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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 973 records · Page 54
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Comparative Performance Evaluation of Large Language Models for Extracting Molecular Interactions and Pathway Knowledge

Understanding the interactions and regulatory relationships among biomolecules is essential for deciphering complex biological systems and elucidating the mechanisms behind diverse biological functions. Traditionally, the collection of such molecular interaction data has relied on expert curation, a process that is both time-consuming and labor-intensive. To address these limitations, this study explores the use of large language models (LLMs) to automate the genome-scale extraction of molecular interaction knowledge. Here, we evaluate the performance of various LLMs on key biological tasks, including the identification of protein-protein interactions, detection of genes associated with pathways influenced by low-dose radiation, and inference of gene regulatory relationships. Our findings demonstrate that larger LLMs tend to perform better, particularly in extracting intricate gene and protein interactions. Despite their strengths, these models face challenges in recognizing functionally diverse gene groups and highly correlated regulatory relationships. Through a comprehensive analysis using established molecular interaction and pathway databases, we show that LLMs possess the potential to identify relevant biomolecules and predict their interactions, offering valuable insights and marking a significant step toward AI-driven biological knowledge discovery.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2013

Boosting Big National Lab Data

Introduction: Big data. Love it or hate it, solving the world’s most intractable problems requires the ability to make sense of huge and complex sets of data and do it quickly. Speeding up the process – from hours to minutes or from weeks to days – is key to our success. One major source of such big data are physical experiments. As many will know, these physical experiments are commonly used to solve challenges in fields such as energy security, manufacturing, medicine, pharmacology, environmental protection and national security. Experiments use different instruments and sensor types to research for example the validity of new drugs, the base cause for diseases, more efficient energy sources, new materials for every day goods, effective methods for environmental cleanup, the optimal ingredients composition for chocolate or determine how to preserve valuable antics. This is done by experimentally determining the structure, properties and processes that govern biological systems, chemical processes and materials. The speed and quality at which we can acquire new insights from experiments directly influences the rate of scientific progress, industrial innovation and competitiveness. And gaining new groundbreaking insights, faster, is key to the economic success of our nations. Recent years have seen incredible advances in sensor technologies, from house size detector systems in large experiments such as the Large Hadron Collider and the ‘Eye of Gaia’ billion pixel camera detector to high throughput genome sequencing. These developments have led to an exponential increase in data volumes, rates and variety produced by instruments used for experimental work. This increase is coinciding with a need to analyze the experimental results at the time they are collected. This speed is required to optimize the data taking and quality, and also to enable new adaptive experiments, where the sample is manipulated as it is observed, e.g. a substance is injected into a tissue sample and the gradual effect is observed as more of the substance is injected, providing better insights into the natural processes that are occurring, as well as result driven sampling adjustment to capture particularly interesting features --- as they emerge. The Department of Energy’s Pacific Northwest National Laboratory (PNNL) is recognized for it’s expertise in the development of new measurement techniques and their application to challenges of national importance. So it was obvious to us to address the need for in-situ analysis of large scale experimental data. We have a wide range of experimental instruments on site, in facilities such as DOE’s national scientific user facility, the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL). Commonly, scientists would create an individual analysis pipeline for each of those instruments; but even the same type of instrument would not necessarily share the same analysis tools. With the rapid increase of data volumes and rates we were facing two key challenges: how to bring a wider set of capabilities to bear to achieve in-situ analysis, and how to do so across a wide range of heterogeneous instruments at affordable costs and in a reasonable timeframe. We decided to take an unconventional approach to the problem, rather than developing customized, one-off solutions for specific instruments we wanted to explore if a more common solution could be found that would go beyond shared, basic infrastructures such as data movement and workflow engines.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2016

PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

Project Title: Immunophilins in the assembly and maintenance of photosynthetic electron transport chain in Arabidopsis Applicant: The Regents of the University of California PI: Sheng Luan, University of California at Berkeley Photosynthetic light energy conversion entails coordinated function of complex molecular machines that capture and convert light energy into chemical forms through photosynthetic electron transport chain. Each molecular machine, such as photosystem II (PSII), may consist of dozens of protein subunits and small molecule cofactors. Despite advanced understanding of the structure and function of these complexes, little is known about “How individual proteins and cofactors assemble into a functional machine and how do these molecular machines maintain their structure and function under a highly hazardous lumenal environment.” Our studies on immunophilins have unexpectedly contributed to the understanding of this question. Originally defined as cellular receptors for immunosuppressants, immunophilins have been discovered in a wide range of organisms from bacteria, fungi, plants, to animals. Immunophilins function in protein folding processes as chaperones and foldases. Arabidopsis genome encodes ca. 50 immunophilins. The most striking finding is that 16 immunophilin members are targeted to chloroplast thylakoid lumen, by far the largest group in the lumenal proteome. What is the function of immunophilins in the thylakoid lumen? Our studies have demonstrated critical roles for several immunophilins in the biogenesis and maintenance of photosynthetic complexes such as PSII. These studies have made a critical link between immunophilins and the assembly of photosynthetic machines and thus opened up a new area of research in photosynthesis. Our goal is to dissect the roles of immunophilins and their partners in the assembly and maintenance of the photosynthetic electron transport chain. The specific objectives for this funding period will be: 1. To dissect the mechanism of action for CYP38. Our studies suggest an “autoinhibitory” model for CYP38 action. We plan to test this model and determine the mechanistic details for CYP38 function by biochemical and genetic procedures. 2. To determine the mechanism of action of FKBP20-2/FKBP16-3. Studies on FBKP16-3 and FKBP20-2 suggest that they may work together as a redox-dependent duo in PSII assembly. We will test this hypothesis using biochemical and genetic tools. 3. To determine the function of lumenal FKBPs by multi-gene mutagenesis approach. Using new genetic knockout (KO) procedures especially CRISPR/CAS9 system, we plan to generate multi-gene KO models that will likely provide vital information on those FKBPs with functional redundancy. 4. Functional analysis of thylakoid lumen network. We will focus our effort on objectives 1-3. Objective 4 represents a long-term goal to establish a more comprehensive network of proteins in the thylakoid lumen and their function in the assembly and maintenance of photosynthetic complexes. I hypothesize that the 16 immunophilin-type chaperones and foldases in the thylakoid lumen constitute an “assembly line” for the various components in the photosynthetic electron transport chain. Understanding this assembly line will enable further engineering of more efficient photosynthetic machines to capture more light energy and enhance plant productivity. Furthermore, it is envisioned that such molecular “assembly line” may be rebuilt to produce artificial photosynthesis in vitro or in non-photosynthetic organisms. These are highly relevant to the mission of “Photosynthetic Systems” and “Physical Biosciences” programs to “enhance our understanding of energy capture, conversion, and/or storage.” During the funding period, we have performed a number of experiments under the proposed objectives and obtained several new results. We have found in Objective 1 that CYP38 is associated to the thylokoid membrane with a larger complex that might be PSII supercomplex. Under objective 2, we have found that FKBP16-2 interacted with PSB27 that was further pursuited and published a research article in PNAS (attached). Under Objective 3, we have identified several mutants of other FKBPs in the thyalkoid lumen that should be further studied if future funding is available. Under Objective 4, we have started to build a network of lumenal proteins that play a number of roles in photosynthesis. For example, the CYP37 and CYP28 are linked to chloroplast signaling to nucleus, critical for controlling plant response to high light and adaptation to climate change. Unfortunately these studies have been terminated due to funding shortage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Fabricated ecosystem workshop: bridging laboratory to field science. Workshop report.

Lawrence Berkeley National Laboratory (Berkeley Lab) scientists held a workshop at the DOE BER Genomic Sciences PI meeting on the use of Fabricated Ecosystems in Washington D.C. on February 25th, 2020. Ecosystem fabrication is an approach to creating controlled microbial, soil and plant ecologies within a laboratory setting that enable discovery and dissection of environmental variables, activities, and interactions. At Berkeley Lab, we are developing two systems which span spatial and temporal scales — the EcoFAB and the EcoPOD. The participants of the workshop discussed the potential applications and challenges of using fabricated ecosystems as tools to tackle BER-relevant scientific questions. Specifically, they identified (1) research challenges that would benefit from ready access to this infrastructure (2) identified and prioritized technical challenges that currently limit these systems and (3) discussed how to use them to bridge the gap between lab and field research. To ensure that the fabricated ecosystems , in particular the larger-scale EcoPODs, are of use to as much of the BER community as possible, the workshop participants made the following recommendations: 1. Produce publicly available datasets for a set of control variables. Use for benchmarking, and assessing reproducibility between systems. 2. Implement data standards. 3. Develop/leverage nano/micro sensors and in situ root imaging. 4. Encourage the development of interdisciplinary teams to develop EcoPOD experiments. 5. Develop fabricated ecosystems with size and complexity that sits between the EcoFAB and EcoPOD to accelerate use and access.

42 ENGINEERING↗
DOE OSTI2022

Predicting stress response and improved protein overproduction in Bacillus subtilis

Abstract Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis , i JT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed i JT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like i JT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Unbalancing Symbiotic Nitrogen Fixation: Can We Make Effectiveness More Effective?

One of the most critical uses of carbon fuels is in generating nitrogen fertilizer. Atmospheric dinitrogen is too stable to be used directly by plants, so plants need other chemical forms of nitrogen. Nitrogen fertilizer production uses ~4% of the world’s natural gas. Making N fertilizers leverages methane’s energy content by enabling additional energy to be captured into food and fiber through increased photosynthesis. Manufacturing fertilizer is essential—without it there would only be enough combined nitrogen to feed ~3 billion of the world’s ~7 billion people. How to alter this addiction is not obvious, but the best chance to secure substantial and sustainable amounts of future N might be through symbiotic nitrogen fixation (SNF). SNF describes mutualistic interactions where nitrogen-fixing bacteria provide fixed nitrogen to their plant hosts, freeing them from the need for nitrogen fertilizers. SNF already occurs on a large scale; "biological nitrogen” contributes about 5 Tg N/yr to American agriculture, a quarter of the nitrogen needed. Improving SNF and replacing inorganic fertilizers are both important goals in establishing sustainable and more energy-efficient farming. SNF has been used in agriculture for millennia, largely by using legumes like beans or alfalfa in mixed cropping systems. Legume SNF occurs in root nodules, organs that develop after a growing root is infected by bacteria called rhizobia. In the plant cells of a legume nodule, specialized forms of rhizobia fix nitrogen and can produce enough ammonia to supply the growing plant. The exchange of photosynthetically derived plant carbon compounds for nitrogen reduced by the bacteria is the engine that drives the symbiosis. Important details about the organization of development and metabolism in SNF remain to be determined, such as how plant and bacterial metabolism are coordinated as nitrogen is being fixed and what governs the overall level of nitrogen fixation. Most bacterial mutations that affect SNF completely block fixation (e.g. are Fix – ), providing a limited window into the feedback interactions that must be part of the process. We have investigated an unusual mutant of Sinorhizobium meliloti, a symbiotic partner of alfalfa, that fixes dinitrogen at a normal rate (i.e., it is Fix + ) but is not effective in supporting plant growth (i.e., it is ineffective or Eff – ). We have shown that the mutant has a specific mutation in the glnD gene, which codes for a protein that regulates the bacterial response to nitrogen stress, among other key pathways. A Fix + Eff – phenotype contains an unavoidable puzzle—how is it possible for the bacteria to fix nitrogen at a normal rate and without benefiting the nitrogen starved plant host? After eliminating other possibilities, we proposed that the bacteria synthesize a nitrogen-containing compound that the plant can’t use so that acquiring this compound does not relieve plant nitrogen stress. Extracts of nodules made by glnD mutant strains have high concentrations of pyruvate canaline oxime (PCO), a derivative of the plant defensive compound canaline, a toxic analog of ornithine, an amino acid. Many legumes produce canaline and the related arginine mimic canavanine. For reasons we do not yet understand, the mutation in glnD appears to trigger significant overproduction of canaline in a defense response that leads to substantial synthesis of PCO. We expected the bacteria to be able to use PCO, but we have not been able to grow S. meliloti on PCO under several conditions. PCO thus appears to be a metabolic dead end for both the plant and bacteria. We also examined bacterial catabolic pathways that degrade arginine and canavanine and showed that mutation of these did not alter the symbiosis in an obvious way. In related experiments to examine the metabolic development of root nodules, we carried out an ambitious experiment to simultaneously measure metabolites and protein changes during nodule maturation. For these experiments we used a related symbiotic interaction between S. medicae and Medicago truncatula. M. truncatula has a simpler genome than alfalfa so we could identify plant proteins based on predictions from the DNA sequence. Nodules formed on Medicago grow linearly, with the least mature tissues at the tip. Because development of nitrogen fixation is accompanied by the production of the pigmented plant oxygen carrier protein leghemoglobin, we were able to locate and manually separate the tip, transitional, and mature nitrogen-fixing regions from each other. Advances in proteomic and metabolomic techniques allowed us to analyze these tissues from a pool of as few as 10 nodules. We observed correlated transitions of various enzymes in the plant and bacterial proteomes from those characteristic of free-living metabolism to the microaerobic metabolism of the nitrogen-fixing tissues. The metabolome could not be separated into plant and bacterial domains but was consistent with this overall transition. A subsequent paper examined proteolysis in nodule bacteria. Protein turnover in mature regions of the nodule is significant. We were able to show complexes between various proteins and important proteases, using precipitation capture techniques to isolate the proteases and sensitive proteomic analysis to reveal the associated proteins.

09 BIOMASS FUELS↗
DOE OSTI2019

Protein expression and gene editing in monocots using foxtail mosaic virus vectors

Abstract Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis , and Nicotiana benthamiana . This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Insights into genetic determinants of volatile fatty acid catabolism in Cupriavidus necator H16

The soil bacterium Cupriavidus necator H16 is a promising host for upgrading waste-derived volatile fatty acids (VFAs) into renewable biochemicals. While bacterial VFA metabolic pathways are well understood, the C. necator genome encodes multiple enzymes for each catabolic step, and the degree of substrate specificity among these homologs is currently unknown. To gain insight into the catabolism of VFA substrates in C. necator, we performed transcriptomics on cells grown with acetate, propionate, butyrate, valerate, or hexanoate as the sole source of carbon and energy. These data revealed that C. necator upregulates multiple sets of genes putatively involved in substrate activation and β-oxidation in response to VFAs. To better understand this redundancy, we performed biochemical and genetic deletion studies of acyl-CoA synthetase enzymes upregulated during growth on VFA substrates. These results demonstrated the functional redundancy of the C. necator VFA catabolism and led to the identification of a gene cluster, H16_B1332-H16_B1337, that contains several genes that are important for the efficient catabolism of hexanoate. Constitutive expression of a second copy of these hexanoate catabolism genes did not improve growth of C. necator on hexanoate, suggesting that other factors (e.g., redox, transport, or toxicity) may be limiting for growth. Collectively, this work provides new insight into how C. necator uses metabolic regulation to effectively utilize VFA substrates and uncovers the important role of the gene cluster H16_B1332-H16_B1337 in the catabolism of hexanoate.

09 BIOMASS FUELS↗
DOE OSTI2023

Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene

Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has “A” at position 254 bp, whereas nDart1 homologs have “G”, which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗
DOE OSTI2025

CRISPR/Cas9 editing of p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE 1 in maize alters phenolic metabolism, lignin structure, and lignin-first biomass processing

Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. Furthermore, the reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication

Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. Here in this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (~1.5 kcal mol -1 ). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson–Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (~2.6 kcal mol -1 ), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.

36 MATERIALS SCIENCE↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗
DOE OSTI2014

Saint Joseph's University Institute for Environmental Stewardship

Task A: Examination of the physiological, morphological, and reproductive responses of Panicum virgatum (switchgrass) cultivars identified as potential biofuel producing cultivars as well as naturally-occurring varieties of switchgrass to projected changes in climate for the central portion of the United States. This project was a multi-year project set in a field site located at the Konza Prairie Biological Station near Manhattan, KS USA. The major objective of the study was to understand the physiological and growth responses of the important biofuel grass species, Panicum virgatum (switch grass) to simulated changes in precipitation expected for the Central Plains region of the United States. Population level adaptation to broad-scale regional climates or within-population variation in genome size of this genetically and phenotypically diverse C4 grass species may influence the responses to future precipitation variability associated with climate change. Therefore, we investigated switchgrass responses to water variability between natural populations collected across latitudinal gradient and populations. P. virgatum plants from natural populations originating from Kansas, Oklahoma, and Texas received frequent, small precipitation events (“ambient’) or infrequent, large precipitation events (‘altered”) to simulate contrasting rainfall variability expected from this region. We measured leaf-level physiology, aboveground biomass varied significantly by population origin but did not differ by genome size. Our results suggest that trait variation in P. virgatum is primarily attributed to population-level adaptation across latitudinal gradient, not genome size, and that neither population-level adaptation nor genome size may be important predictors of P. virgatum responses to future climatic conditions. Based solely on the data presented here, the most important consideration when deciding what varieties of switchgrass to cultivate for biofuel feedstocks under future climate scenarios is local adaptation and not necessarily genome size as has been hypothesized in the literature. Task B: Installation of an extensive green roof system on the Science Center at Saint Joseph's University for research, research-training and educational outreach activities. An experimental green roof system was designed and installed by an outside contractor (Roofmeadows) on the roof of the Science Center at Saint Joseph's University. The roof system includes four test plots, each with a different drainage system, instrumentation to monitor storm water retention, roof deck temperature, heat flux into and out of the building, rain fall, wind speed and direction, relative humidity and heat emission from the roof system. The vegetative roof was planted with 26 species of plants, distributed throughout the roof area, to assess species/variety growth and coverage characteristics, both in terms of the different drain layer systems, and in terms of the different exposures along the north to south axis of the building. Analysis of the drain layer performance, in terms of storm water retention, shows that the aggregate (stone) drainage layer system performed the best, with the moisture management mat system second, and the geotextile drain layer and reservoir sheet layer systems coming in last. This information is of value in the planning and design of vegetative roof systems since the different types of drainage layer systems have different installation costs and different weights. The different drainage layer systems also seem to be having an impact on plant growth and spread with the test plot with the reservoir sheet layer actually having the poorest plant coverage and plant spread of all areas of the roof studied. Plant growth performance analysis is ongoing, but significant differences have been observed in the third growing season ('13) along the north to south axis, with most species doing better towards the northern end of the roof (in terms of percent ground coverage and plant spread and reproduction). Interestingly, plant growth in all four of the test plots was reduced relative to the lower areas of the roof (the lower area was ca. 2 inches lower than the test plots, due to the space needed for sensors under the plots. The lower roof area uses an aggregate drain layer comparable to that in the third test plot), even when accounting for the north to south differences. The reasons for these differences are not clear and studies are underway to examine the impact of wind scour, drainage rates, temperature, and other factors. This information will be of value to planners of extensive vegetative roof systems in the Philadelphia (and broader) region, since plant growth and roof system overall performance is influenced by local climate, making broad generalizations of performance difficult. Task C: Education and community outreach efforts by the IES involving conferences at SJU, presentations by faculty and students off campus, and educational signage. The Institute for Environmental Stewardship hosted three storm water management workshops on the SJU campus in Philadelphia, in collaboration with the Lower Merion Conservancy, a not-for-profit organization located in Montgomery County, PA. These workshops were free and open to the public. The three workshops (held each year in March) drew more than 200 participants total. The presenters included local and state government agencies, not for profit organizations involved in storm water and open space preservation, designers, engineers, planners and others. Feedback was uniformly positive and we plan to continue the workshops for the foreseeable future. Educational signage has been installed at four locations on campus to explain campus infrastructure related to storm water (rain gardens, vegetative roof and green facades), as well as detailed signage installed on the Science Center roof for the vegetative roof system. More than 100 people (from in and outside of SJU) have thus far participated in tours of the roof system. A digital signage system has been installed in the adjacent library and this system provides information about the vegetative roof project and other efforts. A web camera system for the roof has also been installed and the video will be simulcast to the digital signage and with web site (www.sju.edu/ies) in the near future.

09 BIOMASS FUELS↗
DOE OSTI2016

Exascale Virtualized and Programmable Distributed Cyber Resource Control: Final Scientific Technical Report

Extreme-science drives the need for distributed exascale processing and communications that are carefully, yet flexibly, managed. Exponential growth of data for scientific simulations, experimental data, collaborative data analyses, remote visualization and GRID computing requirements of scientists in fields as diverse as high energy physics, climate change, genomics, fusion, synchrotron radiation, material science, medicine, and other scientific disciplines cannot be accommodated by simply applying existing transport protocols to faster pipes. Further, scientific challenges today demand diverse research teams, heightening the need for and increasing the complexity of collaboration. To address these issues within the network layer and physical layer, we have performed a number of research activities surrounding effective allocation and management of elastic optical network (EON) resources, particularly focusing on FlexGrid transponders. FlexGrid transponders support the opportunity to build Layer-1 connections at a wide range of bandwidths and to reconfigure them rapidly. The new flexibility supports complex new ways of using the physical layer that must be carefully managed and hidden from the scientist end-users. FlexGrid networks utilize flexible (or elastic) spectral bandwidths for each data link without using fixed wavelength grids. The flexibility in spectrum allocation brings many appealing features to network operations. Current networks are designed for the worst case impairments in transmission performance and the assigned spectrum is over-provisioned. In contrast, the FlexGrid networks can operate with the highest spectral efficiency and minimum bandwidth for the given traffic demand while meeting the minimum quality of transmission (QoT) requirement. Two primary focuses of our research are: (1) resource and spectrum allocation (RSA) for IP traffic over EONs, and (2) RSA for cross-domain optical networks. Previous work concentrates primarily on large file transfers within a single domain. Adding support for IP traffic changes the nature of the RSA problem: instead of choosing to accept or deny each request for network support, IP traffic is inherently elastic and thus lends itself to a bandwidth maximization formulation. We developed a number of algorithms that could be easily deployed within existing and new FlexGrid networks, leading to networks that better support scientific collaboration. Cross-domain RSA research is essential to support large-scale FlexGrid networks, since configuration information is generally not shared or coordinated across domains. The results presented here are in their early stages. They are technically feasible and practical, but still require coordination among organizations and equipment owners and a higher-layer framework for managing network requests.

42 ENGINEERING↗
DOE OSTI2025

Rewiring Aromatic Compound Consumption: Chromosomal Amplification and Evolution of a Foreign Pathway in Acinetobacter baylyi ADP1

Rational engineering strategies that seek to harness the remarkable diversity of microbial metabolism can be limited by incomplete biological knowledge. As described here, a novel approach to address this challenge involved replacing a native pathway for degrading lignin-derived aromatic compounds via ortho cleavage of protocatechuate in Acinetobacter baylyi ADP1 with a foreign meta-cleavage pathway that uses different enzymes, metabolites, and redox carriers. This alteration may improve lignin valorization and coordinate catabolism with bioproduction strategies. When a 14-kbp region of foreign DNA was inserted in the chromosome, the heterologous genes failed to confer growth on target substrates. Regional gene dosage was increased using a synthetic DNA fragment to promote recombination, and higher copy number enabled growth. During adaptive laboratory evolution, compensatory mutations arose that permit growth with one copy of the foreign genes. This complex metabolic remodeling was accomplished without assumptions about the impediments that initially prevented growth. To understand the changes that emerged, a novel transformation assay identified a combination of mutations sufficient for the new phenotype. Three unexpected changes were revealed: loss of one foreign enzyme, loss of one native enzyme, and loss of a two-component transcriptional regulatory system. This study establishes that large multicopy tandem arrays of poorly adapted pathway genes can confer new functions and improve understanding of metabolism.

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

The Global Spectra-Trait Initiative: A database of paired leaf spectroscopy and functional traits associated with leaf photosynthetic capacity (v1.0.0)

The Global Spectra-Trait Initiative (GSTI) aims to generate generalizable spectra trait models using reflectance data to predict leaf traits associated with the photosynthesis capacity of leaves. It comprises a synthesized dataset of leaf trait data, input datasets and code. Leaf traits include the maximum carboxylation rate of rubisco (Vcmax), the maximum electron transport rate (Jmax), the dark respiration, as well as the prediction of leaf nitrogen, leaf mass per area (LMA), and leaf water content (LWC). The dataset comprises >7500 paired observations from around 400 species from a broad range of biomes. This dataset comprises a zip file of the GSTI GitHub repository (https://github.com/plantphys/gsti), the synthesized database (.csv) and database metadata files. This dataset was updated on 2025-12-12 with minor edits to mirror the accepted manuscript version and GitHub release (Version 1.0.0 (ESSD accepted version)). Edits included minor changes to the project documentation on GitHub and removal of 12 duplicate entries from the database.

54 ENVIRONMENTAL SCIENCES↗