FRET Requirements?
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Dimitra Giannakopoulou↗
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FRET Requirements?
Explore the source record for details and available documents.
Dimitra Giannakopoulou↗
Requirements Elicitation, Analysis and Verification Using FRET and CoCoSIM
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Formal Requirements Elicitation Tool↗
Large-Scale Turbulent Simulations of Grid-To-Rod-Fretting
Abstract Not Provided
Nuclear Fuel Cycle & Fuel Materials(11)↗
A Prototypical Workflow for Grid-to-Rod Fretting Analysis with Hydra-TH
Abstract Not Provided
Engineering(42)↗
Application of Advanced Methods to Predict Grid to Rod Fretting in PWRs
Advanced modeling and simulation methods are being developed as part of the US Department of Energy sponsored Nuclear Energy Modeling and Simulation Hub called CASL (Consortium for Advanced Simulation of LWRs). The key participants of the CASL team include Oak Ridge National Laboratory (lead), Idaho National Laboratory, Sandia National Laboratories, Los Alamos National Laboratory, Massachusetts Institute of Technology, North Carolina State University, University of Michigan, Electric Power Research Institute, Tennessee Valley Authority and Westinghouse Electric Corporation. One of the key objectives of the CASL program is to develop multi-physics methods and tools which evaluate neutronic, thermal-hydraulic, structural mechanics and nuclear fuel rod performance in rod bundles to support power uprates, increased burnup/cycle length and life extension for US nuclear plants.
Karoutas, Zeses↗
Development of sensitive FRET sensors and methods of using the same
Intramolecular biosensors are disclosed, including PBP-based biosensors, comprising a ligand binding domain fused to donor and fluorescent moieties that permit detection and measurement of Fluorescence Resonance Energy Transfer upon binding ligand. At least one of the donor and fluorescent moieties may be internally fused to the biosensor such that both ends of the internally fused fluorophore are fixed. In addition, methods of improving the sensitivity of terminally fused biosensors are provided. The biosensors of the invention are useful for the detection and quantification of ligands in vivo and in culture.
Frommer, Wolf B.↗
A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor
Highlights: {yields} A new FRET-based method detects AT1/B2 receptor heterodimerization. {yields} First time application of AT1-Cerulean as a FRET donor. {yields} Method relies on signal peptide-enhanced cell surface delivery of AT1-Cerulean. {yields} A high FRET efficiency revealed efficient heterodimerization of AT1/B2R proteins. {yields} AT1/B2R heterodimers were functionally coupled to desensitization mechanisms. -- Abstract: Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.
60 APPLIED LIFE SCIENCES↗
Flight set 360L009 (STS-36) case and seals component, volume 2
The performance of the 360L009, Ninth Flight, Redesigned Solid Rocket Motors (RSRM) is assessed in respect to case hardware and joint sealing issues as seen from post-fire assessment of the case and seals. In addition, all of the potential anomalies have been classified and summarized. The post-fire assessment of both motors showed the case segments to be in good condition. Field joint fretting of this flight set ranged from light on most of the joints to locally heavy on three of the joints. The LH aft field joint is the most heavily fretted joint to date; this joint had previously fretted areas from STS-27. No new fretting was found in old fretting locations. However, new frets were found at the edges of repaired areas on both the LH center and aft field joints. All fretting was mapped from the inner clevis leg. The capture feature fretting was not mapped due to the difficulty of measuring the fretting in the capture feature region. It is assumed that the capture feature had damage similar to that on the inner clevis leg.
Carlisle, Alan R.↗
Phospholamban mutants compete with wild type for SERCA binding in living cells
Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.
60 APPLIED LIFE SCIENCES↗
Distance Dependence of Förster Resonance Energy Transfer Rates in 2D Perovskite Quantum Wells via Control of Organic Spacer Length
Two-dimensional (2D) semiconductors are attractive candidates for a variety of optoelectronic applications owing to the unique electronic properties that arise from quantum confinement along a single dimension. Incorporating nonradiative mechanisms that enable directed migration of bound charge carriers, such as Forster resonance energy transfer (FRET), could boost device efficiencies provided that FRET rates outpace undesired relaxation pathways. However, predictive models for FRET between distinct 2D states are lacking, particularly with respect to the distance d between a donor and acceptor. We approach FRET in systems with binary mixtures of donor and acceptor 2D perovskite quantum wells (PQWs), and we synthetically tune distances between donor and acceptor by varying alkylammonium spacer cation lengths. FRET rates are monitored using transient absorption spectroscopy and ultrafast photoluminescence, revealing rapid picosecond lifetimes that scale with spacer cation length. We theoretically model these binary mixtures of PQWs, describing the emitters as classical oscillating dipoles. Finally, we find agreement with our empirical lifetimes and then determine the effects of lateral extent and layer thickness, establishing fundamental principles for FRET in 2D materials.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Picosecond energy transfer and multiexciton transfer outpaces Auger recombination in binary CdSe nanoplatelet solids
Fluorescence resonance energy transfer (FRET) enables photosynthetic light harvesting1, wavelength down-conversion in light-emitting diodes (LEDs)2, and optical bio-sensing schemes3. The rate and efficiency of this donor to acceptor transfer of excitation between chromophores dictates the utility of FRET and can unlock new device operation motifs including quantum-funnel solar cells4, non-contact chromophore pumping from a proximal LED5, and drastically reduced gain thresholds6. However, the fastest reported FRET time constants involving spherical quantum dots (QDs) (0.12-1 ns7-9), do not outpace biexciton Auger recombination (0.01-0.1 ns)10, which impedes multiexciton-driven applications including electrically-pumped lasers11 and carrier-multiplication-enhanced photovoltaics.12,13 Few-monolayer thick semiconductor nano-platelets (NPLs) with tens-of-nanometer diameters14 exhibit intense optical transitions14 and hundreds-of-picosecond Auger recombination15,16, but heretofore lack FRET characterizations. We examine binary CdSe NPL solids and show that inter-plate FRET (~6-23 ps, presumably for co-facial arrangements) can occur 15-50 times faster than Auger recombination15,16 and demonstrate multiexcitonic FRET, making such materials ideal candidates for advanced technologies.
Rowland, Clare E.↗
Single-molecule-sensitive fluorescence resonance energy transfer in freely-diffusing attoliter droplets
Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
Optically controllable molecular logic circuits
Molecular logic circuits represent a promising technology for observation and manipulation of biological systems at the molecular level. However, the implementation of molecular logic circuits for temporal and programmable operation remains challenging. In this paper, we demonstrate an optically controllable logic circuit that uses fluorescence resonance energy transfer (FRET) for signaling. The FRET-based signaling process is modulated by both molecular and optical inputs. Based on the distance dependence of FRET, the FRET pathways required to execute molecular logic operations are formed on a DNA nanostructure as a circuit based on its molecular inputs. In addition, the FRET pathways on the DNA nanostructure are controlled optically, using photoswitching fluorescent molecules to instruct the execution of the desired operation and the related timings. The behavior of the circuit can thus be controlled using external optical signals. As an example, a molecular logic circuit capable of executing two different logic operations was studied. The circuit contains functional DNAs and a DNA scaffold to construct two FRET routes for executing Input 1 AND Input 2 and Input 1 AND NOT Input 3 operations on molecular inputs. The circuit produced the correct outputs with all possible combinations of the inputs by following the light signals. Moreover, the operation execution timings were controlled based on light irradiation and the circuit responded to time-dependent inputs. The experimental results demonstrate that the circuit changes the output for the required operations following the input of temporal light signals.
71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
Flight set 360L007 (STS-33R) case and seals, volume 2
The performance of the 360L007, Seventh Flight, redesigned Solid Rocket Motors (RSRM) is assessed with respect to case hardware and joint sealing issues as seen from post-flight assessment of the case and seals. Potential anomalies are classified and summarized. The post-flight assessment of both motors showed the case segments to be in good condition. Field joint fretting indications on this flight set ranged from none on one joint through light (less than 0.003 inch deep) on most of the joints to locally medium (greater than 0.003, less than 0.005 inch deep) on two joints. No new fretting was found in old fretting locations. All fretting was mapped from the inner clevis leg. The capture feature fretting was not mapped due to the difficulty of measuring the fretting in the capture feature region, but it is assumed that the capture feature had similar damage as the inner clevis leg. Post-flight assessment of both motors showed the seal components to be in good condition.
Carlisle, Alan R.↗
Let’s speak FRETish
FRET (https://github.com/NASA-SW-VnV/fret [github.com]) is a framework for the elicitation, formalization and analysis of requirements. FRET allows its user to enter requirements in a structured natural language called FRETish. Requirements written in FRETish are assigned unambiguous semantics. FRET supports its users in understanding this semantics and repairing requirements if applicable, by utilizing a variety of forms for each requirement: natural language description, formal mathematical logics, diagrams, and interactive simulation. FRET exports requirements into forms that can be used by a variety of analysis tools, including state-of-the-art model checkers and runtime monitoring tools. The talk will cover some of the theory behind the framework, present case studies from the aerospace and robotics domains, as well as current work on extending FRET for specifying requirements for software that learns.
FRET↗