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90 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Integrated transcriptomic and metabolomic investigation of the genes and metabolites involved in swine follicular cyst formation

Follicular cysts are a common reproductive disorder in mammals that is usually caused by stress. However, the pathogenesis of follicular cysts in sows remains unclear. To provide new insights into the mechanisms of follicular cyst formation in pigs, we conducted a combined transcriptomic and metabolomic analysis on theca interna and mural granulosa cells of follicular cysts and mature follicles. We identified 2,533 up-regulated and 1,355 down-regulated genes in follicular cysts, compared with mature follicles. These differentially expressed genes were mainly found in signaling pathways related to tumor formation and cortisol synthesis and secretion as shown by Ingenuity Pathway Analysis, which predicted 4,362 upstream regulatory factors. The combined gene expression and pathway analysis identified the following genes as potential biomarkers for porcine follicular cysts: cytochrome P450 family 2 subfamily C polypeptide 18, L-lactate dehydrogenase, carbamoyl-phosphate synthase, fibroblast growth factor 7, integrin binding sialoprotein, interleukin 23 receptor, prolactin receptor, epiregulin, interleukin 1 receptor type II, arginine vasopressin receptor 1A, fibroblast growth factor 10, claudin 7, G Protein Subunit Gamma 3, cholecystokinin B receptor and cytosolic phospholipase A2 . Metabolomics analysis found significant differences in 87 metabolites, which were enriched in unsaturated fatty acid biosynthesis, and sphingolipid signaling pathways. These results provide valuable information on the molecular mechanisms of follicular cyst formation, which may facilitate the development of new therapeutics to prevent and treat follicular cysts.

Dai, Jiage↗
DOE OSTI2024

Transcriptomic Analysis of Arachidonic Acid Pathway Genes Provides Mechanistic Insight into Multi-Organ Inflammatory and Vascular Diseases

Arachidonic acid (AA) metabolites have been associated with several diseases across various organ systems, including the cardiovascular, pulmonary, and renal systems. Lipid mediators generated from AA oxidation have been studied to control macrophages, T-cells, cytokines, and fibroblasts, and regulate inflammatory mediators that induce vascular remodeling and dysfunction. AA is metabolized by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) to generate anti-inflammatory, pro-inflammatory, and pro-resolutory oxidized lipids. As comorbid states such as diabetes, hypertension, and obesity become more prevalent in cardiovascular disease, studying the expression of AA pathway genes and their association with these diseases can provide unique pathophysiological insights. In addition, the AA pathway of oxidized lipids exhibits diverse functions across different organ systems, where a lipid can be both anti-inflammatory and pro-inflammatory depending on the location of metabolic activity. Therefore, we aimed to characterize the gene expression of these lipid enzymes and receptors throughout multi-organ diseases via a transcriptomic meta-analysis using the Gene Expression Omnibus (GEO) Database. In our study, we found that distinct AA pathways were expressed in various comorbid conditions, especially those with prominent inflammatory risk factors. Comorbidities, such as hypertension, diabetes, and obesity appeared to contribute to elevated expression of pro-inflammatory lipid mediator genes. Our results demonstrate that expression of inflammatory AA pathway genes may potentiate and attenuate disease; therefore, we suggest further exploration of these pathways as therapeutic targets to improve outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Competitive Metabolism of Polycyclic Aromatic Hydrocarbons (PAHs): An Assessment Using In Vitro Metabolism and Physiologically Based Pharmacokinetic (PBPK) Modeling

Humans are routinely exposed to complex mixtures such as polycyclic aromatic hydrocarbons (PAHs) rather than to single compounds, as are often assessed for hazards. Cytochrome P450 enzymes (CYPs) metabolize PAHs, and multiple PAHs found in mixtures can compete as substrates for individual CYPs (e.g., CYP1A1, CYP1B1, etc.). The objective of this study was to assess competitive inhibition of metabolism of PAH mixtures in humans and evaluate a key assumption of the Relative Potency Factor approach that common human exposures will not cause interactions among mixture components. To test this objective, we co-incubated binary mixtures of benzo[a]pyrene (BaP) and dibenzo[def,p]chrysene (DBC) in human hepatic microsomes and measured rates of enzymatic BaP and DBC disappearance. We observed competitive inhibition of BaP and DBC metabolism and measured inhibition coefficients (K i ), observing that BaP inhibited DBC metabolism more potently than DBC inhibited BaP metabolism (0.061 vs. 0.44 µM K i , respectively). We developed a physiologically based pharmacokinetic (PBPK) interaction model by integrating PBPK models of DBC and BaP and incorporating measured metabolism inhibition coefficients. The PBPK model predicts significant increases in BaP and DBC concentrations in blood AUCs following high oral doses of PAHs (≥100 mg), five orders of magnitude higher than typical human exposures. We also measured inhibition coefficients of Supermix-10, a mixture of the most abundant PAHs measured at the Portland Harbor Superfund Site, on BaP and DBC metabolism. We observed similar potencies of inhibition coefficients of Supermix-10 compared to BaP and DBC. Overall, results of this study demonstrate that these PAHs compete for the same enzymes and, at high doses, inhibit metabolism and alter internal dosimetry of exposed PAHs. This approach predicts that BaP and DBC exposures required to observe metabolic interaction are much higher than typical human exposures, consistent with assumptions used when applying the Relative Potency Factor approach for PAH mixture risk assessment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evidence for Lignocellulose-Decomposing Enzymes in the Genome and Transcriptome of the Aquatic Hyphomycete Clavariopsis aquatica

Fungi are ecologically outstanding decomposers of lignocellulose. Fungal lignocellulose degradation is prominent in saprotrophic Ascomycota and Basidiomycota of the subkingdom Dikarya. Despite ascomycetes dominating the Dikarya inventory of aquatic environments, genome and transcriptome data relating to enzymes involved in lignocellulose decay remain limited to terrestrial representatives of these phyla. We sequenced the genome of an exclusively aquatic ascomycete (the aquatic hyphomycete Clavariopsis aquatica), documented the presence of genes for the modification of lignocellulose and its constituents, and compared differential gene expression between C. aquatica cultivated on lignocellulosic and sugar-rich substrates. We identified potential peroxidases, laccases, and cytochrome P450 monooxygenases, several of which were differentially expressed when experimentally grown on different substrates. Additionally, we found indications for the regulation of pathways for cellulose and hemicellulose degradation. Our results suggest that C. aquatica is able to modify lignin to some extent, detoxify aromatic lignin constituents, or both. Such characteristics would be expected to facilitate the use of carbohydrate components of lignocellulose as carbon and energy sources.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Maize Terpene Synthase 8 (ZmTPS8) Contributes to a Complex Blend of Fungal-Elicited Antibiotics

In maize (Zea mays), fungal-elicited immune responses include the accumulation of terpene synthase (TPS) and cytochrome P450 monooxygenases (CYP) enzymes resulting in complex antibiotic arrays of sesquiterpenoids and diterpenoids, including α/β-selinene derivatives, zealexins, kauralexins and dolabralexins. To uncover additional antibiotic families, we conducted metabolic profiling of elicited stem tissues in mapping populations, which included B73 × M162W recombinant inbred lines and the Goodman diversity panel. Five candidate sesquiterpenoids associated with a chromosome 1 locus spanning the location of ZmTPS27 and ZmTPS8. Heterologous enzyme co-expression studies of ZmTPS27 in Nicotiana benthamiana resulted in geraniol production while ZmTPS8 yielded α-copaene, δ-cadinene and sesquiterpene alcohols consistent with epi-cubebol, cubebol, copan-3-ol and copaborneol matching the association mapping efforts. ZmTPS8 is an established multiproduct α-copaene synthase; however, ZmTPS8-derived sesquiterpene alcohols are rarely encountered in maize tissues. A genome wide association study further linked an unknown sesquiterpene acid to ZmTPS8 and combined ZmTPS8-ZmCYP71Z19 heterologous enzyme co-expression studies yielded the same product. To consider defensive roles for ZmTPS8, in vitro bioassays with cubebol demonstrated significant antifungal activity against both Fusarium graminearum and Aspergillus parasiticus. As a genetically variable biochemical trait, ZmTPS8 contributes to the cocktail of terpenoid antibiotics present following complex interactions between wounding and fungal elicitation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Transcriptomics outputs and phylogenetic trees used for pathway discovery of diterpenoid alkaloids in Delphinium and Aconitum

Transcriptome assemblies, open reading frames in nucleotide and peptide sequences, clustered transcriptomes and corresponding amino acid files, and expression matrices in TPM and raw counts for RNA-seq datasets from Delphinium grandiflorum, Aconitum plicatum, Aconitum lycoctonum, Aconitum carmichaelii, Aconitum japonicum, Aconitum kusnezoffii, and Aconitum vilmorinianum. Also included are phylogenetic trees for terpene synthases and cytochromes P450 mined from these assemblies.

biosynthesis↗
DOE OSTI2021

Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2021

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant

Abstract Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression.

Plant Sciences↗
DOE OSTI2022

Plasma‐driven biocatalysis: In situ hydrogen peroxide production with an atmospheric pressure plasma jet increases the performance of OleT JE when compared to adding the same molar amount of hydrogen peroxide in bolus

Abstract Enzymes like fatty acid peroxygenase OleT JE are desirable enzymes for the industry. While they require inexpensive hydrogen peroxide for activity, the same hydrogen peroxide also causes overoxidation of their reactive heme center. Here, we generate hydrogen peroxide slowly in situ using the Cooperation in Science and Technology (COST)‐Jet, an atmospheric pressure plasma jet, to avoid overoxidizing OleT JE . The COST‐Jet was operated in helium with a water admixture to provide hydrogen peroxide for OleT JE activity. This helium/water admixture produced the highest enzyme turnover numbers after 2 min of treatment. These turnover numbers were even superior to using an equimolar amount of hydrogen peroxide to treat the enzymes exogenously, showing that this plasma source can provide a reliable amount of reaction mediator to support OleT JE activity.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2021

Selectivity of enzymes involved in the formation of opposite enantiomeric series of p -menthane monoterpenoids in peppermint and Japanese catnip

Peppermint (Mentha x piperita L.) and Japanese catnip (Schizonepeta tenuifolia (Benth.) Briq.) accumulate p-menthane monoterpenoids with identical functionalization patterns but opposite stereochemistry. In the present study, we investigate the enantioselectivity of multiple enzymes involved in monoterpenoid biosynthesis in these species. Based on kinetic assays, mint limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, isopiperitenone reductase, and menthone reductase exhibited significant enantioselectivity toward intermediates of the pathway that proceeds through (-)-4S-limonene. Limonene synthase, isopiperitenol dehydrogenase and isopiperitenone reductase of Japanese catnip preferred intermediates of the pathway that involves (+)-4R-limonene, whereas limonene 3-hydroxylase was not enantioselective, and the activities of pulegone reductase and menthone reductase were too low to acquire meaningful kinetic data. Molecular modeling studies with docked ligands generally supported the experimental data obtained with peppermint enzymes, indicating that the preferred enantiomer was aligned well with the requisite cofactor and amino acid residues implicated in catalysis. A striking example for enantioselectivity was peppermint (-)-menthone reductase, which binds (-)-menthone with exquisite affinity but was predicted to bind (+)-menthone in a non-productive orientation that positions its carbonyl functional group at considerable distance to the NADPH cofactor. Here, the work presented here lays the groundwork for structure-function studies aimed at unraveling how enantioselectivity evolved in closely related species of the Lamiaceae and beyond.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Comparative genome analyses suggest a hemibiotrophic lifestyle and virulence differences for the beech bark disease fungal pathogens Neonectria faginata and Neonectria coccinea

Abstract Neonectria faginata and Neonectria coccinea are the causal agents of the insect-fungus disease complex known as beech bark disease (BBD), known to cause mortality in beech forest stands in North America and Europe. These fungal species have been the focus of extensive ecological and disease management studies, yet less progress has been made toward generating genomic resources for both micro- and macro-evolutionary studies. Here, we report a 42.1 and 42.7 mb highly contiguous genome assemblies of N. faginata and N. coccinea, respectively, obtained using Illumina technology. These species share similar gene number counts (12,941 and 12,991) and percentages of predicted genes with assigned functional categories (64 and 65%). Approximately 32% of the predicted proteomes of both species are homologous to proteins involved in pathogenicity, yet N. coccinea shows a higher number of predicted mitogen-activated protein kinase genes, virulence determinants possibly contributing to differences in disease severity between N. faginata and N. coccinea. A wide range of genes encoding for carbohydrate-active enzymes capable of degradation of complex plant polysaccharides and a small number of predicted secretory effector proteins, secondary metabolite biosynthesis clusters and cytochrome oxidase P450 genes were also found. This arsenal of enzymes and effectors correlates with, and reflects, the hemibiotrophic lifestyle of these two fungal pathogens. Phylogenomic analysis and timetree estimations indicated that the N. faginata and N. coccinea species divergence may have occurred at ∼4.1 million years ago. Differences were also observed in the annotated mitochondrial genomes as they were found to be 81.7 kb (N. faginata) and 43.2 kb (N. coccinea) in size. The mitochondrial DNA expansion observed in N. faginata is attributed to the invasion of introns into diverse intra- and intergenic locations. These first draft genomes of N. faginata and N. coccinea serve as valuable tools to increase our understanding of basic genetics, evolutionary mechanisms and molecular physiology of these two nectriaceous plant pathogenic species.

Salgado-Salazar, Catalina↗
DOE OSTI2024

Aryl hydrocarbon receptor-dependent toxicity by retene requires metabolic competence

Polycyclic aromatic hydrocarbons (PAHs) are a class of organic compounds frequently detected in the environment with widely varying toxicities. Many PAHs activate the aryl hydrocarbon receptor (AHR), inducing the expression of a battery of genes, including xenobiotic metabolizing enzymes like cytochrome P450s (CYPs); however, not all PAHs act via this mechanism. We screened several parent and substituted PAHs in in vitro AHR activation assays to classify their unique activity. Retene (1-methyl-7-isopropylphenanthrene) displays Ahr2-dependent teratogenicity in zebrafish, but did not activate human AHR or zebrafish Ahr2, suggesting a retene metabolite activates Ahr2 in zebrafish to induce developmental toxicity. To investigate the role of metabolism in retene toxicity, studies were performed to determine the functional role of cyp1a, cyp1b1, and the microbiome in retene toxicity, identify the zebrafish window of susceptibility, and measure retene uptake, loss, and metabolite formation in vivo. Cyp1a-null fish were generated using CRISPR-Cas9. Cyp1a-null fish showed increased sensitivity to retene toxicity, whereas Cyp1b1-null fish were less susceptible, and microbiome elimination had no significant effect. Zebrafish required exposure to retene between 24 and 48 hours post fertilization (hpf) to exhibit toxicity. After static exposure, retene concentrations in zebrafish embryos increased until 24 hpf, peaked between 24 and 36 hpf, and decreased rapidly thereafter. We detected retene metabolites at 36 and 48 hpf, indicating metabolic onset preceding toxicity. This study highlights the value of combining molecular and systems biology approaches with mechanistic and predictive toxicology to interrogate the role of biotransformation in AHR-dependent toxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

DNA adducts form in mouse lung and liver after oral naphthalene exposure

Naphthalene is a ubiquitous combustion product and environmental contaminant with known human exposure. Chronic exposure to naphthalene vapor leads to respiratory tumor formation in rodents. Naphthalene forms DNA adducts (precursors to genotoxicity) in tissue explants but it is unclear if this occurs in vivo. Wild-type C57BL/6 mice were orally exposed to 50 mg/kg 14 C-naphthalene. Naphthalene-DNA adducts were detected by accelerator mass spectrometry at 2- to 72-h post-exposure in both lung and liver, with decreasing abundance over time. Adducts persisted even at 72-h after exposure, which indicates possible evasion of DNA repair and potential to contribute to mutagenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Plant terpene specialized metabolism: complex networks or simple linear pathways?

From the perspectives of pathway evolution, discovery and engineering of plant specialized metabolism, the nature of the biosynthetic routes represents a critical aspect. Classical models depict biosynthesis typically from an end-point angle and as linear, for example, connecting central and specialized metabolism. As the number of functionally elucidated routes increased, the enzymatic foundation of complex plant chemistries became increasingly well understood. The perception of linear pathway models has been severely challenged. With a focus on plant terpenoid specialized metabolism, we review here illustrative examples supporting that plants have evolved complex networks driving chemical diversification. The completion of several diterpene, sesquiterpene and monoterpene routes shows complex formation of scaffolds and their subsequent functionalization. These networks show that branch points, including multiple sub-routes, mean that metabolic grids are the rule rather than the exception. This concept presents significant implications for biotechnological production.

60 APPLIED LIFE SCIENCES↗