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227 records · Page 13
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DOE OSTI2020

An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(β1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

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

A large accessory protein interactome is rewired across environments

To characterize how protein-protein interaction (PPI) networks change, we quantified the relative PPI abundance of 1.6 million protein pairs in the yeast Saccharomyces cerevisiae across 9 growth conditions, with replication, for a total of 44 million measurements. Our multi-condition screen identified 13,764 pairwise PPIs, a 3-fold increase over PPIs identified in one condition. A few 'immutable' PPIs are present across all conditions, while most 'mutable' PPIs are rarely observed. Immutable PPIs aggregate into highly connected 'core' network modules, with most network remodeling occurring within a loosely connected 'accessory' module. Mutable PPIs are less likely to co-express, co-localize, and be explained by simple mass action kinetics, and more likely to contain proteins with intrinsically disordered regions, implying that environment-dependent association and binding is critical to cellular adaptation. Our results show that protein interactomes are larger than previously thought and contain highly dynamic regions that reorganize to drive or respond to cellular changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Quantitation of amiodarone and N-desethylamiodarone in single HepG2 cells by single-cell printing-liquid vortex capture-mass spectrometry

Quantitative measure of a drug and its associated metabolite(s) with single-cell resolution is often limited by sampling throughput or other compromises that limit broad use. Here, we demonstrate the use of single-cell printing-liquid vortex capture-mass spectrometry (SCP-LVC-MS) to quantitatively measure the intracellular concentrations of amiodarone (AMIO) and its metabolite, N-desethylamiodarone (NDEA), from thousands of single cells across several AMIO incubation concentrations ranging from 0 to 10 μM. Concentrations obtained by SCP-LVC-MS were validated through comparison with average assays and traditional measurement of cells in bulk. Average of SCP-LVC-MS measurements and aggregate vial collection assay the concentrations differed by < 5%. Both AMIO and NDEA had clear log-normal distributions with similar standard deviation of concentrations in the cell population. The mean of both AMIO and NDEA intracellular concentrations were positively correlated with AMIO incubation concentration, increasing from 0.026 to 0.520 and 0.0055 to 0.048 mM for AMIO and NDEA, respectively. The standard deviation of AMIO and NDEA log-normal distribution fits were relatively similar in value across incubation concentrations, 0.15–0.19 log 10 (mM), and exhibited a linear trend with respect to each other. The single cell-resolved conversion ratio of AMIO to NDEA increased with decreasing incubation concentration, 7 ± 2%, 18 ± 3%, and 20 ± 7% for 10.0, 1.0, and 0.1 μM AMIO incubation concentrations, respectively. Finally, association with simultaneously measured lipids had several ions with statistically significant difference in intensity but no clear correlations with AMIO intracellular content was observed.

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

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in Journal Pre-proof chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Lastly, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification of small-molecule protein–protein interaction inhibitors for NKG2D

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ + , and CD8 + T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, in this study, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein–protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure–activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Human in vitro metabolism of an environmental mixture of polycyclic aromatic hydrocarbons (PAH) found at the Portland Harbor Superfund Site

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants that pose health risks to humans. Toxicity testing approaches of PAHs have evolved from traditional rodent models to New Approach Methodologies (NAMs), such as high-throughput screening in zebrafish, enabling rapid evaluation of chemical hazards. However, translating toxicity findings from laboratory systems to humans remains difficult due to complexity and species-specific differences. Chemical dosimetry modeling offers a quantitative framework to bridge this gap, but its accuracy depends on robust knowledge of PAH metabolism. The objective of this study was to measure human metabolism rates of Supermix-10, the ten most abundant PAHs found at the Portland Harbor Superfund Site, to support development of human pharmacokinetic models. We incubated individual PAHs from Supermix-10 in pooled human liver microsomes and quantified parent PAH disappearance using high-performance liquid chromatography (HPLC) with UV and florescent detection. To assess the potential of mixture interactions, we also measured metabolism of all 10 compounds in an equimolar mixture and compared rates of parent disappearance to those observed for individual PAHs. All Supermix-10 PAHs demonstrated rapid parent compound disappearance in human hepatic microsomes. PAHs grouped into three metabolism patterns: high metabolism rates and capacity (2-methylnaphthalene, acenaphthylene, fluorene, naphthalene), high affinity metabolism that rapidly achieves low-level saturation (benzo[a]anthracene, chrysene), and moderate metabolism rates and capacity (fluoranthene, pyrene, retene, phenanthrene). Smaller PAHs exhibited faster metabolism, and higher metabolism rates correlated inversely with molecular weight. When incubated in an equimolar mixture, Supermix-10 demonstrated significantly slower metabolism (47–89 %) compared to metabolism of individual PAHs at the same concentration. These findings enhance our understanding of PAH metabolism in humans and demonstrate significant mixture interactions under the conditions tested. Furthermore, our findings offer insights into the metabolic behavior of Supermix-10 and provide critical metabolism rate data to support the development of physiological based pharmacokinetic (PBPK) models. Dosimetry models can translate PAH chemical dosimetry from high-throughput testing platforms, like zebrafish and cellular system assays, to human exposures enhancing the accuracy and reliability of PAH risk assessments.

2-methylnaphthalene↗
DOE OSTI2020

A Preliminary Radiological Risk Assessment Model for Disposition of Remote-Handled Transuranic Wastes at Los Alamos National Laboratory Area G - 20116

The U.S. Department of Energy (DOE) operates a low-level radioactive waste (LLW) disposal site at Material Disposal Area G, in Los Alamos, New Mexico, USA. Area G has been the primary LLW disposal site for Los Alamos National Laboratory (LANL) since the 1960's. In addition to LLW, Area G is host to a variety of other wastes, the disposition of which must be determined before closure of the site. A probabilistic Radiological Risk Assessment (RRA) for Area G is used in order to support decision making regarding some wastes that are not addressed in the extant Area G Performance Assessment (PA) and Composite Analysis (CA). Between 1979 and 1987, 33 special shafts were augered into the Bandelier Tuff at Area G. This volcanic tuff is present across Pajarito Plateau on the eastern slopes of the Jemez Mountains, and varies widely in its consistency, from weakly indurated non-welded layers to welded layers that uphold the mesa cliffs of the Plateau. These mesas are home to LANL, Area G, and the townsites of Los Alamos and White Rock, with residences about 1400 m from Area G. The 33 Shafts were lined with steel casing, and contain remote-handled (RH) transuranic wastes (TRU) resulting from experiments and analysis performed in special glove boxes at the Chemistry and Metallurgy Research (CMR) facility at LANL. Some of these wastes originated as used nuclear fuel. The purpose of the Area G RRA is to evaluate the potential future risk to humans and the environment from the RH TRU in the 33 Shafts in the context of the risk associated with the surrounding wastes at Area G. The analysis is responsive to expectations outlined in DOE Order 458.1, Radiation Protection of the Public and the Environment, and is informed by the Manual and Guidance accompanying DOE O 435.1, Radioactive Waste Management. Because the waste meets the definition of TRU, the regulatory context necessarily takes into consideration the regulation governing the disposal of TRU from the U.S. Environmental Protection Agency (EPA): 40 CFR 191, Environmental Radiation Protection Standards for Management and Disposal of Spent Nuclear Fuel, High-Level and Transuranic Radioactive Wastes. Given the broader regulatory context for the RRA, the analysis is subject to different assumptions from those made in the existing DOE O 435.1 PA and CA, such as allowing for future occupation of the site. The analysis begins with a comprehensive evaluation of features, events, processes, and exposure scenarios (FEPS) for Area G and the wastes it contains. These FEPSs are screened to eliminate from further consideration those of extremely low probability and/or consequence, and a conceptual site model (CSM) is subsequently developed. The scope and structure of the Area G RRA Model is informed by this CSM, and the Area G RRA Model is developed using the GoldSim systems analysis modeling platform. This paper presents the initial version of a defensible, transparent, and reasonably realistic model, which is based on the state of knowledge of the wastes, the site, and the FEPSs that govern contaminant transport from wastes into the environment and subsequent exposures to humans and other biota. Probabilistic model input distributions represent uncertainties inherent in the real and modeled systems. The results of the Area G RRA Model inform decisions regarding the disposition of the RH TRU in the 33 Shafts. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2024

A Structurally Diverse Compound Screening Library to Identify Substrates for Diamine, Polyamine, and Related Acetyltransferases

Spermidine/spermine N-acetyltransferases (SSATs) and other types of polyamine acetyltransferases (PAATs) acetylate diamines and/or polyamines. These enzymes are evolutionarily related and belong to the Gcn5-related N-acetyltransferase (GNAT) superfamily, yet we lack a fundamental understanding of their substrate specificity and/or promiscuity toward different compounds. Many of these enzymes are known or are predicted to acetylate polyamines, but in the cell there are other types of compounds that contain moieties derived from polyamines that may be the native substrates for these enzymes. To learn more about the identity of substrates that are acetylated, we selected and screened 17 different GNAT enzymes for activity toward a set of structurally diverse compounds that contained different types of amine moieties (e.g., aminopropyl, aminobutyl, etc.). These compounds included diamines, triamines, and polyamines containing primary amino groups, and they had structural diversity with variation of the chain length and presence or absence of internal amino groups and other functional groups. We found 12 of the 17 enzymes acetylated at least one of the compounds. Some enzymes were selective toward acetylating only one compound while others exhibited substrate promiscuity toward numerous compounds. Our experimental results ultimately allowed us to pinpoint specific substrates that could be further investigated to more fully understand substrate specificity versus promiscuity of GNAT enzymes and the role of acetylated small molecules in cells.

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

Reactor and fuel cycle performance of light water reactor fuel with 235 U enrichments above 5%

Recent advances in nuclear fuel materials research, particularly on the topic of accident-tolerant fuels, have brought up potential opportunities for expanding the operating envelope of existing light water reactors. As many of the performance improvements offered by these technologies may be most fully realized by increasing fuel enrichment beyond the standard 5% limit, this paper examines the potential reactor performance and fuel cycle performance of low-enriched uranium oxide fueled light water reactors by generically considering pressurized water reactors with 235 U enrichment from 5 to 7%. Advanced cladding, including accident-tolerant cladding, has the potential to increase fuel burnup limits related to hydrogen in the cladding that coincide with those limits associated with end-of-life reactivity. Therefore, higher enrichment will be necessary in order to realize the higher fuel burnups. This work includes evaluation of the fuel cycle length, discharge burnup, reactivity coefficients, and fuel cycle performance, including radioactive waste and environmental impact metrics per unit energy generated. The analysis was performed using the evaluation metrics from the US Department of Energy Office of Nuclear Energy Fuel Cycle Evaluation and Screening Study. The reactor performance and safety analysis show that enrichments between 5 and 7% would have similar fuel temperature and moderator temperature coefficients. However, the soluble boron coefficient would decrease in magnitude, requiring more corrosive boric acid in the coolant or other methods of reactivity control during the fuel cycle. At these higher enrichments the maximum burnup at the rim of the fuel pellet would increase by almost a factor of two, which is expected to impact the formation of high-burnup structure in the fuel and the corresponding thermo-mechanical fuel properties. The fuel cycle performance assessment shows that increasing enrichment reduces the quantity of high-level waste disposed per unit energy generated, but it increases the natural resource requirements normalized to a gigawatt-electricity-per-year basis. Another impact is the slightly higher discharge burnup, resulting in somewhat different activity levels of the spent nuclear fuel and high-level waste radioactivity at 100 and 100,000 years after fuel discharge. The environmental impacts—including land use, water use, carbon emission, and radiological exposure—are of the same magnitude per unit energy generated. Finally, however, the impacts are distributed differently. Less than 5% enrichment has marginally more impact on the back-end of the fuel cycle, and greater than 5% enrichment has marginally more impact on the front-end of the fuel cycle. Ultimately, no neutronic or reactor safety hindrances to employing light water reactor fuel with enrichments greater than 5% are identified; given the achievable reactor performance benefits with advanced fuels, further practical exploration of increased enrichment fuel is recommended.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
DOE OSTI2026

Engineering and Application of a Thermostable MHETase for PET Depolymerization

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗