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NASA NTRS1983

Electron Transport in Paracoccus Halodenitrificans and the Role of Ubiquinone

The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm). When the membranes were extracted with n-pentane, NADH oxidase activity was lost. Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes. Succinate oxidation was not inhibited by dicoumarol or HQNO but was affected by ultraviolet irradiation or n-pentane extraction. However, the addition of the ubiquinone fraction to the n-pentane-extracted membranes did not restore enzyme activity. These observations suggested the reducing equivalents from succinate entered the respiratory chain on the oxygen side of the HQNO-sensitive site and probably did not proceed through a quinone.

Hochstein, L. I.↗
NASA NTRS1984

Electron transport in Paracoccus halodenitrificans and the role of Ubiquinone

The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm). When the membranes were extracted with n-pentane, NADH oxidase activity was lost. Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes. Succinate oxidation was not inhibited by dicoumarol or HQNO but was affected by ultraviolet irradiation or n-pentane extraction. However, the addition of the ubiquinone fraction to the n-pentane-extracted membranes did not restore enzyme activity. These observations suggested the reducing equivalents from succinate entered the respiratory chain on the oxygen side of the HQNO-sensitive site and probably did not proceed through a quinone.

Hochstein, L. I.↗
DOE OSTI2025

Evolution of carbapenemase activity in the class C β-lactamase ADC-1

Antibiotic resistance in bacteria poses a significant threat to public health. Among dozens of available antimicrobial agents, carbapenems are used as drugs of choice for the treatment of serious infections caused by pathogens resistant to other antibiotics. However, their usefulness has been severely compromised due to the emergence and wide spread of carbapenem-resistant clinical isolates worldwide. High-level resistance to carbapenems in bacteria is mediated by the production of β-lactamases from three molecular classes, A, B, and D, but not by class C enzymes. In this study, we selected a triple mutant of the intrinsic class C Acinetobacter-derived cephalosporinase ADC-1 (ADC-1 TM ) that confers high-level resistance to the carbapenems meropenem, ertapenem, and doripenem. Kinetic experiments demonstrated that the apparent binding affinity, along with the acylation and deacylation rates, were all improved for the mutant enzyme. X-ray crystallography, molecular docking, and molecular dynamics simulations revealed that the amino acid substitutions in ADC-1 TM produce significant changes in the enzyme active site architecture and binding mode of the carbapenem ertapenem. These changes allow for better positioning of a deacylating water for nucleophilic attack, thus explaining the significantly improved rate of ertapenem deacylation by ADC-1 TM . In this study, we showed for the first time that a class C β-lactamase can produce high-level resistance to carbapenem antibiotics, which underlines the potential for enzymes of this class to evolve such resistance and could further exacerbate the problem of antibiotic resistance in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1995

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

NASA Discipline Plant Biology↗
DOE OSTI2025

Nicotinamide Cofactor Biomimetics: Design and Structure Activity Relationships

Biocatalysis is an attractive methodology due to its sustainable metrics, combined with its vaunted regio-, chemo-, and stereoselectivity. Among biocatalysts, oxidoreductases catalyze redox transformations on a wide variety of substrates and are thus particularly attractive for synthetic applications. Many oxidoreductases depend on nicotinamide-based cofactors; however, the high cost and poor atom economy of such cofactors can negate the advantage of biosynthetic processes. The native cofactors (i.e., NADH or NADPH) can be replaced by nicotinamide cofactor biomimetics (NCBs) to address these issues. Additionally, NCBs can enhance enzyme activity due to structures with expanded redox properties. These capabilities open possibilities for reactivity and biorthogonality. Thus, the field has responded with new NCB structures, including variation in their electronic and molecular recognition properties. This perspective focuses on structure-activity relationships of simple NCBs, including their function, stability, and other properties, along with methods for their regeneration in biosynthesis.

agricultural chemistry↗
DOE OSTI2025

Comparative transcriptomics of CAZy enzymes in white- and brown-rot agaricomycetes: Evolutionary insights into lignocellulose degradation and the relevance of GH16 glycoside hydrolase functional divergence

White-rot and brown-rot fungi (WRF and BRF, respectively) decompose lignocellulose, the main structural component of plant biomass, through distinct mechanisms. This study examines the transcriptomic responses of three WRF species (Pleurotus ostreatus, Phanerochaete chrysosporium, and Heterobasidion irregulare) and two BRF species (Fomitopsis schrenkii and Rhodonia placenta) grown on poplar wood (W) and glucose (G) as sole carbon sources. RNA-seq analysis revealed upregulation of carbohydrate-active enzymes (CAZymes) linked to lignocellulose degradation. WRF displayed a broader enzymatic repertoire, whereas BRF employed a more selective approach. Among these responses, GH16 glycoside hydrolases were consistently upregulated across all species, including BRF. Since GH16 enzymes are involved in both plant hemicellulose modification and cell wall remodeling, their wood-induced expression may reflect multiple processes rather than a single conserved wood-decay mechanism. Structural and phylogenetic analyses revealed species-specific divergence, consistent with potential functional specialization. These findings broaden our understanding of fungal enzymatic strategies and highlight GH16 enzymes as candidates for further study. Beyond the immediate context of wood decay, this work has broader implications for fungal ecology, evolutionary biology, and biotechnological applications such as biomass conversion and sustainable bioenergy.

Fungal ecological strategies↗
DOE OSTI2026

Evaluating the limitations of Bayesian metabolic control analysis

Bayesian Metabolic Control Analysis (BMCA) is a promising framework for inferring metabolic control coefficients in data-limited scenarios, combining Bayesian inference with linear-logarithmic (lin-log) rate laws. These metabolic control coefficients quantify how changes in enzyme activities affect steady-state fluxes and metabolite concentrations across a metabolic network. However, its predictive accuracy and limitations remain underexplored. This study systematically evaluates BMCA’s ability to infer elasticity values, flux control coefficients (FCC), and concentration control coefficients (CCC) under varying data availability conditions using three synthetic metabolic network models. We demonstrate that BMCA predictions are highly dependent on the inclusion of flux and enzyme concentration data, with the omission of these datasets leading to severe inaccuracies. In our synthetic, enzyme-perturbation datasets, external metabolite concentrations had minimal impact and, in some cases, their exclusion improved predictions; when external-nutrient perturbations were introduced and those concentrations were observed, gains were at most modest. Additionally, we find that posterior estimation with both ADVI and HMC can underestimate large-magnitude elasticities in our synthetic settings, with ADVI showing somewhat higher variance under strong up-regulation; thus, recovering |elasticity| ≳ 1.5 remains challenging regardless of the inference engine. ADVI also fails to accurately infer allosteric interactions, even when regulatory effects are strong. While BMCA maintains reasonable accuracy in partially recovering the rankings of the highest FCC values, its estimates of absolute values remain constrained by prior assumptions and data limitations. Our findings reveal the BMCA algorithm’s strengths and weaknesses, providing guidance on its application in metabolic engineering, and highlighting the need for methodological refinements to enhance its predictive capabilities.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2015

NASA's Rodent Research Project on ISS: Validation of a New Platform for Conducting Biomedical and Basic Research into the Consequences of Long Duration Habitation in Space

Rodent research has played a key role in advancing biomedical discoveries both on Earth and in space. The National Research Counsel’s Decadal survey(1) emphasized the importance of expanding NASAs life sciences research to perform long duration, rodent experiments on the International Space Station (ISS). To accomplish this objective, flight hardware, operations, and science capabilities were developed at NASA ARC to support both commercial and government-sponsored rodent research.Rodent Research-1 (RR-1) was the first mission in which animals were delivered and maintained in the ISS for a long duration mission in modified Animal Enclosure Module hardware. Both RR validation and commercial science objectives were pursued on the RR-1 mission. Adult female mice (20 total Flight, FLT) were launched Sept 21, 2014 in RR hardware within a Dragon Capsule (SpaceX4), then after 4 days in transit, were transferred for habitation on the ISS for 17 days (commercial) or 33 days (validation), when animals were euthanized and select tissues recovered on orbit. Various controls groups consisted of: 1) Basal mice from the same cohorts as FLT mice, but tissues were recovered at time of launch, 2) Vivarium (VIV) were housed in standard cages 3) Ground Controls (GC) were housed in flight hardware within an environmental chamber at Kennedy Space Center. The health and behavior of all mice on the ISS were monitored by video feed on a daily basis. Mice were euthanized by injection of Euthasol, then either fast frozen intact or dissected to preserve livers (fast frozen) and spleens (RNAlater). Samples were stored at ≤ -80˚C until their return to Earth for later analyses.Hardware performed nominally throughout the mission and the planned in-flight science operations were completed successfully. FLT mice appeared generally more physically active on orbit than respective GC groups. After 33 days on the ISS, mean body weights of FLT mice did not differ from GC, with both groups showing a 6% rise compared to time of launch, while VIV mice showed an 8% rise over the same period. Importantly, there were no significant differences in body weights between groups at the end of 33 days on the ISS, providing an indication that the RR hardware supported the health of the mice both on Earth and in space. Based on the preliminary data obtained from the livers and spleens of mice after 17 days on the ISS, purified RNA was of high quality (RIN values of spleen: FLT=9.48 +0.40, GC=9.28 +0.44, n=5/group); therefore, RNA quality from samples retrieved on orbit was acceptable for even the most demanding transcriptomic analyses. In addition, liver enzyme activity levels (units/mg protein) of FLT mice (after 17d on ISS) and all control mice were similar in magnitude to samples that were optimally prepared by freezing in liquid nitrogen in the laboratory (enzymes analyzed included catalase, glutathione reductase and glyceraldehyde-3-phosphate dehydrogenase). Validation analyses still in progress include behavior and tissue biochemistries, as well as optimization of science return by post-flight recovery of tissues for biospecimen sharing and global expression analyses.Together, these preliminary findings demonstrate new capability for supporting long duration rodent research on the ISS to achieve both basic science and biomedical objectives.

Globus, R. K.↗
DOE OSTI2026

Three pairs of fungal Trametes strains isolated from distinct geographic origins show conserved genomic features and adaptive response to plant biomass

The genomes of white-rot fungi hold extended repertoires of enzymes active on virtually all the chemical bonds that intertwine lignocellulose polymers, and several Trametes species have been identified as powerful tools for biorefinery or bioremediation. However, only few studies have addressed the intra-species polymorphism one would expect from fungal strains collected in contrasted environments. We compared the genome sequence of pairs of strains collected in different geographic areas, for each of three fungal species. Using an updated list of the predicted functions for fungal ligno- and cellulolytic enzymes (CAZymes), we observed a high conservation of the gene repertoires among the six strains. We compared the adaptative response of the fungi grown on crystalline cellulose, wheat straw, aspen or pine sawdust by transcriptomics and secretomics. The gene regulation profiles were determined by the species and the substrates, rather than the strain. The secretomes did not show marked differences in the sets of secreted CAZymes after 3 day-growth on the substrates. We identified five transcription factor genes and two sesquiterpenoid synthesis genes induced during growth on lignocellulose. Wider studies using larger sets of strains will be necessary to evaluate the genericity of our findings, and to assess the phenotype diversity one could expect from geographic diversity as compared to taxonomic diversity in Trametes fungi.

Drula, E. [French National Research Institute for ↗
DOE OSTI2026

Harnessing plant‐based platform for low‐cost cellulosic sugar recovery from bioenergy crops

The price of pure cellulase enzyme for the recovery of fermentable cellulosic sugars is one of the major challenges that limit the commercialization of second-generation biofuels and bio-based products. This work shows a means to greatly reduce the cost of cellulases. The abundant capacity of plants to synthesize and hyperaccumulate transgenic proteins in their plastids has been demonstrated for two gene constructs using either NPT II or Tet C to regulate the expression of bacterial cellulase Cel6A. Previously, the expression of Cel6A to 20–35% of total soluble leaf protein in tobacco was shown in two consecutive field trials. Moving forward, the key challenge was the extraction of active enzymes from the transgenic tobacco leaves hyperaccumulating bacterial cellulases in a cost-effective manner. The study showed that unpurified crude extracts of NPT II and Tet C transgenic tobacco leaves recovered ~23% and ~29% w/w cellulosic sugars from energycane bagasse, respectively. A supplementation of as low as 25% of purified commercial cellulase improved the glucose recovery by 2.9 times (~85% w/w) compared to 100% crude extract, which is comparable to the glucose recovery obtained by commercial cellulases, thereby suggesting a reduced requirement of commercial cellulases leading to a cost reduction of 75% for biorefineries. Assuming a stable 40% cellulase yield in total soluble protein under field conditions and multiple harvests (one to three) a year, the study estimates that the potential cost for saccharification of 1 t of lignocellulosic biomass can be reduced to 67–200 USD by using crude leaf extracts of transgenic tobacco.

biofuel↗
DOE OSTI2024

Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks

Bioproduction from one-carbon compounds, such as formate, is an attractive prospect due to reduced energy requirements and the possibility for using CO 2 as a sustainable feedstock. Formate-fixing pathways engineered using Escherichia coli lysate-based cell-free expression (CFE) biocatalysts have the potential to route 100% of feedstock carbon toward chemical synthesis but are undermined by siphoning of in-pathway metabolites and cofactors by the CFE background metabolism. To address this limitation, we engineer a CFE-based thermophilic multienzyme biocatalyst for the synthesis of serine and glycine from formate, bicarbonate, and ammonia. After expression of the thermophilic formate-to-serine pathway in a one-pot reaction, the mesophilic E. coli CFE background machinery is removed by simple heat denaturation, eliminating the siphoning of cofactors, inpathway metabolites, and products. After bioprocess optimization, including pathway gene expression duration and chemical synthesis temperature, we achieve near stoichiometric conversion of formate and bicarbonate to serine and glycine, reaching 97% of stoichiometric yield. The use of a moderately thermophilic biocatalyst allowed chemical synthesis to take place at mesophilic temperatures, enabling the balance of optimal enzyme activity with minimal metabolite/cofactor thermal degradation. In a fed-batch experiment, the biocatalyst shows sustained chemical synthesis rates for 8 h, paving the way toward a continuous bioprocess. Finally, a sensitivity analysis of cofactor usage revealed that the most expensive cofactors, THF and NADPH, can be reduced by 5-fold without significantly lowering product yields. To the best of our knowledge, this is the first instance of expressing a thermophilic pathway in an E. coli lysate-based CFE system to generate a thermophilic biocatalyst for use at mesophilic temperatures. The CFEbased thermophilic formate-to-serine biocatalyst triples the combined serine and glycine yield previously obtained by a CFE-based mesophilic formate-to-serine biocatalyst (30%), and quadruple the yield obtained by a purified enzyme system (22%). Ultimately, this work opens the door to using E. coli lysate-based CFE for thermophilic biocatalyst generation to achieve high chemical synthesis yields.

bacteria↗
DOE OSTI2025

Modification and analysis of context-specific genome-scale metabolic models: methane-utilizing microbial chassis as a case study

ABSTRACT Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a graphical user interface that integrates COBRApy, EscherPy, and RIPTiDe, Python-based tools within the BioUML platform, and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected for Methylotuvimicrobium alcaliphilum 20Z R , a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimized i IA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggests higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1 metabolism, a homolog of the formaldehyde oxidation enzyme ( fae1-2 ), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of the tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase ( fdhAB ) and fae homologs ( fae1-2 and fae3 ) were investigated via mutagenesis. The phenotype of the f dhAB mutant followed the model prediction. Furthermore, a more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate. M. alcaliphilum 20Z R mutants lacking fae1-2 did not display any significant defects in methane or methanol-dependent growth. However, contrary to fae1, the fae1-2 homolog failed to restore the formaldehyde-activating enzyme function in complementation tests. Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes. IMPORTANCE The interrogation of various types of data is a routine strategy to explore the relationship between genotype and phenotype. An efficient approach for integrating and cross-comparing experimental multi-scale data in the context of whole-genome-based metabolic network reconstruction becomes a powerful tool that facilitates fundamental and applied research discoveries. The present study describes the reconstruction of a context-specific (CS) model for the methane-utilizing bacterium, Methylotuvimicrobium alcaliphilum 20Z R . M. alcaliphilum 20Z R is becoming an attractive microbial platform for the production of biofuels, chemicals, pharmaceuticals, and bio-sorbents for capturing atmospheric methane. We demonstrate that this pipeline can help reconstruct metabolic models that are similar to manually curated networks. Furthermore, the model is able to highlight previously overlooked pathways, thus advancing fundamental knowledge of non-model microbial systems or promoting their development toward biotechnological or environmental implementations.

Kulyashov, M. A.↗
DOE OSTI2026

Data for Zheng et al. (2025), "AquaMEND: Reconciling multiple impacts of salinization on soil carbon biogeochemistry"

Soil salinization, exacerbated by climate change, poses a global threat to coastal ecosystems and soil function. Salinity affects soil carbon cycling by directly impacting microbial activity and indirectly altering soil physicochemical properties, but current models inadequately represent these complexities. This dataset contains the observational and modeling data from Zheng et al. (2025), which described a process-based modeling framework that couples soil solution chemistry with microbial carbon cycling reactions to study the impacts of soil salinization. This conceptual model is implemented numerically into the open-source geochemical program PHREEQC 3.0 (Parkhurst and Appelo, 2013). This dataset consists of: - Figure2_AquaMEND_salinity_buffer: Contains model simulation outputs to assess the impact of three different cation exchange and surface complexation processes on salinity buffering (Fig. 2 from Zheng et al. 2025). - Figure3_Salinity_function: Contains salinity function fitting for literature data (Fig. 3 from Zheng et al. 2025). - Figure4_AquaMEND_microbial_mechanisms: Contains model simulation outputs for testing various microbial process-based hypotheses related to soil salinization, including microbial mortality, carbon use efficiency (CUE), extracellular enzyme activity, and other microbial mechanisms (Fig. 4 from Zheng et al. 2025). - Figure5_AquaMEND_Redox: Contains on model simulation outputs to evaluate shifts among key redox processes, such as aerobic respiration, sulfate reduction, and methanogenesis (Fig.5 from Zheng et al. 2025). - Figure6_AquaMEND_sorption: Contains on model simulation outputs for investigating the effects of salinity on dissolved organic matter (DOM) sorption and desorption processes (Fig. 6 from Zheng et al. 2025). - Figure7_AquaMEND_process_couple: Contains on model simulation outputs for exploring coupled biotic-abiotic processes and their interactions (Fig. 7 from Zheng et al. 2025). - data: Includes datasets used to develop salinity response functions and evaluate salinity buffering capacity. Datasets for MEND model calibration. - database: Contains the `.dat` file required by PHREEQC for model execution. - README.md: A Markdown plain text file describing the computational tools and directories. Files are a mixture of plain text CSV (comma-separated value) and plain text *.dat files written by the model; no special software is required to read them.

EARTH SCIENCE > AGRICULTURE > SOILS > SOIL SALINIT↗
NASA NTRS1989

Characteristics and Preliminary Observations of the Influence of Electromyostimulation on the Size and Function of Human Skeletal Muscle During 30 Days of Simulated Microgravity

During 30 days (d) of bedrest, the practicality of using Elec- troMyoStimulation (EMS) as a deterrent to atrophy and strength loss of lower limb musculature was examined. An EMS system was developed that provided variable but quantifiable levels of EMS, and measured torque. The dominant log of three male subjects was stimulated twice daily in a 3-d on/1-d off cycle during bedrest. The non-dominant leg of each subject acted as a control. A stimulator, using a 0.3 ms monophasic 60 Hz pulse waveform, activated muscle tissue for 4 s. The output waveform from the stimulator was sequenced to the Knee Extensors (KE), Knee Flex- ors (KF), Ankle Extensors (AE), and Ankle Flexors (AF), and caused three isometric contractions of each muscle group per minute. Subject tolerance determined EMS Intensity. Each muscle group received four 5-min bouts of EMS each session with a 10 -min rest between bouts. EMS and torque levels for each muscle action were recorded directly an a computer. Overall average EMS Intensity was 197, 197, 195, and 188 mA for the KE, KF, AF, and AE, respectively. Overall average torque development for these muscle groups was 70, 16, 12, and 27 Nm, respectively. EMS intensity doubled during the study, and average torque increased 2.5 times. Average maximum torque throughout a session reached 54% of maximal voluntary for the KE and 29% for the KF. Reductions in leg volume, muscle compartment size, cross-sectional area of slow and fast-twitch fibers, strength, and aerobic enzyme activities, and increased log compliance were attenuated in the legs which received EMS during bedrest. These results indicate that similar EMS levels induce different torques among different muscle groups and that repeated exposure to EMS increases tolerance and torque development. Longer orien- tation periods, therefore, may enhance its effectiveness. Our preliminary data suggest that the efficacy of EMS as an effective countermeasure for muscle atrophy and strength loss during long duration space travel warrants further investigation.

Duvoisin, Marc R.↗
NASA NTRS2000

Development and Application of Computational/In Vitro Toxicological Methods for Chemical Hazard Risk Reduction of New Materials for Advanced Weapon Systems

The development of quantitative structure-activity relationship (QSAR) is essential for reducing the chemical hazards of new weapon systems. The current collaboration between HEST (toxicology research and testing), MLPJ (computational chemistry) and PRS (computational chemistry, new propellant synthesis) is focusing R&D efforts on basic research goals that will rapidly transition to useful products for propellant development. Computational methods are being investigated that will assist in forecasting cellular toxicological end-points. Models developed from these chemical structure-toxicity relationships are useful for the prediction of the toxicological endpoints of new related compounds. Research is focusing on the evaluation tools to be used for the discovery of such relationships and the development of models of the mechanisms of action. Combinations of computational chemistry techniques, in vitro toxicity methods, and statistical correlations, will be employed to develop and explore potential predictive relationships; results for series of molecular systems that demonstrate the viability of this approach are reported. A number of hydrazine salts have been synthesized for evaluation. Computational chemistry methods are being used to elucidate the mechanism of action of these salts. Toxicity endpoints such as viability (LDH) and changes in enzyme activity (glutahoione peroxidase and catalase) are being experimentally measured as indicators of cellular damage. Extrapolation from computational/in vitro studies to human toxicity, is the ultimate goal. The product of this program will be a predictive tool to assist in the development of new, less toxic propellants.

Frazier, John M.↗
DOE OSTI2024

Screening a knowledge‐based library of low molecular weight compounds against the proline biosynthetic enzyme 1‐pyrroline‐5‐carboxylate 1 ( PYCR1)

Abstract Δ 1 ‐pyrroline‐5‐carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H‐dependent reduction of Δ 1 ‐pyrroline‐5‐carboxylate toL‐proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of thePYCR1gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X‐ray crystallography and kinetic assays to determine affinity parameters. The library was counter‐screened against human Δ 1 ‐pyrroline‐5‐carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 μM or lower. Among these, (S)‐tetrahydro‐2H‐pyran‐2‐carboxylic acid (70 μM) has higher affinity than the current best tool compoundN‐formyl‐l‐proline, is 30 times more specific for PYCR1 over human Δ 1 ‐pyrroline‐5‐carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure‐affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1‐hydroxyethane‐1‐sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed withl‐pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Comparative genomics provides insights into the cold adaptation of endophytic fungi associated with Deschampsia antarctica

Endophytic fungi from Deschampsia antarctica , the southernmost flowering plant, provide insights into the cold adaptation mechanisms of plant-associated fungi in extreme environments. This study presents the genome sequences and comparative analysis of eight fungal isolates from D. antarctica leaves. These Antarctic fungal isolates were analyzed alongside 121 plant-associated fungal genomes to uncover signatures of adaptation and endophytic specialization. Antarctic endophytes show striking patterns, including reduced genome size (∼26.3 Mb on average), streamlined gene content (∼8844 genes), and notably small secretomes (∼288 proteins). Despite this reduced gene repertoire, they maintain a robust set of genes encoding carbohydrate-active enzymes (CAZymes) but lack those for lignin and bacterial cell wall degradation, indicating a symbiotic lifestyle that avoids host damage and predation. One isolate, Alternaria sp. UNIPAMPA017 stood out, with 26% of its genome occupied by transposable elements. Lifestyle, rather than phylogeny, was the main driver of CAZyme and secretome profiles, underscoring ecological convergence. Compared to endophytes from Arabidopsis and Populus, D. antarctica endophytes harbor fewer pectin-degrading enzymes, reflecting their adaptation to the cell wall structure of their monocot host. Together, these fungi reveal a pattern of genomic reduction and functional fine-tuning, hallmarks of life adapted to persist in cold, nutrient-scarce niches.

Ascomycota↗