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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 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase

The “Histidine-brace” (His-brace) copper-binding site, composed of Cu(His) 2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV–Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.

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

Dual transposon sequencing profiles the genetic interaction landscape in bacteria

Gene redundancy complicates systematic characterization of gene function as single-gene deletions may not produce discernible phenotypes. We report dual transposon sequencing (dual Tn-seq), a platform for assaying the fitness of a comprehensive double mutant pool in parallel. Dual Tn-seq couples random barcode transposon site sequencing with the Cre-lox system, enabling deep sampling of 73% of the 1.3 million possible double gene deletions in Streptococcus pneumoniae. The genetic interactions identified span a wide range of biochemical processes, revealing new factors in presumably well-studied pathways, exemplified by a cytidine triphosphate synthase PyrJ. Moreover, this approach should permit further investigation of growth condition–specific genetic interactions. Because dual Tn-seq does not require the construction of a large array of single mutants, it should be readily adaptable to various microorganisms.

CTP synthesis↗
DOE OSTI2023

Bovine Rectoanal Junction In Vitro Organ Culture Model System to Study Shiga Toxin-Producing Escherichia coli Adherence

Studies evaluating the interactions between Shiga toxin-producing Escherichia coli O157:H7 (O157) and the bovine recto–anal junction (RAJ) have been limited to either in vitro analyses of bacteria, cells, or nucleic acids at the RAJ, providing limited information. Alternatively, expensive in vivo studies in animals have been conducted. Therefore, our objective was to develop a comprehensive in vitro organ culture system of the RAJ (RAJ-IVOC) that accurately represents all cell types present in the RAJ. This system would enable studies that yield results similar to those observed in vivo. Pieces of RAJ tissue, obtained from unrelated cattle necropsies, were assembled and subjected to various tests in order to determine the optimal conditions for assaying bacterial adherence in a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Tissue integrity was assessed using cell viability, structural cell markers, and histopathology, while the adherence of bacteria was evaluated via microscopy and culture methods. DNA fingerprinting verified the recovered bacteria against the inoculum. When the RAJ-IVOC was assembled in Dulbecco’s Modified Eagle Medium, maintained at a temperature of 39 °C with 5% CO 2 and gentle shaking for a duration of 3–4 h, it successfully preserved tissue integrity and reproduced the expected adherence phenotype of the bacteria being tested. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nucleic Acid-Based Detection Protease Activity

Proteases include clinically relevant markers for clotting disorders, certain cancers as well as toxins. Assays for protease activity often use designed peptides mimicking natural substrates and detection with colorometric and fluorescence-based detection that is difficult to multiplex without expensive and resource demanding instruments. This work demonstrates detection of proteolytic activity using PCR and sequencing-readable reporter molecules. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. Thrombin, an essential component of the clotting cascade, was used as a model system for testing peptide substrate recognition and release of a designed oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing.

Wunschel, David S [Pacific Northwest National Labo↗
DOE OSTI2023

eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’

Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from P. deltoides WV94 and hybrid poplar clone ‘52-225’ (P. trichocarpa × P. deltoides clone ‘52-225’). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in P. deltoides WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in P. deltoides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Design of a High-Assay Low-Enriched Uranium Tri-Structural Isotropic Critical Experiment for Advanced Reactor Validation

High-assay low-enriched uranium (HALEU) fuel is a key component of many small modular reactor designs. Critical experiments are an important way to understand the neutronic performance of systems by obtaining nuclear data validations through measurements. Data reduce uncertainty and risk by showing that systems respond as predicted to changes such as temperature, subsequently advancing the overall technology readiness level of the materials within. Numerous critical experiments have been performed at the National Criticality Experiments Research Center (NCERC) operated by Los Alamos National Laboratory at the Nevada National Security Site since it became operational in 2011. However, the first experiment with HALEU fuel did not occur until 2024. Through extensive engineering, the experiment described in this paper was successfully designed and executed for the Comet vertical lift assembly at NCERC to perform measurements with HALEU tri-structural isotropic fuel that will assist in validation of nuclear data and computational modeling of small modular reactors for years to come.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗
DOE OSTI2021

High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics

Membrane proteins play essential roles in cellular function and metabolism. Nonetheless, biophysical and structural studies of membrane proteins are impeded by the difficulty of their expression in and purification from heterologous cell-based systems. As an alternative to these cell-based systems, cell-free protein synthesis has proven to be an exquisite method for screening membrane protein targets in a variety of lipidic mimetics. Here we report a high-throughput screening workflow and applied it to screen 61eukaryotic membrane protein targets. For each target, we tested its expression in lipidic mimetics: two detergents, two liposomes, and two nanodiscs. We show that 35 membrane proteins (57%) can be expressed in a soluble fraction in at least one of the mimetics with the two detergents performing significantly better than nanodiscs and liposomes, in that order. Using the established cell-free workflow, we studied the production and biophysical assays for mitochondrial pyruvate carrier (MPC) complexes. Our studies show that the complexes produced in cell-free are functional competent in complex formation and substrate binding. Our results highlight the utility of using cell-free systems for screening and production of eukaryotic membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro

Persistence is a bet-hedging strategy in bacterial populations that increases antibiotic tolerance and leads to the establishment of latent infections. In this study, we demonstrated that a synthetic non-toxic taxane-based reversal agent (tRA), developed as an inhibitor of ABC transporter systems in mammalian cancer cells, enhanced antibiotic killing of persister populations from different pathogens, including Burkholderia, Pseudomonas, Francisella, and Yersinia. Acting as an inhibitor of bacterial efflux at 100 nM, tRA99020 enhanced antibiotic efficiency and suppressed the production of natural products of Burkholderia species polyketide synthase (PKS) function. We demonstrate that the metabolites produced by PKS in response to stress by different antibiotics act as inhibitors of mammalian histone deacetylase activity and stimulate cell death. Applying a single-molecule fluorescence in situ hybridization (smFISH) assay, we analyzed on a single-cell level the activation profiles of the persistence regulating pks gene in Burkholderia thailandensis treated with tRA99020 and antibiotics. We posit that a multi-pronged approach encompassing antibiotic therapies and inhibition of efflux systems and fatty acid catabolism will be required for efficient eradication of persistent bacterial populations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A rapid assay for assessing bacterial effects on Arabidopsis thermotolerance

Abstract Background The role of beneficial microbes in mitigating plant abiotic stress has received considerable attention. However, the lack of a reproducible and relatively high-throughput screen for microbial contributions to plant thermotolerance has greatly limited progress in this area, this slows the discovery of novel beneficial isolates and the processes by which they operate. Results We designed a rapid phenotyping method to assess the effects of bacteria on plant host thermotolerance. After testing multiple growth conditions, a hydroponic system was selected and used to optimize an Arabidopsis heat shock regime and phenotypic evaluation. Arabidopsis seedlings germinated on a PTFE mesh disc were floated onto a 6-well plate containing liquid MS media, then subjected to heat shock at 45 °C for various duration. To characterize phenotype, plants were harvested after four days of recovery to measure chlorophyll content. The method was extended to include bacterial isolates and to quantify bacterial contributions to host plant thermotolerance. As an exemplar, the method was used to screen 25 strains of the plant growth promoting Variovorax spp. for enhanced plant thermotolerance. A follow-up study demonstrated the reproducibility of this assay and led to the discovery of a novel beneficial interaction. Conclusions This method enables rapid screening of individual bacterial strains for beneficial effects on host plant thermotolerance. The throughput and reproducibility of the system is ideal for testing many genetic variants of Arabidopsis and bacterial strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Knocksideways in plants: an inducible system for in planta visualization of protein interactions

Understanding protein-protein interactions (PPIs) is key to unlocking the secrets of various cellular processes. While many tools have been developed to detect PPIs, such as the yeast two-hybrid assay, bimolecular fluorescence complementation, and affinity purification, there is great demand for additional methods to complement existing ones. As all approaches have limitations, evidence from at least two independent assays is required to validate PPIs. In a Breakthrough Report, Joanna Winkler and colleagues present here a promising method, named “knocksideways in plants” (KSP), for inducible visualization of PPIs in planta.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Radiation Monitoring for Volatilized Zinc Contamination Using Gamma-Ray Imaging and Spectroscopy

Gamma-ray imaging is a tool that has grown in importance in the applications of non-destructive assay (NDA) for radioactive survey and analysis of nuclear facilities. Imaging techniques have shown great promise in providing valuable information involving radioactive waste management and contamination prevention. For the application studied in this work, 65 Zn has been identified as a radioactive contaminant during tritium extraction. Due to the volatile nature of 65 Zn under the pressure and temperature changes during extraction operations, 65 Zn can easily travel through components of the extraction system as vapor, making it difficult to trap. Previous research involving the development of a filtration system showed that the 65 Zn can be trapped, mitigating product contamination. However, during the extraction process, direct analysis of the equipment to confirm that zinc contamination is trapped in the filter and has not spread to other components is impractical. Here, in this situation, the need to assay the location of the contamination with little-to-no interference with operations is vital. In this work, we demonstrate the use of a commercialized 3D position-sensitive CdZnTe (CZT) gamma-ray imaging spectrometer to provide analysis of the 65 Zn contamination. Onsite measurements during an extraction process are studied to assess the location and migration of the 65 Zn. The results obtained from real-time glovebox monitoring demonstrate the feasibility of gamma-ray imaging for localizing the contamination and providing a preliminary qualitative assessment that is intended to be used in future work quantifying the contamination build-up and activity over time.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2020

Green synthesis and characterization of nontoxic L-methionine capped silver and gold nanoparticles

The simple green method for synthesis of stable L-Methionine (L-Met) capped silver (Ag@LM NPs) and gold (Au@LM NPs) nanoparticles (NPs) without adding any additional reduction agent or stabilizer was developed. Colloidal dispersions were characterized by UV–Vis spectrophotometry. The size and spherical shape of NPs were evaluated by transmission electron microscopy. Their surface covering was confirmed by atomic force microscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and zeta potential measurements. Density functional theory calculations pointed that the preferential adsorption mode of L-Met on both Ag and Au surfaces was a vertical binding geometry via –NH2 group, while horizontal binding mode via [sbnd]S[sbnd] and –NH2 groups is also possible. The genotoxicity (evaluated by the micronucleus assay) of NPs, as well as their effects on some oxidative stress parameters (catalase activity, malondialdehyde level), were assessed in vitro using human peripheral blood cells as a model system. The influence of NPs on the morphology of lymphocyte cells studied using atomic force microscopy revealed that the membrane of cells remained unaffected after the treatment with NPs. When considering the effects of NPs on catalase activity and malondialdehyde level, neither particle type promoted oxidative stress. However, the treatment of lymphocytes with Ag@LM NPs induced a concentration-dependent enhancement of the micronuclei incidence and suppression of the cell proliferation while Au@LM NPs promoted cell proliferation, with no significant effects on micronuclei formation. The Ag@LM NPs were more prone to induce DNA damage than Au@LM NPs, which makes the latter type more suitable for further studies in nano-medicine. © 2019.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Genotype-Specific Neutralization of Norovirus Is Mediated by Antibodies Against the Protruding Domain of the Major Capsid Protein

Abstract Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

Ford-Siltz, Lauren A.↗
DOE OSTI2025

Persistence of bacterial-mediated anti-rotifer protection in preliminary outdoor cultivation trial for Microchloropsis salina

Outdoor algal cultivation systems are susceptible to a wide variety of deleterious species. In previously published studies, we observed protection using microbial consortia at laboratory scale cultures; Microchloropsis salina in the presence of microbial consortia were protected from grazing from the marine rotifer, Brachionus plicatilis. Our objective for the present work was to determine if this protection conferred by microbial consortia in controlled laboratory experiments would persist in an open, outdoor multi-liter cultivation system. We found that algal protection did persist as evidenced by the presence of fewer motile rotifers and decreased rotifer-associated egg counts for the consortia-treated outdoor cultures. Due to the low temperature and light conditions that reduced growth of the algae outdoors, we performed an indoor laboratory assay which also confirmed the persistence of algal protection. Lastly, the lower numbers of motile rotifers and fewer rotifer-associated eggs in the consortia-treated algal cultures suggests a possible protective mechanism by the consortia through interfering with the rotifer lifecycle or reproduction. Finally, these initial results support the possibility that low cost, prophylactic treatments with microbial consortia can protect algae from deleterious species in outdoor cultivation systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biosensor and optogenetics for systems biology of yeast branched-chain alcohol production and tolerance

In this project we combined synthetic biology, systems biology, protein engineering and metabolic engineering to study, control, and improve the production of branched chain alcohols (BCAs), a class of advanced biofuels preferred by the DOE, in the yeast Saccharomyces cerevisiae. This involved the development and application of optogenetic systems as a new modality of dynamic control of native and engineered metabolic pathways, using light as inducible or repressible agent. The optogenetic systems include gene circuits for light control of gene expression, as well as light-assembled synthetic organelles and photo-switchable protein binders to control metabolic and protein function with light at the protein level. In addition, we developed the first genetically encoded biosensor for BCA production in yeast, which we used to design high throughput assays to identify highly productive strains, pathways, and enzymes. We also showed that this biosensor can be functionally co-expressed with optogenetic circuits in the same strain, raising the possibility of establishing, for the first time, computer-interfaced closed-loop controls of engineered metabolic pathways. The yeast gene deletion library was also utilized to conduct the first systems-level study on BCA toxicity in yeast, which uncovered key fundamental principles of yeast sensitivity and tolerance to these alcohols, allowing us to design highly tolerant strains with increased BCA production. This project, thus comprises the development of several new technologies, which we integrated to make new discoveries on the dynamics of BCA production and their mechanisms of cellular toxicity and tolerance, as well as to establish new paradigms to engineer and control metabolic pathways and microbial fermentations with light, for the production of BCAs and other products of interest to the DOE.

2-metyl-1-butanol↗
DOE OSTI2025

New NDA Methods for Thorium Fuel Cycle Safeguards (Final Report)

This project developed portable Neutron Resonance Transmission Analysis (pNRTA) as a new non-destructive assay (NDA) method for thorium fuel cycles safeguards and other applications where multiple isotopes must be measured when present together. pNRTA leverages epithermal neutron resonances to assay multiple safeguards-relevant isotopes (e.g., 233 U and 235 U) when they are present together in a sample. Existing techniques are challenged by this task, driving the need for new active interrogation methods. With selected detectors, pNRTA works in high gamma-ray backgrounds from fission and activation products and 232 U progeny expected in thorium fuel cycle samples. This project leveraged a pNRTA system developed at Pacific Northwest National Laboratory (PNNL) and collaboration with the Massachusetts Institute of Technology (MIT). The system uses a commercially available deuterium-tritium (DT) neutron generator at short standoff (2 m). Key achievements in this project included: first-of-a-kind pNRTA quantitative measurements of 233 U oxide samples, an assessment of neutron detector technologies suitable for pNRTA in high gamma-ray background environments, experimentally demonstrating quantitative assay of samples containing 233 U and 235 U, and modeling studies showing the applicability of pNRTA to a wide range of material forms. Further, a custom algorithm was developed at MIT, which provided mean bias of 9% and relative standard deviation of 36% in assaying 233 U, 235 U, 238 U, and 232 Th content in eight measured samples. These outcomes form a solid technical basis for pNRTA as a new promising capability for international safeguards verification that is portable, non-destructive, quantitative, and isotopic specific.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2024

Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.

Silva, Vanessa (ORCID:0000000194068433)↗