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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein

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.

74 ATOMIC AND MOLECULAR PHYSICS↗
NASA NTRS1973

Mars: Fretted and chaotic terrains

Fretted Martian terrain is characterized by smooth, flat, lowland areas separated from a cratered upland by abrupt escarpments of complex planimetric configuration and a maximum estimated height approaching 1 to 2 km. It is the product of some unusual erosive or abstractive process that has created steep escarpments. Chaotic terrain differs from fretted terrain in having a rough floor topography featuring a haphazard jumble of large angular blocks, and by arc-shaped slump blocks on its bounding escarpments. Its existence has now been confirmed by Mariner 9 pictures, and the characteristics, location, and areal extent of chaotic terrain have been more accurately and completely defined.

Sharp, R. P.↗
NASA NTRS1976

Selected fretting-wear-resistant coatings for titanium - 6-percent-aluminum - 4-percent-vanadium alloy

A titanium - 6-percent-aluminum - 4-percent-vanadium alloy (Ti-6Al-4V) was subjected to fretting-wear exposures against uncoated Ti-6Al-4V as a baseline and against various coatings and surface treatments applied to Ti-6Al-4V. The coatings evaluated included plasma-sprayed tungsten carbide with 12 percent cobalt, aluminum oxide with 13 percent titanium oxide, chromium oxide, and aluminum bronze with 10 percent aromatic polyester; polymer-bonded polyimide, polyimide with graphite fluoride, polyimide with molybdenum disulfide (MoS2), and methyl phenyl silicone bonded MoS2, preoxidation surface treatment, a nitride surface treatment, and a sputtered MoS2 coating. Results of wear measurements on both the coated and uncoated surfaces after 300,000 fretting cycles indicated that the polyimide coating was the most wear resistant and caused the least wear to the uncoated mating surface.

Bill, R. C.↗
NASA NTRS1985

Selected fretting-wear-resistant coatings for Ti-6 pct Al-4 pct V alloy

The ability of several wear-resistant coatings to reduce fretting in the Ti-6Al-4V alloy is investigated. The experimental apparatus and procedures for evaluating fretting in uncoated Ti-6Al-4V alloy and in the alloy with plasma-sprayed coatings, polymer-bonded coating, and surface treatments are described. The wear volume and wear rate for the alloys are measured and compared. It is concluded that Al2O3 with 13 percent TiO2, preoxidation and nitride surface treatments, and MoS2 sputtering result in wear-resistant surfaces; however, the polyimide coating is the most wear resistant coating in both dry and moist air, and it causes the least wear to the uncoated alloy surface.

Bill, R. C.↗
NASA NTRSDate not supplied

From Partial to Global Assume-Guarantee Contracts: Compositional Realizability Analysis in FRET

Realizability checking refers to the formal procedure that aims to determine whether an implementation exists, always complying to a set of requirements, regardless of the stimuli provided by the system’s environment. Such a check is essential to ensure that the specification does not allow behavior that can force the system to violate safety constraints. In this paper, we present an approach that decomposes realizability checking into smaller, more tractable problems. More specifically, our approach automatically partitions specifications into sets of non-interfering requirements. We prove that checking whether a specification is realizable reduces to checking that each partition is realizable.We have integrated realizability checking and implemented our decomposition approach within the open-source Formal Requirements ElicitationTool (FRET). A FRET user may check the realizability of a specification monolithically or compositionally. We evaluate our approach by comparing monolithic and compositional checking and showcase the strengths of our decomposition approach on a variety of industrial-level case studies

Anastasia Mavridou↗
NASA NTRS2023

Using FRET to Create, Analyze and Monitor Requirements for a Lift Plus Cruise Case Study

In this technical report we provide information on the use of the NASA Formal RequirementsElicitation Tool (FRET) to create requirements for a Lift Plus Cruise (LPC) aircraft case study. Furthermore, we provide details on using FRET to translate these requirements into an appropriate format for the Copilot tool, enabling their usage to perform runtime verification on a synthesized LPC system.

Formal Requirements Elicitation Tool↗
NASA NTRSDate not supplied

Verifying PLC Programs via Monitors: Extending the Integration of FRET and PLCverif

Verification of Programmable Logic Controller (PLC) programs requires reasoning about propositions qualified in terms of time. CERN’s PLCverif, an open-source tool for the analysis of safety-critical PLC systems, uses Linear Temporal Logic (LTL) for the specification of properties. Until now, PLCverif depended on third-party tools that accept LTL specifications to perform verification. However, our experience with industrial PLC programs shows that, to overcome analysis limitations, a wide range of techniques are needed to successfully verify complex properties. In this paper, we extend PLCverif to enable PLC program verification of pure-past LTL (PLTL) safety properties with assertion-based verification tools. To this end, we take an algorithm from the runtime-monitoring domain, apply it to bounded model checking of PLC programs, and implement it in PLCverif. We extend the integration of NASA’s Formal Requirements Elicitation Tool (FRET) into PLCverif to use PLTL properties generated with FRET. In addition, we leverage the program structure induced by the PLC scan-cycle for a state-space reduction. Finally, we expose the algorithm to a real-world case study of critical systems at CERN.

Formal verification↗
DOE OSTI2015

Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

The regulation of RNA transcription is central to cellular function. Changes in gene expression drive differentiation and cellular responses to events such as injury. RNA trafficking can also have a large impact on protein expression and its localization. Thus, the ability to image RNA transcription and trafficking in real time and in living cells is a worthwhile goal that has been difficult to achieve. The availability of “light-up” aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light-up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. As a result, high background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH)

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

in situ hybridization (ISH)↗
NASA NTRS2021

Compositional Realizability Checking within FRET

A set of requirements for a reactive system is realizable if, for any sequence of inputs that satisfy the assumptions on the environment, the guarantees always hold. Realizability checking is essential to ensure that an implementation can be constructed that satisfies the requirements. We propose a framework that supports users in the non-trivial task of developing realizable requirements. Our framework uses architectural information to automatically de-compose a set of requirements into subsets that can be analyzed separately, and therefore more efficiently. It then integrates existing algorithms in order to detect unrealizability, identify minimal sets of conflicting requirements, and compute counterexamples. The capability to focus on minimal conflict sets is key for localizing and correcting the sources of unrealizability. Our approach supports this process by enabling users to interactively visualize and explore the produced conflict sets and counterexamples. We have implemented our framework in the open-source Formal Requirements Elicitation Tool (FRET), and have used it on a variety of industrial-level case studies, showcasing the strengths of our approach in terms of raw performance, as well as diagnostic potential.

FRET↗
DOE OSTI2025

Steric Modulation of Protein‐Mediated Nanoparticle Assembly: Controlling Cluster Size, Polydispersity, and FRET Responses by Rebalancing Short‐ and Long‐Range Interactions

Understanding and manipulating protein-nanoparticle interactions is of broad interest to fields ranging from nanomedicine to the biological fabrication of functional hierarchical materials. This study investigates how steric forces introduced by a pegylated derivative of superfolder green fluorescent protein (sfGFP) that is monofunctional for silica binding modulate the delicate interplay of long-range (electrostatic and van der Waals) and short-range (protein-mediated) interactions in pH-responsive silica nanoparticle (SiNP) assembly by bifunctional silica-binding sfGFP. Increasing the length of the PEG segment and pre-incubating SiNPs with increasing concentrations of pegylated proteins enables precise control over cluster size within the 800–1450 nm range with a sixfold decrease in polydispersity index to a remarkable 0.1 endpoint. Weakening short-range attractive interactions via mutagenesis extends this control to clusters in the 50–250 nm range and reveals that the Förster resonance energy transfer (FRET) efficiency of clusters scales linearly with cluster diameter below 230 nm but increases only by 15% as clusters grow to 1450 nm. Furthermore, these findings enable the development of a system that provides an optical readout to dynamic changes in solution conditions enacted by a combination of pH adjustment and ion charge screening.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2020

A novel fiber-fretting test for tribological characterization of the fiber/matrix interface

Ceramic matrix composites exhibit excellent high temperature properties and are candidate materials for structural applications from jet engines to nuclear fuel cladding. The strength and deformation behavior of these composites are intrinsically tied to the properties of the fiber/matrix interface. This research introduces a novel technique to evaluate the fundamental friction and wear characteristics of these interfaces. A case study on SiC f /PyC/SiC m composites applies in situ fiber fretting to investigate the kinetic friction coefficient and cyclic wear characteristics of the interphase. The experimental methodology is presented in explicit detail to encourage other researchers to explore the capabilities of this technique. Additionally, testing was carried out as a function of fiber roughness, PyC thickness (≈10, 500, 1200 nm), cycle count (10, 100, 1000), and frequency (1, 5, and 10 Hz). The friction coefficient and wear behavior changed with cycle length due to a transition from adhesive to abrasive mechanisms. SEM fractography provided insights to tribological evolution from adhesive sliding to three-body abrasive wear. The structural disorder at the tribo-surface relating to graphitic content and dangling bond density was investigated using Raman spectroscopy. Properties are contextualized with comparison to uniaxial tensile behavior of the parent composite.

36 MATERIALS SCIENCE↗
DOE OSTI2015

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

Significance The relationship between proteins unfolded under physiological conditions and those unfolded by chemical denaturation remains controversial. Specifically, although FRET studies suggest that unfolded proteins invariably contract with falling denaturant levels, scattering studies argue that they do not. Here, we explore the origins of this discrepancy using PEG as a negative control. Scattering indicates that, as expected, the polymer’s dimensions are denaturant-independent. The dye-labeled polymer, nevertheless, exhibits denaturant-dependent changes in measured transfer efficiency similar to those seen for unfolded proteins. This similarity raises questions regarding the interpretation of such changes as being indicative of hydrophobic or hydrogen bond-driven collapse.

Watkins, Herschel M.↗
DOE OSTI2016

FRET sensor-based quantification of intracellular trehalose in mammalian cells

Abstract Trehalose acts as a stress protectant and an autophagy inducer in mammalian cells. The molecular mechanisms of action remain obscure because intracellular trehalose at micromolar level is difficult to quantitate. Here, we show a novel trehalose monitoring technology based on FRET. FLIPsuc90μ∆1Venus sensor expressed in mammalian cells enables to quickly and non-destructively detect an infinitesimal amount of intracellular trehalose.

Kikuta, Shingo↗
DOE OSTI2019

Epigenetic biomarker screening by FLIM-FRET for combination therapy in ER+ breast cancer

Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER-dependent genes could serve as potential therapeutic targets. We described here a Fluorescence lifetime imaging-based Förster resonance energy transfer (FLIM-FRET) methodology for biomarker screening that could facilitate combination therapy based on our study. We screened 11 epigenetic-related markers which include oxidative forms of DNA methylation, histone modifications, and methyl-binding domain proteins. Among them, we identified H4K12acetylation (H4K12ac) and H3K27 acetylation (H3K27ac) as potential epigenetic therapeutic targets. When histone acetyltransferase inhibitor targeting H4K12ac and H3K27ac was combined with tamoxifen, an enhanced therapeutic outcome was observed against ER-positive breast cancer both in vitro and in vivo. Together, we demonstrate a single molecule approach as an effective screening tool for devising targeted epigenetic therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BP SS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BP SS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BP SS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BP SS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R 0 ) and fluorophore orientation factor (κ 2 ) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Verification of the Coupled Fluid/Solid Transfer in a CASL Grid-to-Rod-Fretting Simulation: A Technical Brief on the Analysis of Convergence Behavior and Demonstration of Software Tools for Verification

For a CASL grid-to-rod fretting problem, Sandia's Percept software was used in conjunction with the Sierra Mechanics suite to analyze the convergence behavior of the data transfer from a fluid simulation to a solid mechanics simulation. An analytic function, with properties relatively close to numerically computed fluid approximations, was chosen to represent the pressure solution in the fluid domain. The analytic pressure was interpolated on a sequence of grids on the fluid domain, and transferred onto a separate sequence of grids in the solid domain. The error in the resulting pressure in the solid domain was measured with respect to the analytic pressure. The error in pressure approached zero as both the fluid and solids meshes were refined. The convergence of the transfer algorithm was limited by whether the source grid resolution was the same or finer than the target grid resolution. In addition, using a feature coverage analysis, we found gaps in the solid mechanics code verification test suite directly relevant to the prototype CASL GTRF simulations.

99 GENERAL AND MISCELLANEOUS↗