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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 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Rational Control of Off‐State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement**

Abstract Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non‐fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off ‐state conformations, trans1 and trans2 , located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off ‐state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high‐level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue‐ and red‐shifts of the absorption bands associated to trans1 and trans2 , respectively. Thus, due to elimination of trans2 , the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two‐fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2‐V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

A fluorescent-protein spin qubit

Quantum bits (qubits) are two-level quantum systems that support initialization, readout and coherent control1. Optically addressable spin qubits form the foundation of an emerging generation of nanoscale sensors. The engineering of these qubits has mainly focused on solid-state systems. However, fluorescent proteins, rather than exogenous fluorescent probes, have become the gold standard for in vivo microscopy because of their genetic encodability. Although fluorescent proteins possess a metastable triplet state, they have not been investigated as qubits. Here we realize an optically addressable spin qubit in enhanced yellow fluorescent protein. A near-infrared laser pulse enables triggered readout of the triplet state with up to 20% spin contrast. Using coherent microwave control of the enhanced-yellow-fluorescent-protein spin at liquid-nitrogen temperatures, we measure a (16 ± 2) μs coherence time under Carr–Purcell–Meiboom–Gill decoupling. We express the qubit in mammalian cells, maintaining contrast and coherent control despite the complex intracellular environment. Finally, we demonstrate optically detected magnetic resonance in bacterial cells at room temperature with contrast up to 8%. Our results introduce fluorescent proteins as a powerful qubit platform that paves the way for applications in the life sciences, such as nanoscale field sensing and spin-based imaging modalities.

Feder, Jacob S. [Univ. of Chicago, IL (United Stat↗
DOE OSTI2023

Resolution-enhanced X-ray fluorescence microscopy via deep residual networks

Abstract Multimodal hard X-ray scanning probe microscopy has been extensively used to study functional materials providing multiple contrast mechanisms. For instance, combining ptychography with X-ray fluorescence (XRF) microscopy reveals structural and chemical properties simultaneously. While ptychography can achieve diffraction-limited spatial resolution, the resolution of XRF is limited by the X-ray probe size. Here, we develop a machine learning (ML) model to overcome this problem by decoupling the impact of the X-ray probe from the XRF signal. The enhanced spatial resolution was observed for both simulated and experimental XRF data, showing superior performance over the state-of-the-art scanning XRF method with different nano-sized X-ray probes. Enhanced spatial resolutions were also observed for the accompanying XRF tomography reconstructions. Using this probe profile deconvolution with the proposed ML solution to enhance the spatial resolution of XRF microscopy will be broadly applicable across both functional materials and biological imaging with XRF and other related application areas.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Carbon dots from surface-capping/passivation of small carbon nanoparticles with nanoscale titanium dioxide

Carbon dots are classically defined as small carbon nanoparticles (CNPs) with effective surface passivation, which has been accomplished predominantly by surface organic functionalization. In the current work, the passivation is achieved by the surface coating of CNPs with nanoscale TiO 2 for CNP/TiO 2 core/shell nanostructures, which are analogous to conventional semiconductor core/shell quantum dots (QDs). The TiO 2 capping of CNPs results in substantial enhancements in the fluorescence quantum yields, also analogous to the similar enhancements famously known for the semiconductor QDs. In conclusion, mechanistic implications of the findings, including the associated further validation on the classical definition of carbon dots, are discussed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Amorphous Ag 2-x Cu x S quantum dots: “all-in-one” theranostic nanomedicines for near-infrared fluorescence/photoacoustics dual-modal-imaging-guided photothermal therapy

Integrating dual-modal imaging and photothermal effect within a single nanosystem is an enormous challenge for current nanomedicine application. In this study, non-toxic amorphous Ag 2-x Cu x S quantum dots (QDs) possessing near-infrared fluorescence (NIRF), enhanced photothermal and photoacoustic (PA) performance, as “one-in-all” nanomedicines, are designed and obtained. Cu doping in amorphous Ag 2 S QDs causes red-shift of NIRF to the lowest tissue absorption wavelength in so called “first biowindow” (~820 nm) and improves photothermal conversion efficiency to 44.0% simultaneously. Density functional theory simulation results suggest the fluorescence red-shift and enhanced photothermal/photoacoustics properties can be attributed to the generation of intragap states introduced by Cu doping. The amorphous Ag 2-x Cu x S QDs exhibit high long-term biocompatibility. Under a single laser irradiation with relatively low power density, an effective tumor ablation induced by high photothermal effect of amorphous Ag 2-x Cu x S QDs is achieved in vivo. Simultaneously, the PTT process can be guided by high quality NIRF/PA dual-modal-imaging.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Simultaneous enhancement of thermally activated delayed fluorescence and photoluminescence quantum yield via homoconjugation

A critical challenge facing thermally activated delayed fluorescence (TADF) is to facilitate rapid and efficient electronic transitions while ensuring a narrow singlet–triplet energy gap (ΔE ST ) in a single luminophore. We present a TADF-active iptycene that clearly demonstrates that homoconjugation can be harnessed as a viable design strategy towards answering this challenge. A homoconjugated analogue of an established quinoxaline-based TADF luminophore has been produced by fusing three of these luminophores together across a shared triptycene core. Homoconjugation was confirmed by electrochemistry, and as a direct consequence of this phenomenon we observed synergistic improvements to photoluminescence quantum yield (Φ PL ), radiative rate of singlet decay ($k^{S}_{r}$), delayed fluorescence lifetime (τ TADF ), and rate of reverse intersystem crossing (k rISC ), all while narrowing the ΔE ST . The enhancement is rationalised with TD-DFT calculations including spin–orbit coupling (SOC). A facile synthesis, the ubiquity of the pyrazine motif in state-of-the-art TADF materials of all colours, and the extent of the overall performance enhancement leads to a great potential for generality.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Lignin-based carbon quantum dots with high fluorescence performance prepared by supercritical catalysis and solvothermal treatment for tumor-targeted labeling

The poor fluorescence performance of lignin-based carbon quantum dots (L-CQDs) prepared using the bottom-up method has hindered their development. Here, in this study, a two-step strategy was proposed to efficiently enhance the fluorescence properties of L-CQDs. Lignin was first cracked using an ethanol supercritical/noble metal catalyst; then, the L-CQDs were prepared with the cracked lignin fragments as carbon precursors without adding any modified reagents. Compared with the OL-CQDs prepared by the traditional one-pot hydrothermal method, the L-CQDs-1 prepared from CL-1 containing much low molecular weight compounds, and have photoluminescence (increased from 63 to 975) and excellent up-conversion photoluminescence (enhanced by 16.3 to 963), which significantly enhanced by about 15 times and 60 times, respectively. They can emit bright blue fluorescence under both ultraviolet and near-infrared light irradiation owing to a large amount of surface defects caused by the rich compound composition. When L-CQDs-1 were combined with, and quenched by folic acid (FA), the prepared FA@L-CQDs-1 show the ability to target and label tumor cells. This study opens new avenues for the preparation of L-CQDs with high fluorescence performance using lignocellulosic material without heteroatom additives.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Subsurface Spectroscopy in Heterogeneous Materials Using Self-Healing Laser Beams

Self-healing optical beams are a class of propagation modes that can recover their beam shapes after distortion or partial blockage. This self-healing property makes them attractive for use in applications involving turbid media as they can—in theory—penetrate further into these materials than standard Gaussian beams. In this paper, we characterize the propagation of two different self-healing beams (Bessel and Airy) through a solid scattering material with different scatterer concentrations and find that both beams do recover after scattering for samples below a threshold scatterer concentration. Additionally, we test the applicability of both beam shapes for improved sub-surface spectroscopy in heterogeneous materials using fluorescent particles and find that there is an average fluorescence intensity enhancement of 1.3× using self-healing beams versus a standard Gaussian beam.

47 OTHER INSTRUMENTATION↗
DOE OSTI2021

Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well!performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full alpha-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acety coA to alpha-santalene was compartmentalized into the mitochondria, leading to a 3.7 fold improvement in the production of alpha-santalene. Furthermore, the newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. For this work, we found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5 G43R + S49T+S54P+S80A+Y88F . Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei

Abstract Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.

09 BIOMASS FUELS↗
DOE OSTI2021

Data for Cloning and Characterization of a Panel of Mitochondrial Targeting Sequences for Compartmentalization Engineering in Saccharomyces cerevisiae

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl-CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl-coA to α-santalene was compartmentalized into the mitochondria, leading to a 3.7-fold improvement in the production of α-santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

Conversion↗
DOE OSTI2022

Implementation of a Practical Teaching Course on Protein Engineering

Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.

Gomes, Luciana (ORCID:0000000289921097)↗
DOE OSTI2022

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h−1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h−1 and 2-fold higher for 128 L h−1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h−1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

Soares, Alexandra↗
DOE OSTI2023

Arabinogalactan Structures of Repetitive Serine-Hydroxyproline Glycomodule Expressed by Arabidopsis Cell Suspension Cultures

Arabinogalactan-proteins (AGPs) are members of the hydroxyproline-rich glycoprotein (HRGP) superfamily. They are heavily glycosylated with arabinogalactans, which are usually composed of a β-1,3-linked galactan backbone with 6-O-linked galactosyl, oligo-1,6-galactosyl, or 1,6-galactan side chains that are further decorated with arabinosyl, glucuronosyl, rhamnosyl, and/or fucosyl residues. Here, our work with Hyp-O-polysaccharides isolated from (Ser-Hyp)32-EGFP (enhanced green fluorescent protein) fusion glycoproteins overexpressed in transgenic Arabidopsis suspension culture is consistent with the common structural features of AGPs isolated from tobacco. In addition, this work confirms the presence of β-1,6-linkage on the galactan backbone identified previously in AGP fusion glycoproteins expressed in tobacco suspension culture. Furthermore, the AGPs expressed in Arabidopsis suspension culture lack terminal-rhamnosyl residues and have a much lower level of glucuronosylation compared with those expressed in tobacco suspension culture. These differences not only suggest the presence of distinct glycosyl transferases for AGP glycosylation in the two systems, but also indicate the existence of minimum AG structures for type II AG functional features.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Fluorescent CQD-Doped Styrene Acrylic Emulsion Coating Film with Enhanced Optical Properties

Styrene acrylic emulsions (SAEs) have emerged as a promising material for water-based coatings. However, they are still limited by their own defects in practical applications, poor weatherability, and degradation of performance at lower or higher temperatures. Here, we introduce a facile approach to producing fluorescent carbon quantum dots (CQDs) from wood processing residues and fabricating fluorescent CQD/SAE coating films via emulsion-casting. The addition of the fluorescent CQDs enhanced the optical performance of the CQD/SAE coating films. The fluorescent CQDs were prepared via a hydrothermal approach and were obtained after heating at 180 °C for 6 h at a reaction concentration of 50 mg/mL. The synthesized CQDs resulted in a high fluorescence, and the CQDs had an average size of 1.63 nm. Various concentrations of the fluorescent CQDs were doped into the SAE coating film, which improved its optical properties. We also characterized and discussed the products and then explored their optical properties. This study presents the potential of fluorescent CQD/SAE coating films for applications in anti-counterfeiting coatings, fluorescent adhesives, and papermaking.

36 MATERIALS SCIENCE↗