposited coating. Fretting tests are performed using a ball-on-flat configuration. The evolution of the kinetic friction coefficient is determined, along with slip ratio. Experimental results show that the steady-state friction coefficient between ceramic balls (Si3N4 and ZrO2 and an electro-deposited coating is about 0.06, lower than the value between AISI 52100 ball and the coating. After a steady-state sliding, the transition of the friction coefficient is varied with a ball. The friction coefficient for ZrO2 balls became a critical value after higher fretting cycles than those for Si3N4 and AISI 52100 balls. In addition, it is identified that two parameters can describe the transition of the friction coefficient. Finally, the evolution of the friction coefficient is expressed as an exponential or a power-law form.

  10. Development of ERK Activity Sensor, an in vitro, FRET-based sensor of Extracellular Regulated Kinase activity

    Directory of Open Access Journals (Sweden)

    Alberola-Ila José


    Full Text Available Abstract Background Study of ERK activation has thus far relied on biochemical assays that are limited to the use of phospho-specific antibodies and radioactivity in vitro, and analysis of whole cell populations in vivo. As with many systems, fluorescence resonance energy transfer (FRET can be utilized to make highly sensitive detectors of molecular activity. Here we introduce FRET-based ERK Activity Sensors, which utilize variants of Enhanced Green Fluorescent Protein fused by an ERK-specific peptide linker to detect ERK2 activity. Results ERK Activity Sensors display varying changes in FRET upon phosphorylation by active ERK2 in vitro depending on the composition of ERK-specific peptide linker sequences derived from known in vivo ERK targets, Ets1 and Elk1. Analysis of point mutations reveals specific residues involved in ERK binding and phosphorylation of ERK Activity Sensor 3. ERK2 also shows high in vitro specificity for these sensors over two other major MAP Kinases, p38 and pSAPK/JNK. Conclusion EAS's are a convenient, non-radioactive alternative to study ERK dynamics in vitro. They can be utilized to study ERK activity in real-time. This new technology can be applied to studying ERK kinetics in vitro, analysis of ERK activity in whole cell extracts, and high-throughput screening technologies.

  11. The Martain drainage system and the origin of valley networks and fretted channels (United States)

    Carr, Michael H.


    Outflow channels provide strong evidence for abundant water near the Martian surface and an extensive groundwater system. Collapse of the surface into some channels suggests massive subsurface erosion and/or solution in addition to erosion by flow across the surface. Flat floors, steep walls, longitudinal striae and ridges, downstream deflection of striae from channel walls, and lack of river channels suggest that fretted channels form dominantly by mass wasting. A two-stage process is proposed. In the first stage, extension of valleys heads is favored by seepage of groundwater into debris shed from slopes. The debris moves downstream, aided by interstitial groundwater at the base of the debris, possibly with high pore pressures. In the second stage, because of climate change or a lower heat flow, groundwater can no longer seep into the debris flows in the valleys, their movement almost stops, and more viscous ice-lubricated debris aprons form. Almost all uplands at elevations greater than +1 km are dissected by valley networks, although the drainage densities are orders of magnitude less than is typical for the Earth. The valley networks resemble terrestrial river systems in planimetric shape, but U-shaped and rectangular-shaped cross sections, levee- like peripheral ridges, median ridges, patterns of branching and rejoining, and flat floors without river channels suggest that the networks may not be true analogs to terrestrial river valleys. It is proposed that they, like the fretted channels, formed mainly by mass wasting, aided by groundwater seepage into the mass-wasted debris. Movements of only millimeters to centimeters per year are needed to explain the channel lengths. Most valley formation ceased early at low latitudes because of progressive dehydration of the near surface, the result of sublimation of water and/or drainage of groundwater to regions of lower elevations. Valley formation persisted to later dates where aided by steep slopes, as on crater

  12. Non-Fullerene Electron Acceptors for Use in Organic Solar Cells

    KAUST Repository

    Nielsen, Christian B.


    The active layer in a solution processed organic photovoltaic device comprises a light absorbing electron donor semiconductor, typically a polymer, and an electron accepting fullerene acceptor. Although there has been huge effort targeted to optimize the absorbing, energetic, and transport properties of the donor material, fullerenes remain as the exclusive electron acceptor in all high performance devices. Very recently, some new non-fullerene acceptors have been demonstrated to outperform fullerenes in comparative devices. This Account describes this progress, discussing molecular design considerations and the structure–property relationships that are emerging. The motivation to replace fullerene acceptors stems from their synthetic inflexibility, leading to constraints in manipulating frontier energy levels, as well as poor absorption in the solar spectrum range, and an inherent tendency to undergo postfabrication crystallization, resulting in device instability. New acceptors have to address these limitations, providing tunable absorption with high extinction coefficients, thus contributing to device photocurrent. The ability to vary and optimize the lowest unoccupied molecular orbital (LUMO) energy level for a specific donor polymer is also an important requirement, ensuring minimal energy loss on electron transfer and as high an internal voltage as possible. Initially perylene diimide acceptors were evaluated as promising acceptor materials. These electron deficient aromatic molecules can exhibit good electron transport, facilitated by close packed herringbone crystal motifs, and their energy levels can be synthetically tuned. The principal drawback of this class of materials, their tendency to crystallize on too large a length scale for an optimal heterojunction nanostructure, has been shown to be overcome through introduction of conformation twisting through steric effects. This has been primarily achieved by coupling two units together, forming dimers

  13. A rhodanine flanked nonfullerene acceptor for solution-processed organic photovoltaics

    KAUST Repository

    Holliday, Sarah


    A novel small molecule, FBR, bearing 3-ethylrhodanine flanking groups was synthesized as a nonfullerene electron acceptor for solution-processed bulk heterojunction organic photovoltaics (OPV). A straightforward synthesis route was employed, offering the potential for large scale preparation of this material. Inverted OPV devices employing poly(3-hexylthiophene) (P3HT) as the donor polymer and FBR as the acceptor gave power conversion efficiencies (PCE) up to 4.1%. Transient and steady state optical spectroscopies indicated efficient, ultrafast charge generation and efficient photocurrent generation from both donor and acceptor. Ultrafast transient absorption spectroscopy was used to investigate polaron generation efficiency as well as recombination dynamics. It was determined that the P3HT:FBR blend is highly intermixed, leading to increased charge generation relative to comparative devices with P3HT:PC60BM, but also faster recombination due to a nonideal morphology in which, in contrast to P3HT:PC60BM devices, the acceptor does not aggregate enough to create appropriate percolation pathways that prevent fast nongeminate recombination. Despite this nonoptimal morphology the P3HT:FBR devices exhibit better performance than P3HT:PC60BM devices, used as control, demonstrating that this acceptor shows great promise for further optimization.

  14. Physiological response of Desulfurispirillum indicum S5 to arsenate and nitrate as terminal electron acceptors. (United States)

    Rauschenbach, Ines; Bini, Elisabetta; Häggblom, Max M; Yee, Nathan


    The ability of anaerobic prokaryotes to employ different terminal electron acceptors for respiration enables these organisms to flourish in subsurface ecosystems. Desulfurispirillum indicum strain S5 is an obligate anaerobic bacterium that is able to grow by respiring a range of different electron acceptors, including arsenate and nitrate. Here, we examined the growth, electron acceptor utilization, and gene expression of D. indicum growing under arsenate and nitrate-reducing conditions. Consistent with thermodynamic predictions, the experimental results showed that the reduction of nitrate to ammonium yielded higher cell densities than the reduction of arsenate to arsenite. However, D. indicum grew considerably faster by respiration on arsenate compared with nitrate, with doubling times of 4.3 ± 0.2 h and 19.2 ± 2.0 h, respectively. Desulfurispirillum indicum growing on both electron acceptors exhibited the preferential utilization of arsenate before nitrate. The expression of the arsenate reductase gene arrA was up-regulated approximately 100-fold during arsenate reduction, as determined by qRT-PCR. Conversely, the nitrate reductase genes narG and napA were not differentially regulated under the conditions tested. The results of this study suggest that physiology, rather than thermodynamics, controls the growth rates and hierarchy of electron acceptor utilization in D. indicum.

  15. Optimization of propafenone analogues as antimalarial leads. (United States)

    Lowes, David J; Guiguemde, W Armand; Connelly, Michele C; Zhu, Fangyi; Sigal, Martina S; Clark, Julie A; Lemoff, Andrew S; Derisi, Joseph L; Wilson, Emily B; Guy, R Kiplin


    Propafenone, a class Ic antiarrythmic drug, inhibits growth of cultured Plasmodium falciparum. While the drug's potency is significant, further development of propafenone as an antimalarial would require divorcing the antimalarial and cardiac activities as well as improving the pharmacokinetic profile of the drug. A small array of propafenone analogues was designed and synthesized to address the cardiac ion channel and PK liabilities. Testing of this array revealed potent inhibitors of the 3D7 (drug sensitive) and K1 (drug resistant) strains of P. falciparum that possessed significantly reduced ion channel effects and improved metabolic stability. Propafenone analogues are unusual among antimalarial leads in that they are more potent against the multidrug resistant K1 strain of P. falciparum compared to the 3D7 strain.

  16. Spectroscopic study of solar twins and analogues

    CERN Document Server

    Datson, Juliet; Portinari, Laura


    Context. Many large stellar surveys have been and are still being carried out, providing huge amounts of data, for which stellar physical parameters will be derived. Solar twins and analogues provide a means to test the calibration of these stellar catalogues because the Sun is the best-studied star and provides precise fundamental parameters. Solar twins should be centred on the solar values. Aims. This spectroscopic study of solar analogues selected from the Geneva-Copenhagen Survey (GCS) at a resolution of 48,000 provides effective temperatures and metallicities for these stars. We test whether our spectroscopic parameters, as well as the previous photometric calibrations, are properly centred on the Sun. In addition, we search for more solar twins in our sample. Methods. The methods used in this work are based on literature methods for solar twin searches and on methods we developed in previous work to distinguish the metallicity-temperature degeneracies in the differential comparison of spectra of solar ...

  17. Holographic Fluids with Vorticity and Analogue Gravity

    CERN Document Server

    Leigh, Robert G; Petropoulos, P Marios


    We study holographic three-dimensional fluids with vorticity in local equilibrium and discuss their relevance to analogue gravity systems. The Fefferman-Graham expansion leads to the fluid's description in terms of a comoving and rotating Papapetrou-Randers frame. A suitable Lorentz transformation brings the fluid to the non-inertial Zermelo frame, which clarifies its interpretation as moving media for light/sound propagation. We apply our general results to the Lorentzian Kerr-AdS_4 and Taub-NUT-AdS_4 geometries that describe fluids in cyclonic and vortex flows respectively. In the latter case we associate the appearance of closed timelike curves to analogue optical horizons. In addition, we derive the classical rotational Hall viscosity of three-dimensional fluids with vorticity. Our formula remarkably resembles the corresponding result in magnetized plasmas.

  18. Confirmation via Analogue Simulation: A Bayesian Analysis

    CERN Document Server

    Dardashti, Radin; Thebault, Karim P Y; Winsberg, Eric


    Analogue simulation is a novel mode of scientific inference found increasingly within modern physics, and yet all but neglected in the philosophical literature. Experiments conducted upon a table-top 'source system' are taken to provide insight into features of an inaccessible 'target system', based upon a syntactic isomorphism between the relevant modelling frameworks. An important example is the use of acoustic 'dumb hole' systems to simulate gravitational black holes. In a recent paper it was argued that there exists circumstances in which confirmation via analogue simulation can obtain; in particular when the robustness of the isomorphism is established via universality arguments. The current paper supports these claims via an analysis in terms of Bayesian confirmation theory.

  19. Polyamine analogues targeting epigenetic gene regulation. (United States)

    Huang, Yi; Marton, Laurence J; Woster, Patrick M; Casero, Robert A


    Over the past three decades the metabolism and functions of the polyamines have been actively pursued as targets for antineoplastic therapy. Interactions between cationic polyamines and negatively charged nucleic acids play a pivotal role in DNA stabilization and RNA processing that may affect gene expression, translation and protein activity. Our growing understanding of the unique roles that the polyamines play in chromatin regulation, and the discovery of novel proteins homologous with specific regulatory enzymes in polyamine metabolism, have led to our interest in exploring chromatin remodelling enzymes as potential therapeutic targets for specific polyamine analogues. One of our initial efforts focused on utilizing the strong affinity that the polyamines have for chromatin to create a backbone structure, which could be combined with active-site-directed inhibitor moieties of HDACs (histone deacetylases). Specific PAHAs (polyaminohydroxamic acids) and PABAs (polyaminobenzamides) polyamine analogues have demonstrated potent inhibition of the HDACs, re-expression of p21 and significant inhibition of tumour growth. A second means of targeting the chromatin-remodelling enzymes with polyamine analogues was facilitated by the recent identification of flavin-dependent LSD1 (lysine-specific demethylase 1). The existence of this enzyme demonstrated that histone lysine methylation is a dynamic process similar to other histone post-translational modifications. LSD1 specifically catalyses demethylation of mono- and di-methyl Lys4 of histone 3, key positive chromatin marks associated with transcriptional activation. Structural and catalytic similarities between LSD1 and polyamine oxidases facilitated the identification of biguanide, bisguanidine and oligoamine polyamine analogues that are potent inhibitors of LSD1. Cellular inhibition of LSD1 by these unique compounds led to the re-activation of multiple epigenetically silenced genes important in tumorigenesis. The use of

  20. Benchmarking analogue models of brittle thrust wedges (United States)

    Schreurs, Guido; Buiter, Susanne J. H.; Boutelier, Jennifer; Burberry, Caroline; Callot, Jean-Paul; Cavozzi, Cristian; Cerca, Mariano; Chen, Jian-Hong; Cristallini, Ernesto; Cruden, Alexander R.; Cruz, Leonardo; Daniel, Jean-Marc; Da Poian, Gabriela; Garcia, Victor H.; Gomes, Caroline J. S.; Grall, Céline; Guillot, Yannick; Guzmán, Cecilia; Hidayah, Triyani Nur; Hilley, George; Klinkmüller, Matthias; Koyi, Hemin A.; Lu, Chia-Yu; Maillot, Bertrand; Meriaux, Catherine; Nilfouroushan, Faramarz; Pan, Chang-Chih; Pillot, Daniel; Portillo, Rodrigo; Rosenau, Matthias; Schellart, Wouter P.; Schlische, Roy W.; Take, Andy; Vendeville, Bruno; Vergnaud, Marine; Vettori, Matteo; Wang, Shih-Hsien; Withjack, Martha O.; Yagupsky, Daniel; Yamada, Yasuhiro


    We performed a quantitative comparison of brittle thrust wedge experiments to evaluate the variability among analogue models and to appraise the reproducibility and limits of model interpretation. Fifteen analogue modeling laboratories participated in this benchmark initiative. Each laboratory received a shipment of the same type of quartz and corundum sand and all laboratories adhered to a stringent model building protocol and used the same type of foil to cover base and sidewalls of the sandbox. Sieve structure, sifting height, filling rate, and details on off-scraping of excess sand followed prescribed procedures. Our analogue benchmark shows that even for simple plane-strain experiments with prescribed stringent model construction techniques, quantitative model results show variability, most notably for surface slope, thrust spacing and number of forward and backthrusts. One of the sources of the variability in model results is related to slight variations in how sand is deposited in the sandbox. Small changes in sifting height, sifting rate, and scraping will result in slightly heterogeneous material bulk densities, which will affect the mechanical properties of the sand, and will result in lateral and vertical differences in peak and boundary friction angles, as well as cohesion values once the model is constructed. Initial variations in basal friction are inferred to play the most important role in causing model variability. Our comparison shows that the human factor plays a decisive role, and even when one modeler repeats the same experiment, quantitative model results still show variability. Our observations highlight the limits of up-scaling quantitative analogue model results to nature or for making comparisons with numerical models. The frictional behavior of sand is highly sensitive to small variations in material state or experimental set-up, and hence, it will remain difficult to scale quantitative results such as number of thrusts, thrust spacing

  1. Electromagnetic wave analogue of electronic diode


    Shadrivov, Ilya V.; Powell, David A.; Kivshar, Yuri S.; Fedotov, Vassili A.; Zheludev, Nikolay I.


    An electronic diode is a nonlinear semiconductor circuit component that allows conduction of electrical current in one direction only. A component with similar functionality for electromagnetic waves, an electromagnetic isolator, is based on the Faraday effect of the polarization state rotation and is also a key component of optical and microwave systems. Here we demonstrate a chiral electromagnetic diode, which is a direct analogue of an electronic diode: its functionality is underpinned by ...

  2. Synthesis of constrained analogues of tryptophan

    Directory of Open Access Journals (Sweden)

    Elisabetta Rossi


    Full Text Available A Lewis acid-catalysed diastereoselective [4 + 2] cycloaddition of vinylindoles and methyl 2-acetamidoacrylate, leading to methyl 3-acetamido-1,2,3,4-tetrahydrocarbazole-3-carboxylate derivatives, is described. Treatment of the obtained cycloadducts under hydrolytic conditions results in the preparation of a small library of compounds bearing the free amino acid function at C-3 and pertaining to the class of constrained tryptophan analogues.

  3. The Brookhaven electron analogue, 1953--1957

    Energy Technology Data Exchange (ETDEWEB)

    Plotkin, M.


    The following topics are discussed on the Brookhaven electron analogue: L.J. Haworth and E.L. VanHorn letters; Original G.K. Green outline for report; General description; Parameter list; Mechanical Assembly; Alignment; Degaussing; Vacuum System; Injection System; The pulsed inflector; RF System; Ferrite Cavity; Pick-up electrodes and preamplifiers; Radio Frequency power amplifier; Lens supply; Controls and Power; and RF acceleration summary.

  4. Synthesis of a Cyclic Analogue of Galardin

    Institute of Scientific and Technical Information of China (English)

    马大为; 吴问根; 钱静; 郎深慧; 叶其壮


    A cyclic analogue 4 of galardin,a known MMP inhibitor,is designed to improve its selectivity.The synthesis of 4starts from dimethyl(S)-malate using diaselective alkylation and subsequent cyclization and amide formation as key steps.The compound 4 showed MMP inhibitory activity on all MMPs tested with IC50 ranging from 20.1 μM to 104 μM.

  5. Identification of Acceptor States in Li-N Dual-Doped p-Type ZnO Thin Films

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yin-Zhu; LU Jian-Guo; YE Zhi-Zhen; HE Hai-Ping; CHEN Lan-Lan; ZHAO Bing-Hui


    @@ Li-N dual-doped p-type ZnO (ZnO:(Li,N)) thin films are prepared by pulsed laser deposition. The optical properties are studied using temperature-dependent photoluminescence. The Lizn-No complex aceeptor with an energy level of 138 meV is identified from the free-to-neutral-acceptor (e, Ao) emission. The Haynes factor is about 0.087 for the LiZn-NO complex acceptor, with the acceptor bound-exciton binding energy of 12meV. Another deeper acceptor state located at 248meV, also identified from the (e, Ao) emission, is attributed to zinc vacancy acceptor. The two acceptor states might both contribute to the observed p-type conductivity in ZnO:(Li,N).

  6. Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. (United States)

    Jones, Alexander M; Danielson, Jonas Ah; Manojkumar, Shruti N; Lanquar, Viviane; Grossmann, Guido; Frommer, Wolf B


    Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2-800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI:

  7. Feedback-mediated cancer therapy: a FRET-based nanoreporter approach (United States)

    Sarkar, Suproteem K.; Khater, Yashika; Kulkarni, Ashish; Sengupta, Shiladitya


    A theranostic nanoparticle system was developed by integrating a chemotherapeutic agent with an "activatable" fluorescent tracer. The system signals tumor death by monitoring the activity of caspase-3, a product of apoptosis, and can therefore screen the treatment sensitivity of a particular tumor. The polymer nanoparticles (Poly [isobutylene-alt-maleic anhydride]) were formed through reprecipitation and contained paclitaxel, a chemotherapy drug, and fluorescein isothiocyanate, a fluorescent dye. The dye's fluorescence was quenched through Förster resonance energy transfer (FRET) by a quencher that was connected to the dye by a peptide chain. With sizes ranging from 200-250 nm, the nanoparticles were stable for two weeks. The nanoparticles were tested in vitro with responsive Lewis Lung Carcinoma (LLC) cells and taxane-resistant cells. Upon cell death by paclitaxel exposure, caspase-3 cleaved the peptide chain connecting the dye and the quencher, causing the system to fluoresce. When LLC cells were treated with the system, the nanoreporters fluoresced, but when resistant cells were tested, and when the drug was removed from the system, the nanoreporters did not fluoresce. Since the system screens if a drug can successfully kill a particular tumor, it offers a novel and promising approach to personalized medicine.

  8. Graphene quantum dots FRET based sensor for early detection of heart attack in human. (United States)

    Bhatnagar, Deepika; Kumar, Vanish; Kumar, Ashok; Kaur, Inderpreet


    Cardiac immunosensor for early detection of heart attack (myocardial infarction) was developed using amine functionalized graphene quantum dots (afGQDs) conjugated with antibody anti-cardiac Troponin I (anti-cTnI) to detect cardiac marker antigen Troponin I (cTnI) in blood based on fluorescence resonance energy transfer (FRET) between conjugate and graphene (quencher) only in 10 min. The anti-cTnI was covalently conjugated to afGQDs through carbodiimide coupling reaction. The conjugate was characterized by zeta potential UV-vis spectroscopy and field emission scanning electron microscopy (FESEM). The sensing performance of the sensor was studied with respect to changes in the photon count and photoluminescence of GQDs based on interaction of target cTnI with its specific anti-cTnI antibody. The sensor is highly specific and shows negligible response to non-specific antigens. The sensor displayed a linear response to cTnI from 0.001 to 1000 ng mL(-1) with a limit of detection of 0.192 pg mL(-1).

  9. Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor. (United States)

    Nakanishi, Yoichi; Iida, Syuntaro; Ueoka-Nakanishi, Hanayo; Niimi, Tomoaki; Tomioka, Rie; Maeshima, Masayoshi


    Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

  10. Large-eddy simulations of turbulent flow for grid-to-rod fretting in nuclear reactors

    CERN Document Server

    Bakosi, J; Lowrie, R B; Pritchett-Sheats, L A; Nourgaliev, R R


    The grid-to-rod fretting (GTRF) problem in pressurized water reactors is a flow-induced vibration problem that results in wear and failure of the fuel rods in nuclear assemblies. In order to understand the fluid dynamics of GTRF and to build an archival database of turbulence statistics for various configurations, implicit large-eddy simulations of time-dependent single-phase turbulent flow have been performed in 3x3 and 5x5 rod bundles with a single grid spacer. To assess the computational mesh and resolution requirements, a method for quantitative assessment of unstructured meshes with no-slip walls is described. The calculations have been carried out using Hydra-TH, a thermal-hydraulics code developed at Los Alamos for the Consortium for Advanced Simulation of Light water reactors, a United States Department of Energy Innovation Hub. Hydra-TH uses a second-order implicit incremental projection method to solve the single-phase incompressible Navier-Stokes equations. The simulations explicitly resolve the la...

  11. Multirate Simulations of String Vibrations Including Nonlinear Fret-String Interactions Using the Functional Transformation Method

    Directory of Open Access Journals (Sweden)

    R. Rabenstein


    Full Text Available The functional transformation method (FTM is a well-established mathematical method for accurate simulations of multidimensional physical systems from various fields of science, including optics, heat and mass transfer, electrical engineering, and acoustics. This paper applies the FTM to real-time simulations of transversal vibrating strings. First, a physical model of a transversal vibrating lossy and dispersive string is derived. Afterwards, this model is solved with the FTM for two cases: the ideally linearly vibrating string and the string interacting nonlinearly with the frets. It is shown that accurate and stable simulations can be achieved with the discretization of the continuous solution at audio rate. Both simulations can also be performed with a multirate approach with only minor degradations of the simulation accuracy but with preservation of stability. This saves almost 80% of the computational cost for the simulation of a six-string guitar and therefore it is in the range of the computational cost for digital waveguide simulations.

  12. Fretting corrosion behaviour of ball-and-socket joint on dental implants with different prosthodontic alloys. (United States)

    Gil, F J; Canedo, R; Padrós, A; Bañeres, M V; Arano, J M


    The fretting corrosion of five materials for implant suprastructures (cast-titanium, machined-titanium, gold alloy, silver-palladium alloy and chromium-nickel alloy), was investigated in vitro, the materials being galvanically coupled to a titanium ball-and-socket-joint with tetrafluoroethylene under mechanical load. Various electrochemical parameters (E(corr), i(corr), Evans diagrams, polarization resistance and Tafel slopes) were analyzed. The microstructure of the different dental materials was observed before and after corrosion processes by optical and electron microscopy. It can be observed that the mechanical load produces an important decrease of the corrosion resistance. The cast and machined titanium had the most passive current density at a given potential and chromium-nickel alloy had the most active critical current density values. The high gold content alloys have excellent resistance corrosion, although this decreases when the gold content is lower in the alloy. The palladium alloy had a low critical current density due to the presence of gallium in this composition but a selective dissolution of copper-rich phases was observed through energy dispersive X-ray analysis.

  13. Dissecting the effect of anions on Hg2+ detection using a FRET based DNA probe. (United States)

    Kiy, Mehmet Murat; Zaki, Ahmed; Menhaj, Arsalsan Beg; Samadi, Azadeh; Liu, Juewen


    Many biosensors have been developed to detect Hg(2+) using thymine-rich DNA. While sensor response to various cations is often studied to demonstrate selectivity, the effect of anions has been largely overlooked. Anions may compete with DNA for metal binding and thus produce a false negative result. Anions cannot be added alone; the cation part of a salt may cause DNA compaction and other effects, obscuring the role of anions. We find that the sensitivity of a FRET-based Hg(2+) probe is independent of Na(+) concentration. Therefore, by using various sodium salts, any change in sensitivity can be attributed solely to the effect of anions. Halide salts, sulfides, and amines are strong inhibitors; anions containing oxo or hydroxyl groups (e.g. nitrate, sulfate, phosphate, carbonate, acetate, and citrate) do not interfere with Hg(2+) detection even at 100 mM concentration. Mercury hydrolysis and its diffusion into polypropylene containers can also strongly affect the detection results. We conclude that thymine-rich DNA should be useful for Hg(2+) detection in many environmental water samples.

  14. A FRET-based fluorescent probe for mercury ions in water and living cells (United States)

    Zhang, Bo; Ma, Pinyi; Gao, Dejiang; Wang, Xinghua; Sun, Ying; Song, Daqian; Li, Xuwen


    On the basis of fluorescence resonance energy transfer (FRET), a new rhodamine derivative (DRh) was synthesized as a ratiometric fluorescent probe for detecting Hg2 + in water and living cells samples. The recognition properties of the probe DRh with metal ions had been investigated in H2O/CH3CN (9:1, v/v; Tris-HCl 50 mmol L- 1; pH = 7.0) solution by the UV-Vis spectrophotometry and the fluorescence spectrophotometry. The results showed that the probe DRh exhibited the selective recognition of Hg2 +. Upon the addition of Hg2 +, the spirolactam ring of probe DRh was opened. The 1:1 stoichiometric structure between DRh and Hg2 + were supported by Job's plot, MS and DFT theoretical calculations. The linearly fluorescence intensity ratio (I582/I538) is proportional to the concentration of Hg2 + in the range 0-30 μmol L- 1. The limit of detection (LOD) of Hg2 + is 0.008 μmol L- 1 (base on S/N = 3). The present probe was applied to the determination of Hg2 + in neutral water samples and gave recoveries ranging from 104.5 to 107.9%. Furthermore, the fluorescent probe also can be applied as a bioimaging reagent for Hg2 + detection in HeLa cells.

  15. Detailed analysis of complex single molecule FRET data with the software MASH (United States)

    Hadzic, Mélodie C. A. S.; Kowerko, Danny; Börner, Richard; Zelger-Paulus, Susann; Sigel, Roland K. O.


    The processing and analysis of surface-immobilized single molecule FRET (Förster resonance energy transfer) data follows systematic steps (e.g. single molecule localization, clearance of different sources of noise, selection of the conformational and kinetic model, etc.) that require a solid knowledge in optics, photophysics, signal processing and statistics. The present proceeding aims at standardizing and facilitating procedures for single molecule detection by guiding the reader through an optimization protocol for a particular experimental data set. Relevant features were determined from single molecule movies (SMM) imaging Cy3- and Cy5-labeled Sc.ai5γ group II intron molecules synthetically recreated, to test the performances of four different detection algorithms. Up to 120 different parameterizations per method were routinely evaluated to finally establish an optimum detection procedure. The present protocol is adaptable to any movie displaying surface-immobilized molecules, and can be easily reproduced with our home-written software MASH (multifunctional analysis software for heterogeneous data) and script routines (both available in the download section of

  16. Direct interactions of early and late assembling division proteins in Escherichia coli cells resolved by FRET. (United States)

    Alexeeva, Svetlana; Gadella, Theodorus W J; Verheul, Jolanda; Verhoeven, Gertjan S; den Blaauwen, Tanneke


    The bacterial cell division machinery is organized in the so-called divisome composed of highly dynamic but low abundant interacting (membrane-bound) proteins. In order to elucidate the molecular interactions between these proteins, we developed a robust background-insensitive quantitative spectral unmixing method for estimating FRET efficiencies at near endogenous protein levels using fluorescent protein fusions. The assembly of the division machinery of Escherichia coli occurs in two steps that are discrete in time: first the FtsZ-ring and the so-called early localizing proteins that together seem to prepare the division assembly at midcell. Subsequently, the late localizing protein complexes that contain the peptidoglycan-synthesizing proteins PBP1B and FtsI (PBP3) are recruited to the division site, which initiates septation. Physical interactions were observed between members within each group but also between the early and late localizing proteins strongly suggesting that these proteins despite their differential localization in time are linked at the molecular and functional level. Interestingly, we find FtsN, one of the latest proteins in the divisome assembly, interacting with late assembling proteins FtsI and FtsW, but also with early (proto-ring) protein ZapA. This is in line with the recently described role of FtsN in divisome stabilization including the proto-ring elements.

  17. Proficiency of acceptor-donor-acceptor organic dye with spiro-MeOTAD HTM on the photovoltaic performance of dye sensitized solar cell (United States)

    Ramavenkateswari, K.; Venkatachalam, P.


    This work investigates the proficiency of acceptor-donor-acceptor (A-D-A) organic dye Diisopropyl azodicarboxylate (DIAC) as photosensitizer on the photovoltaic parameters of silver (Ag) doped TiO2 photoanode dye-sensitized solar cells (DSSCs) with quasi-solid state electrolyte/hole transport material (HTM) spiro-MeOTAD. TNSs (TiO2 nanosticks) photoanodes are prepared through sol-gel method and hydrothermal technique. X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM) and BET measurement were used to characterize the structure and morphology of TiO2 nanostructures. The Diisopropyl azodicarboxylate organic dye with TNPs-Ag@TNSs composite photoanode structure and spiro-MeOTAD HTM exhibited better power conversion efficiency (PCE).

  18. Thymidine analogues for tracking DNA synthesis. (United States)

    Cavanagh, Brenton L; Walker, Tom; Norazit, Anwar; Meedeniya, Adrian C B


    Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

  19. Thymidine Analogues for Tracking DNA Synthesis

    Directory of Open Access Journals (Sweden)

    Brenton L. Cavanagh


    Full Text Available Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in “tagging DNA synthesis” is the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU. The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using ‘Huisgen’s reaction’ (1,3-dipolar cycloaddition or ‘click chemistry’. This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other “unnatural bases”. These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

  20. Rational design of two-dimensional molecular donor-acceptor nanostructure arrays (United States)

    Zhang, Jia Lin; Zhong, Shu; Zhong, Jian Qiang; Niu, Tian Chao; Hu, Wen Ping; Wee, Andrew Thye Shen; Chen, Wei


    The construction of long-range ordered organic donor-acceptor nanostructure arrays over microscopic areas supported on solid substrates is one of the most challenging tasks towards the realization of molecular nanodevices. They can also be used as ideal model systems to understand light induced charge transfer, charge separation and energy conversion processes and mechanisms at the nanometer scale. The aim of this paper is to highlight recent advances and progress in this topic. Special attention is given to two different strategies for the construction of organic donor-acceptor nanostructure arrays, namely (i) molecular self-assembly on artificially patterned or pre-defined molecular surface nanotemplates and (ii) molecular nanostructure formation steered via directional and selective intermolecular interactions. The interfacial charge transfer and the energy level alignment of these donor-acceptor nanostructures are also discussed.

  1. Effect of cathode electron acceptors on simultaneous anaerobic sulfide and nitrate removal in microbial fuel cell. (United States)

    Cai, Jing; Zheng, Ping; Mahmood, Qaisar


    The current investigation reports the effect of cathode electron acceptors on simultaneous sulfide and nitrate removal in two-chamber microbial fuel cells (MFCs). Potassium permanganate and potassium ferricyanide were common cathode electron acceptors and evaluated for substrate removal and electricity generation. The abiotic MFCs produced electricity through spontaneous electrochemical oxidation of sulfide. In comparison with abiotic MFC, the biotic MFC showed better ability for simultaneous nitrate and sulfide removal along with electricity generation. Keeping external resistance of 1,000 Ω, both MFCs showed good capacities for substrate removal where nitrogen and sulfate were the main end products. The steady voltage with potassium permanganate electrodes was nearly twice that of with potassium ferricyanide. Cyclic voltammetry curves confirmed that the potassium permanganate had higher catalytic activity than potassium ferricyanide. The potassium permanganate may be a suitable choice as cathode electron acceptor for enhanced electricity generation during simultaneous treatment of sulfide and nitrate in MFCs.

  2. External field effects on aging phenomenon of acceptor-doped BaTiO3 ceramics

    Directory of Open Access Journals (Sweden)

    Y. Y. Guo


    Full Text Available Our experiments on ferroelectric aging phenomena of a series of acceptor-doped BaTiO3 ceramics demonstrate that after well-aging, all samples show a similar double hysteresis loop under smaller applied electric field, regardless of ionic radius or ionic valence of the acceptor. However, with increasing the applied electric field, the completely constricted loops gradually start to open, indicating the aging effect becomes weak under larger electric field. The unified microscopic mechanism responsible for the similar aging behavior in different acceptor-doped BaTiO3 ceramics may be that the larger field is considered to kinetically facilitate a part of oxygen vacancies short-range hopping. As a result, the defect dipole field provided by oxygen vacancies and the associated defect dipoles frozen in the original states decreases, thus contributing to the weaker aging effect.

  3. Electroluminescence from charge transfer states in Donor/Acceptor solar cells

    DEFF Research Database (Denmark)

    Sherafatipour, Golenaz; Madsen, Morten

    charge transfer (CT) excitons, which is Coulombically bound interfacial electron- hole pairs residing at the donor/acceptor heterojunctions. The CT state represents an intermediate state between the exciton dissociation and recombination back to the ground state. Since the recombination of photo...... at the donor/acceptor interface is detected. As a less studied system, we examine here the interfacial charge transfer state recombination in DBP:C70 thin-films. The weak EL from the small molecule solar cell biased in the forward direction gives valuable information about the CT state recombination, from...... which the maximum open-circuit voltage can be estimated, and further can be used in the modeling and optimization of the OPV devices. [1] C. Deibe, T. Strobe, and V. Dyakonov, “Role of the charge transfer state in organic donor-acceptor solar cells,” Adv. Mater., vol. 22, pp. 4097–4111, 2010. [2] K...

  4. Neutralization and bonding mechanisms of shallow acceptors at grain boundaries in polycrystalline silicon

    Energy Technology Data Exchange (ETDEWEB)

    Kazmerski, L.L.; Nelson, A.J.; Dhere, R.G.; Yahia, A.; Abou-Elfotouh, F.


    Interactions between shallow acceptors (B, Al, Ga, and In) and hydrogen in polycrystalline Si are investigated. The bonding mechanisms involved in the acceptor neutralization process at grain boundaries are examined using microanalytical techniques. Differences in the incorporation of molecular and atomic hydrogen, and corresponding variations in electrical passivation at grain boundaries, are observed. Low-temperature Auger difference spectroscopy confirms Si--H bonding to dominate B, Ga, and In-doped cases, with no direct acceptor--hydrogen bonding. Al-rich grain boundaries show H-complex and hydroxyl bonding. The data confirm chemical bond strength trends with B

  5. Photoinduced Electron Transfer within Supramolecular Donor-Acceptor Peptide Nanostructures under Aqueous Conditions. (United States)

    Sanders, Allix M; Magnanelli, Timothy J; Bragg, Arthur E; Tovar, John D


    We report the synthesis, self-assembly, and electron transfer capabilities of peptide-based electron donor-acceptor molecules and supramolecular nanostructures. These modified peptides contain π-conjugated oligothiophene electron donor cores that are peripherally substituted with naphthalene diimide electron acceptors installed via imidation of site-specific lysine residues. These molecules self-assemble into one-dimensional nanostructures in aqueous media, as shown through steady-state absorption, photoluminescence, and circular dichroism spectra, as well as transmission electron microscopy. Excitation of the oligothiophene donor moieties results in electron transfer to the acceptor units, ultimately creating polar, charge-separated states that persist for over a nanosecond as observed with transient absorption spectroscopy. This study demonstrates how transient electric fields can be engineered into aqueous nanomaterials of biomedical relevance through external, temporally controlled photonic inputs.

  6. Molecular nitrogen acceptors in ZnO nanowires induced by nitrogen plasma annealing (United States)

    Ton-That, C.; Zhu, L.; Lockrey, M. N.; Phillips, M. R.; Cowie, B. C. C.; Tadich, A.; Thomsen, L.; Khachadorian, S.; Schlichting, S.; Jankowski, N.; Hoffmann, A.


    X-ray absorption near-edge spectroscopy, photoluminescence, cathodoluminescence, and Raman spectroscopy have been used to investigate the chemical states of nitrogen dopants in ZnO nanowires. It is found that nitrogen exists in multiple states: NO,NZn, and loosely bound N2 molecule. The results establish a direct link between a donor-acceptor pair emission at 3.232 eV and the concentration of loosely bound N2. This work confirms that N2 at Zn site is a potential candidate for producing a shallow acceptor state in N-doped ZnO as theoretically predicted by Lambrecht and Boonchun [Phys. Rev. B 87, 195207 (2013), 10.1103/PhysRevB.87.195207]. Additionally, shallow acceptor states arising from NO complexes have been ruled out in this paper.

  7. Rapid Energy Transfer Enabling Control of Emission Polarization in Perylene Bisimide Donor-Acceptor Triads. (United States)

    Menelaou, Christopher; ter Schiphorst, Jeroen; Kendhale, Amol M; Parkinson, Patrick; Debije, Michael G; Schenning, Albertus P H J; Herz, Laura M


    Materials showing rapid intramolecular energy transfer and polarization switching are of interest for both their fundamental photophysics and potential for use in real-world applications. Here, we report two donor-acceptor-donor triad dyes based on perylene-bisimide subunits, with the long axis of the donors arranged either parallel or perpendicular to that of the central acceptor. We observe rapid energy transfer (energy transfer rate for the linearly arranged triad but severely underestimates it for the orthogonal case. We show that the rapid energy transfer arises from a combination of through-bond coupling and through-space transfer between donor and acceptor units. As they allow energy cascading to an excited state with controllable polarization, these triad dyes show high potential for use in luminescent solar concentrator devices.

  8. Ultrafast Non-Förster Intramolecular Donor-Acceptor Excitation Energy Transfer. (United States)

    Athanasopoulos, Stavros; Alfonso Hernandez, Laura; Beljonne, David; Fernandez-Alberti, Sebastian; Tretiak, Sergei


    Ultrafast intramolecular electronic energy transfer in a conjugated donor-acceptor system is simulated using nonadiabatic excited-state molecular dynamics. After initial site-selective photoexcitation of the donor, transition density localization is monitored throughout the S2 → S1 internal conversion process, revealing an efficient unidirectional donor → acceptor energy-transfer process. Detailed analysis of the excited-state trajectories uncovers several salient features of the energy-transfer dynamics. While a weak temperature dependence is observed during the entire electronic energy relaxation, an ultrafast initially temperature-independent process allows the molecular system to approach the S2-S1 potential energy crossing seam within the first ten femtoseconds. Efficient energy transfer occurs in the absence of spectral overlap between the donor and acceptor units and is assisted by a transient delocalization phenomenon of the excited-state wave function acquiring Frenkel-exciton character at the moment of quantum transition.

  9. Identification of Ag-acceptor related photoluminescence in $^{111}\\!$Ag doped CdTe

    CERN Document Server

    Hamann, J; Deicher, M; Filz, T; Ostheimer, V; Schmitz, C; Wolf, H; Wichert, T


    Bridgman-grown, nominally undoped CdTe crystals were doped with Ag by implanting radioactive $^{111}\\!$Ag. Photoluminescence spectra of the crystals show a donor-acceptor pair (DAP) line at 1.491 eV. The decrease of the intensity of this line with a half life of T$_{1/2}$=(7.2$\\pm$0.4) d is in good agreement with the half life of the $\\beta\\!^{-}$-decay of $^{111}\\!$Ag to $^{111}\\!$Cd of 7.45 d. This decrease is not caused by the aging behavior of Ag which was reported in the literature. The data show that the involved acceptor defect contains exactly one Ag atom and confirm the earlier assignment of the acceptor to the AgCd defect. Based on the DAP line at 1.491 eV, the spectra did not reveal a contamination of the CdTe crystals by stable Ag.

  10. Double acceptor in p-type GaAsN grown by chemical beam epitaxy (United States)

    Elleuch, Omar; Wang, Li; Lee, Kan-Hua; Ikeda, Kazuma; Kojima, Nobuaki; Ohshita, Yoshio; Yamaguchi, Masafumi


    The properties of the acceptor states in GaAsN grown by chemical beam epitaxy (CBE) are studied by analyzing their charges based on the Poole-Frenkel model. Deep level transient spectroscopy (DLTS) shows two acceptor levels at 0.11 and 0.19 eV above the valence band maximum. The emission rates of carriers from these states are enhanced with increasing the electric field during the DLTS measurement, which indicates that the energies required for the emission are decreased. By analyzing this field-enhanced emission process, the polarizabilities of the levels at 0.11 and 0.19 eV are found to be -1 (±0.1) and -2 (±0.1), respectively. In addition, these states have almost the same concentration. Therefore, we conclude that they originate from the same defect, acting as a double acceptor in GaAsN film grown by CBE.

  11. Characterization of phosphorus removal bacteria in (AO)2 SBR system by using different electron acceptors

    Institute of Scientific and Technical Information of China (English)

    JIANG Yi-feng; WANG Lin; YU Ying; WANG Bao-zhen; LIU Shuo; SHEN Zheng


    Characteristics of phosphorus removal bacteria were investigated by using three different types of electron acceptors, as well as the positive role of nitrite in phosphorus removal process. An (AO)2 SBR (anaerobic-aerobic-anoxic-aerobic sequencing batch reactor) was thereby employed to enrich denitrifying phosphorus removal bacteria for simultaneously removing phosphorus and nitrogen via anoxic phosphorus uptake. Ammonium oxidation was controlled at the first phase of the nitrification process. Nitrite-inhibition batch tests illustrated that nitrite was not an inhibitor to phosphorus uptake process, but served as an alternative electron acceptor to nitrate and oxygen if the concentration was under the inhibition level of 40mg NO2 - N · L- 1. It implied that in addition to the two well-accepted groups of phosphorus removal bacterium ( one can only utilize oxygen as electron acceptor, P1, while the other can use both oxygen and nitrate as electron acceptor, P2 ), a new group of phosphorus removal bacterium P3, which could use oxygen, nitrate and nitrite as electron acceptor to take up phosphorus were identified in the test system. To understand (AO)2 SBR sludge better, the relative population of the different bacteria in this system, plus another A/O SBR sludge (seed sludge) were respectively estimated by the phosphorus uptake batch tests with either oxygen or nitrate or nitrite as electron acceptor. The results demonstrated that phosphorus removal capability of (AO)2 SBR sludge had a little degradation after A/O sludge was cultivated in the (AO)2 mode over a long period of time. However, denitrifying phosphorus removal bacteria ( P2 and P3 ) was significantly enriched showed by the relative population of the three types of bacteria,which implied that energy for aeration and COD consumption could be reduced in theory.

  12. Local Intermolecular Order Controls Photoinduced Charge Separation at Donor/Acceptor Interfaces in Organic Semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Feier, Hilary M.; Reid, Obadiah G.; Pace, Natalie A.; Park, Jaehong; Bergkamp, Jesse J.; Sellinger, Alan; Gust, Devens; Rumbles, Garry


    How free charge is generated at organic donor-acceptor interfaces is an important question, as the binding energy of the lowest energy (localized) charge transfer states should be too high for the electron and hole to escape each other. Recently, it has been proposed that delocalization of the electronic states participating in charge transfer is crucial, and aggregated or otherwise locally ordered structures of the donor or the acceptor are the precondition for this electronic characteristic. The effect of intermolecular aggregation of both the polymer donor and fullerene acceptor on charge separation is studied. In the first case, the dilute electron acceptor triethylsilylhydroxy-1,4,8,11,15,18,22,25-octabutoxyphthalocyaninatosilicon(IV) (SiPc) is used to eliminate the influence of acceptor aggregation, and control polymer order through side-chain regioregularity, comparing charge generation in 96% regioregular (RR-) poly(3-hexylthiophene) (P3HT) with its regiorandom (RRa-) counterpart. In the second case, ordered phases in the polymer are eliminated by using RRa-P3HT, and phenyl-C61-butyric acid methyl ester (PC61BM) is used as the acceptor, varying its concentration to control aggregation. Time-resolved microwave conductivity, time-resolved photoluminescence, and transient absorption spectroscopy measurements show that while ultrafast charge transfer occurs in all samples, long-lived charge carriers are only produced in films with intermolecular aggregates of either RR-P3HT or PC61BM, and that polymer aggregates are just as effective in this regard as those of fullerenes.

  13. Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Roch, Christina; Kuhn, Joachim; Kleesiek, Knut [Institut fuer Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Universitaetsklinik der Ruhr-Universitaet Bochum, 32545 Bad Oeynhausen (Germany); Goetting, Christian, E-mail: [Institut fuer Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Universitaetsklinik der Ruhr-Universitaet Bochum, 32545 Bad Oeynhausen (Germany)


    The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K{sub m} and V{sub max} values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.

  14. Threshold-like complexation of conjugated polymers with small molecule acceptors in solution within the neighbor-effect model. (United States)

    Sosorev, Andrey Yu; Parashchuk, Olga D; Zapunidi, Sergey A; Kashtanov, Grigoriy S; Golovnin, Ilya V; Kommanaboyina, Srikanth; Perepichka, Igor F; Paraschuk, Dmitry Yu


    In some donor-acceptor blends based on conjugated polymers, a pronounced charge-transfer complex (CTC) forms in the electronic ground state. In contrast to small-molecule donor-acceptor blends, the CTC concentration in polymer:acceptor solution can increase with the acceptor content in a threshold-like way. This threshold-like behavior was earlier attributed to the neighbor effect (NE) in the polymer complexation, i.e., next CTCs are preferentially formed near the existing ones; however, the NE origin is unknown. To address the factors affecting the NE, we record the optical absorption data for blends of the most studied conjugated polymers, poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) and poly(3-hexylthiophene) (P3HT), with electron acceptors of fluorene series, 1,8-dinitro-9,10-antraquinone (), and 7,7,8,8-tetracyanoquinodimethane () in different solvents, and then analyze the data within the NE model. We have found that the NE depends on the polymer and acceptor molecular skeletons and solvent, while it does not depend on the acceptor electron affinity and polymer concentration. We conclude that the NE operates within a single macromolecule and stems from planarization of the polymer chain involved in the CTC with an acceptor molecule; as a result, the probability of further complexation with the next acceptor molecules at the adjacent repeat units increases. The steric and electronic microscopic mechanisms of NE are discussed.

  15. Copper-catalyzed asymmetric conjugate addition of organometallic reagents to extended Michael acceptors

    Directory of Open Access Journals (Sweden)

    Thibault E. Schmid


    Full Text Available The copper-catalyzed asymmetric conjugate addition (ACA of nucleophiles onto polyenic Michael acceptors represents an attractive and powerful methodology for the synthesis of relevant chiral molecules, as it enables in a straightforward manner the sequential generation of two or more stereogenic centers. In the last decade, various chiral copper-based catalysts were evaluated in combination with different nucleophiles and Michael acceptors, and have unambiguously demonstrated their usefulness in the control of the regio- and enantioselectivity of the addition. The aim of this review is to report recent breakthroughs achieved in this challenging field.

  16. Effect of electronic acceptor segments on photophysical properties of low-band-gap ambipolar polymers. (United States)

    Li, Yuanzuo; Cui, Jingang; Zhao, Jianing; Liu, Jinglin; Song, Peng; Ma, Fengcai


    Stimulated by a recent experimental report, charge transfer and photophysical properties of donor-acceptor ambipolar polymer were studied with the quantum chemistry calculation and the developed 3D charge difference density method. The effects of electronic acceptor strength on the structure, energy levels, electron density distribution, ionization potentials, and electron affinities were also obtained to estimate the transporting ability of hole and electron. With the developed 3D charge difference density, one visualizes the charge transfer process, distinguishes the role of molecular units, and finds the relationship between the role of DPP and excitation energy for the three polymers during photo-excitation.

  17. Solution-processable donor-acceptor polymers with modular electronic properties and very narrow bandgaps. (United States)

    Foster, Michael E; Zhang, Benjamin A; Murtagh, Dustin; Liu, Yi; Sfeir, Matthew Y; Wong, Bryan M; Azoulay, Jason D


    Bridgehead imine-substituted cyclopentadithiophene structural units, in combination with highly electronegative acceptors that exhibit progressively delocalized π-systems, afford donor-acceptor (DA) conjugated polymers with broad absorption profiles that span technologically relevant wavelength (λ) ranges from 0.7 electronic properties so as to achieve very narrow optical bandgaps (Eg (opt) < 0.5 eV). This strategy affords modular DA copolymers with broad- and long-wavelength light absorption in the infrared and materials with some of the narrowest bandgaps reported to date.

  18. Doping of germanium and silicon crystals with non-hydrogenic acceptors for far infrared lasers (United States)

    Haller, Eugene E.; Brundermann, Erik


    A method for doping semiconductors used for far infrared lasers with non-hydrogenic acceptors having binding energies larger than the energy of the laser photons. Doping of germanium or silicon crystals with beryllium, zinc or copper. A far infrared laser comprising germanium crystals doped with double or triple acceptor dopants permitting the doped laser to be tuned continuously from 1 to 4 terahertz and to operate in continuous mode. A method for operating semiconductor hole population inversion lasers with a closed cycle refrigerator.

  19. Modular supramolecular approach for co-crystallization of donors and acceptors into ordered networks (United States)

    Stupp, Samuel I.; Stoddart, J. Fraser; Shveyd, Alex K.; Tayi, Alok S.; Sue, Andrew C. H.; Narayanan, Ashwin


    Organic charge-transfer (CT) co-crystals in a mixed stack system are disclosed, wherein a donor molecule (D) and an acceptor molecule (A) occupy alternating positions (DADADA) along the CT axis. A platform is provided which amplifies the molecular recognition of donors and acceptors and produces co-crystals at ambient conditions, wherein the platform comprises (i) a molecular design of the first constituent (.alpha.-complement), (ii) a molecular design of the second compound (.beta.-complement), and (iii) a solvent system that promotes co-crystallization.

  20. Synthesis and Characterization of Organic Dyes Containing Various Donors and Acceptors

    Directory of Open Access Journals (Sweden)

    Wen-Chung Ou-Yang


    Full Text Available New organic dyes comprising carbazole, iminodibenzyl, or phenothiazine moieties, respectively, as the electron donors, and cyanoacetic acid or acrylic acid moieties as the electron acceptors/anchoring groups were synthesized and characterized. The influence of heteroatoms on carbazole, iminodibenzyl and phenothiazine donors, and cyano-substitution on the acid acceptor is evidenced by spectral, electrochemical, photovoltaic experiments, and density functional theory calculations. The phenothiazine dyes show solar-energy-to-electricity conversion efficiency (η of 3.46–5.53%, whereas carbazole andiminodibenzyl dyesshow η of 2.43% and 3.49%, respectively.

  1. Structural instability of N-acceptors in homo- and heteroepitaxially grown ZnO by MBE

    Energy Technology Data Exchange (ETDEWEB)

    Ando, K.; Abe, T.; Taya, T.; Ishihara, Y.; Enomoto, K.; Yamazaki, Y.; Yoshikawa, J.; Fujino, K.; Nakamura, H.; Ohno, T.; Kasada, H. [Department of Information and Electronic Engineering, Graduate School of Engineering, Tottori University, 4-1-1 Koyama-Minami, Tottori 680-8550 (Japan)


    Unique properties of the N-acceptor in homo- and heteroepitaxially grown ZnO by molecular beam epitaxy (MBE) are studied by means of microproving of surface sheet-resistance, Hall-effect measurement, persistent photoconduction (PPC) and thermally stimulated current (TSC). Rapid postanneal of N-doped ZnO is found to induce the change in the conduction type from n-type (as-grown) to p/n-type mixed conduction, forming island structure, and these properties are related to a structural instability of the N-acceptor. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

  2. Exploring Directional LLʹCT in (donor)M(acceptor) Complexes


    Kramer, Wesley William


    The overarching theme of the work presented in this dissertation is the to develope a better understanding of the factors which govern the properties of donor-acceptor (D-A) LL'CT transition metal complexes. Chapter 2 describes a series of (catcholate)Ni(diimine) D-A LL'CT complexes. The spectroscopic and electrochemical properties can be tuned independently through variations in donor or acceptor ligand electronics. Chapter 3 describes the Group 10 series of (cat-tBu2)M(bdi) complexes (M = ...

  3. Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases. (United States)

    Hindsgaul, O; Kaur, K J; Srivastava, G; Blaszczyk-Thurin, M; Crawley, S C; Heerze, L D; Palcic, M M


    The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are

  4. Investigation of acceptor states in ZnO by junction DLTS (United States)

    von Wenckstern, H.; Pickenhain, R.; Schmidt, H.; Brandt, M.; Biehne, G.; Lorenz, M.; Grundmann, M.; Brauer, G.


    We have realized a p-type ZnO surface layer by N + ion implantation of a high quality ZnO wafer and subsequent annealing. The conduction type of this surface layer was revealed by scanning capacitance microscopy. Rectifying current-voltage characteristics for processed devices were coherent with the existence of an internal pn junction. Deep donor- and acceptor-like defects were investigated by junction deep level transient spectroscopy. The donor-like levels correspond to those commonly observed for E1 and E3 defects. The acceptor states resolved have thermal activation energies of about 150 meV and 280 meV, respectively.

  5. A 4% efficient organic solar cell using a fluorinated fused subphthalocyanine dimer as an electron acceptor

    Energy Technology Data Exchange (ETDEWEB)

    Verreet, Bregt; Heremans, Paul [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); ESAT, Katholieke Universiteit Leuven, Kasteelpark Arenberg 10, B-3001 Leuven (Belgium); Rand, Barry P.; Cheyns, David; Hadipour, Afshin; Aernouts, Tom [IMEC, Kapeldreef 75, B-3001 Leuven (Belgium); Medina, Anais; Claessens, Christian G. [Departamento de Quimica Organica, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Torres, Tomas [Departamento de Quimica Organica, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); IMDEA-Nanociencia, Facultad de Ciencias, Ciudad Universitaria de Cantoblanco, 28049 Madrid (Spain)


    Planar bilayer organic solar cells with a fluorinated fused subphthalocyanine dimer (FSubPcDimer) as an acceptor and chloroboron (III) subphthalocyanine (SubPc) as a donor obtain a 60% higher J{sub sc} compared to cells using C{sub 60} as an acceptor, resulting in a power conversion efficiency of 4%. This is obtained thanks to the important contribution to the photocurrent of the low-bandgap FSubPcDimer. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  6. Analogue to Digital and Digital to Analogue Converters (ADCs and DACs): A Review Update

    CERN Document Server

    Pickering, J


    This is a review paper updated from that presented for CAS 2004. Essentially, since then, commercial components have continued to extend their performance boundaries but the basic building blocks and the techniques for choosing the best device and implementing it in a design have not changed. Analogue to digital and digital to analogue converters are crucial components in the continued drive to replace analogue circuitry with more controllable and less costly digital processing. This paper discusses the technologies available to perform in the likely measurement and control applications that arise within accelerators. It covers much of the terminology and 'specmanship' together with an application-oriented analysis of the realisable performance of the various types. Finally, some hints and warnings on system integration problems are given.

  7. Observing single FoF1-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor (United States)

    Heitkamp, Thomas; Deckers-Hebestreit, Gabriele; Börsch, Michael


    Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, ten-stepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

  8. 3D-localization of the a-subunit in F 0F I-ATP synthase by time resolved single-molecule FRET (United States)

    Düser, Monika G.; Zarrabi, Nawid; Bi, Yumin; Zimmermann, Boris; Dunn, Stanley D.; Börsch, Michael


    F °F I-ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F I part to the proton translocation through the F ° part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F ° motor. At present, the relative position of the a-subunit is not known. We examined the rotary movements of the ɛ-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ɛ-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ɛ resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ɛ, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F ° part.

  9. Synthesis of Fe3O4@phenol formaldehyde resin core-shell nanospheres loaded with Au nanoparticles as magnetic FRET nanoprobes for detection of thiols in living cells. (United States)

    Yang, Ping; Xu, Qi-Zhi; Jin, Sheng-Yu; Zhao, Yang; Lu, Yang; Xu, Xue-Wei; Yu, Shu-Hong


    A magnetic, sensitive, and selective fluorescence resonance energy transfer (FRET) probe for detection of thiols in living cells was designed and prepared. The FRET probe consists of an Fe(3)O(4) core, a green-luminescent phenol formaldehyde resin (PFR) shell, and Au nanoparticles (NPs) as FRET quenching agent on the surface of the PFR shell. The Fe(3)O(4) NPs were used as the core and coated with green-luminescent PFR nanoshells by a simple hydrothermal approach. Au NPs were then loaded onto the surface of the PFR shell by electric charge absorption between Fe(3)O(4)@PFR and Au NPs after modifying the Fe(3)O(4)@PFR nanocomposites with polymers to alter the charge of the PFR shell. Thus, a FRET probe can be designed on the basis of the quenching effect of Au NPs on the fluorescence of Fe(3)O(4)@PFR nanocomposites. This magnetic and sensitive FRET probe was used to detect three kinds of primary biological thiols (glutathione, homocysteine, and cysteine) in cells. Such a multifunctional fluorescent probe shows advantages of strong magnetism for sample separation, sensitive response for sample detection, and low toxicity without injury to cellular components.

  10. FRET imaging of calcium signaling in live cells in the microenvironment. (United States)

    Qian, Tongcheng; Lu, Shaoying; Ma, Hongwei; Fang, Jing; Zhong, Wenxuan; Wang, Yingxiao


    The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

  11. Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET. (United States)

    Hengesbach, Martin; Voigts-Hoffmann, Felix; Hofmann, Benjamin; Helm, Mark


    Pseudouridine is the most abundant of more than 100 chemically distinct natural ribonucleotide modifications. Its synthesis consists of an isomerization reaction of a uridine residue in the RNA chain and is catalyzed by pseudouridine synthases. The unusual reaction mechanism has become the object of renewed research effort, frequently involving replacement of the substrate uridines with 5-fluorouracil (f(5)U). f(5)U is known to be a potent inhibitor of pseudouridine synthase activity, but the effect varies among the target pseudouridine synthases. Derivatives of f(5)U have previously been detected, which are thought to be either hydrolysis products of covalent enzyme-RNA adducts, or isomerization intermediates. Here we describe the interaction of pseudouridine synthase 1 (Pus1p) with f(5)U-containing tRNA. The interaction described is specific to Pus1p and position 27 in the tRNA anticodon stem, but the enzyme neither forms a covalent adduct nor stalls at a previously identified reaction intermediate of f(5)U. The f(5)U27 residue, as analyzed by a DNAzyme-based assay using TLC and mass spectrometry, displayed physicochemical properties unaltered by the reversible interaction with Pus1p. Thus, Pus1p binds an f(5)U-containing substrate, but, in contrast to other pseudouridine synthases, leaves the chemical structure of f(5)U unchanged. The specific, but nonproductive, interaction demonstrated here thus constitutes an intermediate of Pus turnover, stalled by the presence of f(5)U in an early state of catalysis. Observation of the interaction of Pus1p with fluorescence-labeled tRNA by a real-time readout of fluorescence anisotropy and FRET revealed significant structural distortion of f(5)U-tRNA structure in the stalled intermediate state of pseudouridine catalysis.

  12. Unspecific membrane protein-lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements. (United States)

    Borrell, Jordi H; Montero, M Teresa; Morros, Antoni; Domènech, Òscar


    In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca(2+) display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (Lα) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid-to-protein ratio of 40. When the lipid-to-protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self-segregated units of LacY, the lower domain is constituted only by phospholipids in gel (Lβ) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid-protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid-LacY interface segregated from the fluid bulk phase where POPG predominates.

  13. The Greenland analogue project. Yearly report 2010

    Energy Technology Data Exchange (ETDEWEB)

    Harper, J.; Brinkerhoff, D.; Johnson, J. [University of Montana, Missoula (United States); Ruskeeniemi, T.; Engstroem, J.; Kukkonen, I. [Geological Survey of Finland (Finland)] [and others


    A four-year field and modelling study of the Greenland ice sheet and subsurface conditions, Greenland Analogue Project (GAP), has been initiated collaboratively by SKB, Posiva and NWMO to advance the understanding of processes associated with glaciation and their impact on the long-term performance of a deep geological repository. The study site encompasses a land terminus portion of the Greenland ice sheet, east of Kangerlussuaq, and is in many ways considered to be an appropriate analogue of the conditions that are expected to prevail in much of Canada and Fennoscandia during future glacial cycles. The project begins in 2009 and is scheduled for completion in 2012. Our current understanding of the hydrological, hydrogeological and hydrogeochemical processes associated with cold climate conditions and glacial cycles, and their impact on the long-term performance of deep geological repositories for spent nuclear fuel, will be significantly improved by studying a modern analogue. The GAP will conduct the first in situ investigations of some of the parameters and processes needed to achieve a better understanding of how an ice sheet may impact a deep repository, and will provide measurements, observations and data that may significantly improve our safety assessments and risk analyses of glaciation scenarios. This report was produced by the GAP team members and presents an overview of the activities within the GAP during the interval January 1 to December 31, 2010, as well as research results obtained during this time frame. Research for the GAP is ongoing, and additional results related to the data presented here may become available in the future and will be presented in subsequent annual reports. (orig.)

  14. Acetylenic dithiafulvene derived donor-pi-acceptor dyads: synthesis, electrochemistry and non-linear optical properties

    DEFF Research Database (Denmark)

    Nielsen, Mogens Brønsted; Petersen, Jan Conrad; Thorup, Niels;


    A selection of donor-acceptor chromophores containing the redox-active dithiafulvene unit about acetylenic and aryl scaffolds has been synthesized. The molecules were studied for their optical, redox and structural properties. Moreover, third-order non-linear optical properties were investigated...

  15. Characterization of donor-acceptor-pair emission in fluorescent 6H-SiC

    DEFF Research Database (Denmark)

    Ou, Yiyu; Jokubavicius, Valdas; Linnarsson, Margareta

    Boron (B)- and nitrogen (N)-codoped 6H-SiC epilayer exhibits strong donor to acceptor pair (DAP) band luminescence which makes it a promising candidate for the white light emitting diode (LED) [1]. To investigate the optimized dopant concentrations, five samples with the same B concentration level...

  16. Limited Cash Flow on Slot Machines: Effects of Prohibition of Note Acceptors on Adolescent Gambling Behaviour (United States)

    Hansen, Marianne; Rossow, Ingeborg


    This study addresses the impact of prohibition of note acceptors on gambling behaviour and gambling problems among Norwegian adolescents. Data comprised school surveys at three time points; 2004 and 2005 (before intervention) and 2006 (after intervention). Net samples comprised 20.000 students aged 13-19 years at each data collection. Identical…

  17. Synthesis of OMS Materials and Investigation of Their Acceptor-Donor Characteristics. (United States)

    Grajek, H; Paciura-Zadrożna, J; Choma, J; Michalski, E; Witkiewicz, Z


    Three ordered mesoporous siliceous (OMS) materials known as MCM41s-unmodified MCM-41C16 ("C16"), and two MCM41s with different surface functionalities: MCM-41C16-SH ("C16-SH") and MCM-41C16-NH2 ("C16-NH2")-were synthesized and studied by inverse gas chromatography in order to determine their acceptor-donor properties. The specific retention volumes of nonpolar and polar probes that were chromatographed on these ordered mesoporous silica adsorbents were evaluated under infinite dilution conditions. Two methods were employed to calculate the standard free energy of adsorption, ΔGads, of each chromatographed probe on the basis its specific retention volume. These ΔGads values were then employed to estimate the van der Waals contribution and the specific contribution of the free surface energy for each MCM41. DN values (donor numbers, based on the Gutmann scale) and AN* values (acceptor numbers, based on the Riddle-Fowkes scale) were employed to determine the values of parameters that characterize the ability of the MCM41s to act as electron acceptors (parameter: KA) and donors (parameter: KD). Considering the different compositions of the probes, each of which has different acceptor-donor properties, a new chromatographic test to supplement the Grob test is suggested.

  18. Dominant effects of first monolayer energetics at donor/acceptor interfaces on organic photovoltaics. (United States)

    Izawa, Seiichiro; Nakano, Kyohei; Suzuki, Kaori; Hashimoto, Kazuhito; Tajima, Keisuke


    Energy levels of the first monolayer are manipulated at donor/acceptor interfaces in planar heterojunction organic photovoltaics by using molecular self-organization. A "cascade" energy landscape allows thermal-activation-free charge generation by photoirradiation, destabilizes the energy of the interfacial charge-transfer state, and suppresses bimolecular charge recombination, resulting in a higher open-circuit voltage and fill factor.

  19. Solution-grown organic single-crystalline donor-acceptor heterojunctions for photovoltaics. (United States)

    Li, Hanying; Fan, Congcheng; Fu, Weifei; Xin, Huolin L; Chen, Hongzheng


    Organic single crystals are ideal candidates for high-performance photovoltaics due to their high charge mobility and long exciton diffusion length; however, they have not been largely considered for photovoltaics due to the practical difficulty in making a heterojunction between donor and acceptor single crystals. Here, we demonstrate that extended single-crystalline heterojunctions with a consistent donor-top and acceptor-bottom structure throughout the substrate can be simply obtained from a mixed solution of C60 (acceptor) and 3,6-bis(5-(4-n-butylphenyl)thiophene-2-yl)-2,5-bis(2-ethylhexyl)pyrrolo[3,4-c]pyrrole-1,4-dione (donor). 46 photovoltaic devices were studied with the power conversion efficiency of (0.255±0.095)% under 1 sun, which is significantly higher than the previously reported value for a vapor-grown organic single-crystalline donor-acceptor heterojunction (0.007%). As such, this work opens a practical avenue for the study of organic photovoltaics based on single crystals.

  20. Organic donor/acceptor photovoltaics : The role of C-60/metal interfaces

    NARCIS (Netherlands)

    Melzer, C; Krasnikov, VV; Hadziioannou, G


    The photovoltaic properties of thin films based on donor/acceptor heterojunctions, prepared by means of either consecutive evaporation or co-evaporation, and sandwiched between asymmetric contacts are investigated. (E,E,E,E)-1,4-bis[(4-styryl)styryl]-2-methoxy-5-(2'-ethylhexoxy)benzene (MEH-OPV5) an

  1. [4 + 2] Annulation of Donor-Acceptor Cyclopropanes with Acetylenes Using 1,2-Zwitterionic Reactivity. (United States)

    Novikov, Roman A; Tarasova, Anna V; Denisov, Dmitry A; Borisov, Denis D; Korolev, Victor A; Timofeev, Vladimir P; Tomilov, Yury V


    A new process for the [4 + 2] annulation of donor-acceptor cyclopropanes with acetylenes under the effect of anhydrous GaCl3 using 1,2-zwitterion reactivity was elaborated. The reaction opens access to substituted dihydronaphthalenes, naphthalenes, and other fused carbocycles. The direction of the reaction can be efficiently controlled by temperature.

  2. Organic Materials in the Undergraduate Laboratory: Microscale Synthesis and Investigation of a Donor-Acceptor Molecule (United States)

    Pappenfus, Ted M.; Schliep, Karl B.; Dissanayake, Anudaththa; Ludden, Trevor; Nieto-Ortega, Belen; Lopez Navarrete, Juan T.; Ruiz Delgado, M. Carmen; Casado, Juan


    A series of experiments for undergraduate courses (e.g., organic, physical) have been developed in the area of small molecule organic materials. These experiments focus on understanding the electronic and redox properties of a donor-acceptor molecule that is prepared in a convenient one-step microscale reaction. The resulting intensely colored…

  3. Effects of alternative electron acceptors and temperature on methanogenesis in rice paddy soils

    NARCIS (Netherlands)

    Bodegom, van P.M.; Stams, A.J.M.


    Soil slurries collected from rice paddies were incubated anaerobically at different temperatures. Changes in the composition of electron acceptors and electron donors in the slurries were monitored daily. During the incubation NO3- was reduced first, followed by Fe3 and SO42- reduction and methane p

  4. Comparison of oxygen and hypochlorite as cathodic electron acceptor in microbial fuel cells. (United States)

    Jadhav, D A; Ghadge, A N; Mondal, Debika; Ghangrekar, M M


    Effect of oxygen and sodium hypochlorite (NaOCl) as cathodic electron acceptors on performance of a clayware microbial fuel cell (MFC) was evaluated in this study. Maximum power density of 6.57 W/m(3) was obtained with NaOCl as catholyte, which is about 9 times higher than oxygen being used as an electron acceptor. Voltammetry and Tafel analysis further supported the faster reduction kinetics lead to increase in power output and reduction in internal resistance of MFC operated with NaOCl as an electron acceptor. Using NaOCl as catholyte, higher exchange current density of 10.91 and 11.52 mA/m(2) and lower charge transfer resistance of 0.58 and 0.56 kΩ m(2) was observed for anode and cathode, respectively. Higher organic matter removal of about 90% with 25% Coulombic efficiency was achieved using NaOCl as catholyte. Higher internal resistance, lower cathode potential and slow reduction kinetics deteriorated performance of MFC using oxygen as cathodic electron acceptor.

  5. Rhodanine dye-based small molecule acceptors for organic photovoltaic cells. (United States)

    Kim, Yujeong; Song, Chang Eun; Moon, Sang-Jin; Lim, Eunhee


    The solution-processable small molecules based on carbazole or fluorene containing rhodanine dyes at both ends were synthesized and introduced as acceptors in organic photovoltaic cells. The high energy levels of their lowest unoccupied molecular orbitals resulted in a power conversion efficiency of 3.08% and an open circuit voltage of up to 1.03 V.

  6. Quantitative measurements of magnetic polaron binding on acceptors in CdMnTe alloys (United States)

    Nhung, Tran Hong; Planel, R.


    The acceptor binding energy is measured as a function of Temperature and composition in Cd1-x Mnx Te alloys, by time resolved spectroscopy. The Bound magnetic polaron effect is measured and compared with a theory accouting for magnetic saturation and fluctuations.

  7. Mechanism and Dynamics of Charge Transfer in Donor-Bridge-Acceptor Systems

    NARCIS (Netherlands)

    Gorczak-Vos, N.


    Photoinduced charge transfer in organic materials is a fundamental process in various biological and technological areas. Donor-bridge-acceptor (DBA) molecules are used as model systems in numerous theoretical and experimental work to systematically study and unravel the underlying mechanisms of cha

  8. 2004 Electron Donor Acceptor Interactions Gordon Conference - August 8-13, 2004

    Energy Technology Data Exchange (ETDEWEB)



    The 2004 Gordon Conference on Donor/Acceptor Interactions will take place at Salve Regina University in Newport, Rhode Island on August 8-13, 2004. The conference will be devoted to the consequences of charge interaction and charge motion in molecular and materials systems.

  9. Technical Considerations in Magnetic Analogue Models

    CERN Document Server

    Adams, Patrick W M


    The analogy between vorticity and magnetic fields has been a subject of interest to researchers for a considerable period of time, mainly because of the structural similarities between the systems of equations that govern the evolution of the two fields. We recently presented the analysis of magnetic fields and hydrodynamics vorticity fields and argued for a formal theory of analogue magnetism. This article provides in depth technical details of the relevant considerations for the simulation procedures and extends the analyses to a range of fluids.

  10. Analogues of Euler and Poisson Summation Formulae

    Indian Academy of Sciences (India)

    Vivek V Rane


    Euler–Maclaurin and Poisson analogues of the summations $\\sum_{a < n ≤ b}(n)f(n), \\sum_{a < n ≤ b}d(n) f(n), \\sum_{a < n ≤ b}d(n)(n) f(n)$ have been obtained in a unified manner, where (()) is a periodic complex sequence; () is the divisor function and () is a sufficiently smooth function on [, ]. We also state a generalised Abel's summation formula, generalised Euler's summation formula and Euler's summation formula in several variables.

  11. Analogue cosmology in a hadronic fluid

    Directory of Open Access Journals (Sweden)

    Bilić Neven


    Full Text Available Analog gravity models of general relativity seem promising routes to providing laboratory tests of the foundation of quantum field theory in curved space-time. In contrast to general relativity, where geometry of a spacetime is determined by the Einstein equations, in analog models geometry and evolution of analog spacetime are determined by the equations of fluid mechanics. In this paper we study the analogue gravity model based on massless pions propagating in a expanding hadronic fluid. The analog expanding spacetime takes the form of an FRW universe, with the apparent and trapping horizons defined in the standard way.

  12. Satellite television analogue and digital reception techniques

    CERN Document Server

    Benoit, Herve


    Satellite television is part of the lives of millions of television viewers worldwide and its influence is set to increase significantly with the launch of digital satellite television services.This comprehensive reference book, written by the author of the highly successful 'Digital Television', provides a technical overview of both analogue and digital satellite TV. Written concisely and thoroughly, it covers all aspects of satellite TV necessary to understand its operation and installation. It also covers the evolution of satellite television, and contains a detailed glossary of tec

  13. Study of microbial perchlorate reduction: Considering of multiple pH, electron acceptors and donors

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Xing [Key Laboratory of Water Pollution Control and Recycling (Shandong), School of Environmental Science and Engineering, Shandong University, Jinan 250100 (China); Gao, Baoyu, E-mail: [Key Laboratory of Water Pollution Control and Recycling (Shandong), School of Environmental Science and Engineering, Shandong University, Jinan 250100 (China); Jin, Bo [School of Chemical Engineering, The University of Adelaide, Adelaide SA 5005,Australia (Australia); Zhen, Hu [Key Laboratory of Water Pollution Control and Recycling (Shandong), School of Environmental Science and Engineering, Shandong University, Jinan 250100 (China); Wang, Xiaoyi [CSIRO Land and Water, Gate 5, Waite Road, Urrbrae, SA 5064 (Australia); Dai, Ming [School of Chemical Engineering, The University of Adelaide, Adelaide SA 5005,Australia (Australia)


    Graphical abstract: Schemes of perchlorate reduction in ClO{sub 4}{sup −}/ClO{sub 3}{sup −}–NO{sub 3}{sup −} e{sup −}acceptor systems. - Highlights: • We created a multiple electron acceptor/donor system for ClO{sub 4}{sup −} reduction. • Nitrate reduction was inhibited when using perchlorate-grown Azospira sp. KJ. • Reduction proceeded as an order of ClO{sub 3}{sup −}, ClO{sub 4}{sup −}and NO{sub 3}{sup −}. • Oxidation of acetate was inhibited by succinate in acetate–succinate series. - Abstract: Bioremediation of perchlorate-cotaminated water by a heterotrophic perchlorate reducing bacterium creates a multiple electron acceptor-donor system. We experimentally determined the perchlorate reduction by Azospira sp. KJ at multiple pH, electron acceptors and donors systems; this was the aim of this study. Perchlorate reduction was drastically inhibited at the pH 6.0, and the maximum reduction of perchlorate by Azospira sp. KJ was observed at pH value of 8.0. Perchlorate reduction was retarded in ClO{sub 4}{sup −}–ClO{sub 3}{sup −}, ClO{sub 4}{sup −}–ClO{sub 3}{sup −}–NO{sub 3}{sup −},and ClO{sub 4}{sup −}–NO{sub 3}{sup −} acceptor systems, while being completely inhibited by the additional O{sub 2} in the ClO{sub 4}{sup −}–O{sub 2} acceptor system. The reduction proceeded as an order of ClO{sub 3}{sup −}, ClO{sub 4}{sup −}, and NO{sub 3}{sup −} in the ClO{sub 4}{sup −}–ClO{sub 3}{sup −}–NO{sub 3}{sup −} system. K{sub S,}v{sub max}, and q{sub max} obtained at different e{sup −} acceptor and donor conditions are calculated as 140.5–190.6 mg/L, 8.7–13.2 mg-perchlorate/L-h, and 0.094–0.16 mg-perchlorate/mg-DW-h, respectively.

  14. BRET and Time-resolved FRET strategy to study GPCR oligomerization: from cell lines toward native tissues

    Directory of Open Access Journals (Sweden)

    Martin eCottet


    Full Text Available The concept of oligomerization of G protein-coupled receptor (GPCR opens new perspectives regarding physiological function regulation. The capacity of one GPCR to modify its binding and coupling properties by interacting with a second one can be at the origin of regulations unsuspected two decades ago. Although the concept is interesting, its validation at a physiological level is challenging and probably explains why receptor oligomerization is still a matter of debate.Demonstrating the direct interactions between two proteins is not trivial since few techniques present a spatial resolution allowing to conclude to close interactions. Resonance energy transfer (RET strategies are actually the most convenient ones. During the last two decades, two of them, the Bioluminescent Resonance Energy Transfer (BRET and Time-resolved Fluorescence Energy Transfer (TR-FRET have been widely used since they exhibit high signal-to-noise ratio. Most of the experiments based on GPCR labeling have been performed in cell lines and it has been shown that all GPCRs have the propensity to form homo- or hetero-oligomers. However, whether these data can be extrapolated to GPCRs expressed in native tissues and explain receptor functioning in real life, remains an open question. Native tissues impose different constraints since GPCR sequences cannot be modified. Recently a fluorescent ligand-based GPCR labeling strategy combined to TR-FRET approach has been successfully used to prove the existence of GPCR oligomerization in native tissues.Although the RET based strategies are generally quite simple to implement, precautions have to be taken before concluding to the absence or the existence of specific interactions between receptors. For example, one should exclude the possibility of collision of receptors diffusing throughout the membrane leading to a specific FRET signal. We will review the advantages and the limits of different approaches and discuss the consequent

  15. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions. (United States)

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P


    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.

  16. U.S. Nuclear Regulatory Commission natural analogue research program

    Energy Technology Data Exchange (ETDEWEB)

    Kovach, L.A.; Ott, W.R. [Nuclear Regulatory Commission, Washington, DC (United States)


    This article describes the natural analogue research program of the U.S. Nuclear Regulatory Commission (US NRC). It contains information on the regulatory context and organizational structure of the high-level radioactive waste research program plan. It also includes information on the conditions and processes constraining selection of natural analogues, describes initiatives of the US NRC, and describes the role of analogues in the licensing process.

  17. The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro. (United States)

    Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L


    FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest.

  18. A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using PNA Probe and Water-Soluble Blue Light Emitting Polymer

    Directory of Open Access Journals (Sweden)

    Nidhi Mathur


    Full Text Available A reliable, fast, and low-cost biosensor for medical diagnostics using DNA sequence detection has been developed and tested for the detection of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium hexylfluorenyl-2, 7-diyl-alt-co- (1,4-phenylene] dibromide salt (PFP has been taken as cationic conjugated polymer (CCP and PNA attached with fluorescein dye (PNAC∗ as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA having complementary sequence, Forster resonance energy transfer (FRET may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.

  19. New conjugated molecular scaffolds based on [2,2]paracyclophane as electron acceptors for organic photovoltaic cells. (United States)

    Yang, Yang; Zhang, Guanxin; Yu, Chenmin; He, Chang; Wang, Jianguo; Chen, Xin; Yao, Jingjing; Liu, Zitong; Zhang, Deqing


    Two conjugated molecules with a [2,2]paracyclophane core were designed as non-fullerene electron acceptors for photovoltaic cells. Using as the donor, a high power conversion efficiency (2.69%) is achieved for the blending thin film of with , which is relatively high for solution-processed OPVs based on small molecular non-fullerene acceptors and as the electron donor.

  20. Unusual low-energy near-infrared bands for ferrocenyl-naphthalimide donor-acceptor dyads with aromatic spacer groups

    DEFF Research Database (Denmark)

    Tagg, Tei; Kjærgaard, Henrik Grum; Lane, Joseph R.


    Time-dependent density functional theory (TDDFT) calculations for a series of donor-spacer-acceptor (D-S-A) molecules with phenyl (1), biphenyl (2), and anthryl (3) spacers interpolated between the ferrocenylalkene donor and -C≡C-4-naphthalimido acceptor components predicted the presence of weak,...