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At least 1,243 records · Page 69
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DOE OSTI2024

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr + and Olig2 + cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

CamRegBase: a gene regulation database for the biofuel crop, Camelina sativa

Abstract Camelina is an annual oilseed plant from the Brassicaceae family that is gaining momentum as a biofuel winter cover crop. However, a significant limitation in further enhancing its utility as a producer of oils that can be used as biofuels, jet fuels or bio-based products is the absence of a repository for all the gene expression and regulatory information that is being rapidly generated by the community. Here, we provide CamRegBase (https://camregbase.org/) as a one-stop resource to access Camelina information on gene expression and co-expression, transcription factors, lipid associated genes and genome-wide orthologs in the close-relative reference plant Arabidopsis. We envision this as a resource of curated information for users, as well as a repository of new gene regulation information.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Expanding the synthetic biology toolbox of Cupriavidus necator for establishing fatty acid production

Abstract The Gram-negative betaproteobacterium Cupriavidus necator is a chemolithotroph that can convert carbon dioxide into biomass. Cupriavidus necator has been engineered to produce a variety of high-value chemicals in the past. However, there is still a lack of a well-characterized toolbox for gene expression and genome engineering. Development and optimization of biosynthetic pathways in metabolically engineered microorganisms necessitates control of gene expression via functional genetic elements such as promoters, ribosome binding sites (RBSs), and codon optimization. In this work, a set of inducible and constitutive promoters were validated and characterized in C. necator, and a library of RBSs was designed and tested to show a 50-fold range of expression for green fluorescent protein (gfp). The effect of codon optimization on gene expression in C. necator was studied by expressing gfp and mCherry genes with varied codon-adaptation indices and was validated by expressing codon-optimized variants of a C12-specific fatty acid thioesterase to produce dodecanoic acid. We discuss further hurdles that will need to be overcome for C. necator to be widely used for biosynthetic processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors

Abstract Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation. We develop a novel variant of the catalytically-deactivated Cas13d enzyme dCasRx by fusing it to translation initiation factor IF3. We demonstrate dCasRx-IF3’s ability to enhance expression 21.3-fold above dCasRx when both are targeted to the start of the 5′ untranslated region of mRNA encoding red fluorescent protein in Escherichia coli. Activation of translation is location-dependent, and we show dCasRx-IF3 represses translation when targeted to the ribosomal binding site, rather than enhancing it. We provide evidence that dCasRx-IF3 targeting enhances mRNA stability relative to dCasRx, providing mechanistic insights into how this new tool functions to enhance gene expression. We also demonstrate targeted upregulation of native LacZ 2.6-fold, showing dCasRx-IF3’s ability to enhance expression of endogenous genes. dCasRx-IF3 requires no additional host modification to influence gene expression. This work outlines a novel approach, CRISPR-RNAa, for post-transcriptional control of translation to activate gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. Here, the condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1–7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in β-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A maize semi-dwarf mutant reveals a GRAS transcription factor involved in brassinosteroid signaling

Brassinosteroids (BR) and gibberellins (GA) regulate plant height and leaf angle in maize (Zea mays). Mutants with defects in BR or GA biosynthesis or signaling identify components of these pathways and enhance our knowledge about plant growth and development. In this study, we characterized three recessive mutant alleles of GRAS transcription factor 42 (gras42) in maize, a GRAS transcription factor gene orthologous to the DWARF AND LOW TILLERING (DLT) gene of rice (Oryza sativa). These maize mutants exhibited semi-dwarf stature, shorter and wider leaves, and more upright leaf angle. Transcriptome analysis revealed a role for GRAS42 as a determinant of BR signaling. Analysis of the expression consequences from loss of GRAS42 in the gras42-mu1021149 mutant indicated a weak loss of BR signaling in the mutant, consistent with its previously demonstrated role in BR signaling in rice. Loss of BR signaling was also evident by the enhancement of weak BR biosynthetic mutant alleles in double mutants of nana plant1-1 and gras42-mu1021149. The gras42-mu1021149 mutant had little effect on GA-regulated gene expression, suggesting that GRAS42 is not a regulator of core GA signaling genes in maize. Single-cell expression data identified gras42 expressed among cells in the G2/M phase of the cell cycle consistent with its previously demonstrated role in cell cycle gene expression in Arabidopsis (Arabidopsis thaliana). Cis-acting natural variation controlling GRAS42 transcript accumulation was identified by expression genome-wide association study (eGWAS) in maize. Our results demonstrate a conserved role for GRAS42/SCARECROW-LIKE 28 (SCL28)/DLT in BR signaling, clarify the role of this gene in GA signaling, and suggest mechanisms of tillering and leaf angle control by BR.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Complete replacement of Arabidopsis oil-producing enzymes with heterologous diacylglycerol acyltransferases

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) share responsibility for triacylglycerol (TAG) biosynthesis, and their selectivities control TAG fatty acid (FA) compositions. For rational metabolic engineering of seed oils, replacing endogenous TAG biosynthesis with exogenous enzymes containing different substrate FA selectivities is desirable; however, the dgat1-1/pdat1-2 double mutant is pollen lethal. Here, we evaluated the ability of 3 DGAT1s, from phylogenetically diverse plants with distinct TAG assembly processes, to completely replace endogenous TAG biosynthesis in Arabidopsis ( Arabidopsis thaliana ). We transformed dgat1-1 mutant plants with expression constructs for DGAT1 s from Camelina sativa , Physaria fendleri , and castor ( Ricinus communis ). Transgene expression was properly “contextualized” by using a previously determined minimum necessary expression unit containing the promoter/5′ UTR and first intron of native AtDGAT1 ; both of these DNA elements are essential for pollen expression. Next, we crossed homozygous lines with a DGAT1/DGAT1/PDAT1/pdat1-2 parent. C. sativa and P. fendleri DGAT1s restored the FA compositions and transcriptional differences of dgat1-1 to near wild-type and rescued the dgat1-1/pdat1-2 pollen lethality. R. communis DGAT1 was active in dgat1-1 seeds but produced unique oil profiles and alterations in the expression of lipid metabolic genes; it also failed to rescue dgat1-1/pdat1-2 lethality. This study confirms that the promoter and first intron of AtDGAT1 can modulate the expression of foreign DGAT1 genes to fit the correct spatiotemporal profile necessary for completely replacing endogenous TAG biosynthesis. Furthermore, it demonstrates an additional layer of unexpected enzyme incompatibility between oilseed lineages, which may complicate bioengineering approaches that seek to replace essential genes with orthologs.

McGuire, Sean T. [Washington State Univ., Pullman,↗
DOE OSTI2021

Bioenergy sorghum’s deep roots: A key to sustainable biomass production on annual cropland

Abstract Bioenergy sorghum has high biomass yield potential, drought resilience, good nitrogen use efficiency, and a root system that contributes to the accumulation of soil organic carbon. In this study, field grown bioenergy sorghum root systems were analyzed during the growing season to characterize their depth, biomass, morphology, anatomy, and gene expression profiles. Bioenergy sorghum roots grew continuously during a 155‐day growing season producing ~175 nodal roots, accumulating ~7 Mg of dry biomass per hectare, and reaching >2 m deep in the soil profile. Nodal roots within 20 cm of the stem were 1–5 mm in diameter, whereas roots deeper in soil profiles were enriched in lateral roots with small diameters (~30–500 µm) enabling growth through soil macropores. In older field‐grown plants, roots with intact endodermal, vascular and inner root tissues were surrounded by degraded or aerenchyma‐filled epidermal and cortical cell layers. Transcriptome analysis of nodal, surface, and deep roots identified >2,500 differentially expressed genes involved in root growth, transport, adaptation, defense, and AMF–root interaction. Deep roots (180–240 cm) differentially expressed genes that regulate lateral root growth. Surface roots (0–20 cm) located mid‐row differentially expressed genes involved in nitrate transport, whereas ammonium transport genes were expressed in surface and deep roots and genes involved in phosphate transport were expressed in nodal, surface, and deep roots. Overall, bioenergy sorghum's long growing season enables root systems to grow deeper and accumulate more biomass than annual grain crops such as maize, attributes that could help restore annual cropland soil organic carbon levels and improve soil productivity. Deep roots active in nutrient transport are positioned to take‐up fertilizer leached deep into soil profiles mitigating potential nutrient run‐off. Bioenergy sorghum's large and deep root system is a key to sustainable production of biofuels, biopower, and bioproducts on annual cropland.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Triacylglycerol, total fatty acid, and biomass accumulation of metabolically engineered energycane grown under field conditions confirms its potential as feedstock for drop‐in fuel production

Abstract Metabolic engineering for hyperaccumulation of lipids in vegetative tissues of high biomass crops promises a step change in oil yields for the production of advanced biofuels. Energycane is the ideal feedstock for this approach due to its exceptional biomass production and persistence under marginal conditions. Here, we evaluated metabolically engineered energycane with constitutive expression of the lipogenic factors WRINKLED 1 ( WRI 1), DIACYLGLYCEROL ACYLTRANSFERASE 1 ( DGAT 1), and OLEOSIN 1 ( OLE 1) for the accumulation of triacylglycerol (TAG), total fatty acid (TFA), and biomass under field conditions at the University of Florida‐IFAS experiment station near Citra, Florida. TAG and TFA accumulation were highest in leaves (up to 9.9% and 12.9% of DW, respectively), followed by juice from crushed stems, stems, and roots. TAG and TFA accumulation increased up to harvest time and correlated highest with OLE 1 and DGAT 1 expression. Biomass dry weight, TAG, and TFA content differed greatly depending on DGAT 1 and OLE 1 expression in transgenic lines with similar WRI 1 expression. Biomass did not significantly differ between WT and line L2 with DAGT 1 and OLE 1 expressed at low levels and TAG and TFA accumulating to 12‐ and 1.6‐fold that of WT leaves, respectively. In contrast, line L13, with intron‐mediated enhancement of DGAT 1 expression, displayed a 245‐ to 330‐fold increase in TAG and a 4.75‐ to 6.45‐fold increase in TFA content compared with WT leaves and a biomass reduction of 52%. These results provide the basis for developing novel feedstocks for expanding plant lipid production and point to new prospects for advanced biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bioenergy sorghum stem growth regulation: intercalary meristem localization, development, and gene regulatory network analysis

SUMMARY Bioenergy sorghum is a highly productive drought tolerant C 4 grass that accumulates 80% of its harvestable biomass in approximately 4 m length stems. Stem internode growth is regulated by development, shading, and hormones that modulate cell proliferation in intercalary meristems (IMs). In this study, sorghum stem IMs were localized above the pulvinus at the base of elongating internodes using magnetic resonance imaging, microscopy, and transcriptome analysis. A change in cell morphology/organization occurred at the junction between the pulvinus and internode where LATERAL ORGAN BOUNDARIES ( SbLOB ), a boundary layer gene, was expressed. Inactivation of an AGCVIII kinase in DDYM ( dw2 ) resulted in decreased SbLOB expression, disrupted IM localization, and reduced internode cell proliferation. Transcriptome analysis identified approximately 1000 genes involved in cell proliferation, hormone signaling, and other functions selectively upregulated in the IM compared with a non‐meristematic stem tissue. This cohort of genes is expressed in apical dome stem tissues before localization of the IM at the base of elongating internodes. Gene regulatory network analysis identified connections between genes involved in hormone signaling and cell proliferation. The results indicate that gibberellic acid induces accumulation of growth regulatory factors (GRFs) known to interact with ANGUSTIFOLIA (SbAN3), a master regulator of cell proliferation. GRF:AN3 was predicted to induce SbARF3/ETT expression and regulate SbAN3 expression in an auxin‐dependent manner. GRFs and ARFs regulate genes involved in cytokinin and brassinosteroid signaling and cell proliferation. The results provide a molecular framework for understanding how hormone signaling regulates the expression of genes involved in cell proliferation in the stem IM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

maslov-group/FUN-PROSE

mRNA levels of all genes in a genome is a critical piece of information defining the overall state of the cell in a given environmental condition. Being able to reconstruct such condition-specific expression in fungal genomes is particularly important to metabolically engineer these organisms to produce desired chemicals in industrially scalable conditions. Most previous deep learning approaches focused on predicting the average expression levels of a gene based on its promoter sequence, ignoring its variation across different conditions. Here we present FUN-PROSE—a deep learning model trained to predict differential expression of individual genes across various conditions using their promoter sequences and expression levels of all transcription factors. We train and test our model on three fungal species and get the correlation between predicted and observed condition-specific gene expression as high as 0.85. We then interpret our model to extract promoter sequence motifs responsible for variable expression of individual genes. We also carried out input feature importance analysis to connect individual transcription factors to their gene targets. A sizeable fraction of both sequence motifs and TF-gene interactions learned by our model agree with previously known biological information, while the rest corresponds to either novel biological facts or indirect correlations.

Liu, Simon↗
DOE OSTI2020

Thermal chains and entrainment in cumulus updrafts, Part 1: Theoretical description

Recent studies have shown that cumulus updrafts often consist of a succession of discrete rising thermals with spherical vortex-like circulations. Herein, a theory is developed for why this “thermal chain” structure occurs. Theoretical expressions are obtained for a passive tracer, buoyancy, and vertical velocity in axisymmetric moist updrafts. Analysis of these expressions suggests that the thermal chain structure arises from enhanced lateral mixing associated with intrusions of dry environmental air below an updraft’s vertical velocity maximum. This dry air entrainment reduces buoyancy locally. Consequently, the updraft flow above levels of locally reduced buoyancy separates from below, leading to a breakdown of the updraft into successive discrete thermals. The range of conditions in which thermal chains exist is also analyzed from the theoretical expressions. A transition in updraft structure from isolated rising thermal, to thermal chain, to starting plume occurs with increases in updraft width, environmental relative humidity, and/or convective available potential energy. Corresponding expressions for the bulk fractional entrainment rate ε are also obtained. These expressions indicate rather complicated entrainment behavior of ascending updrafts, with local enhancement of ε up to a factor of ~2 associated with the aforementioned environmental air intrusions, consistent with recent large eddy simulation (LES) studies. These locally large entrainment rates contribute significantly to overall updraft dilution in thermal chain-like updrafts, while other regions within the updraft can remain relatively undilute. Part 2 of this study compares results from the theoretical expressions to idealized numerical simulations and LES.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Linking crop traits to transcriptome differences in a progeny population of tetraploid potato

Background: Potato is the third most consumed crop in the world. Breeding for traits such as yield, product quality and pathogen resistance are main priorities. Identifying molecular signatures of these and other important traits is important in future breeding efforts. In this study, a progeny population from a cross between a breeding line, SW93–1015, and a cultivar, Désirée, was studied by trait analysis and RNA-seq in order to develop understanding of segregating traits at the molecular level and identify transcripts with expressional correlation to these traits. Transcript markers with predictive value for field performance applicable under controlled environments would be of great value for plant breeding.Results: A total of 34 progeny lines from SW93–1015 and Désirée were phenotyped for 17 different traits in a field in Nordic climate conditions and controlled climate settings. A master transcriptome was constructed with all 34 progeny lines and the parents through a de novo assembly of RNA-seq reads. Gene expression data obtained in a controlled environment from the 34 lines was correlated to traits by different similarity indices, including Pearson and Spearman, as well as DUO, which calculates the co-occurrence between high and low values for gene expression and trait. Our study linked transcripts to traits such as yield, growth rate, high laying tubers, late and tuber blight, tuber greening and early flowering. We found several transcripts associated to late blight resistance and transcripts encoding receptors were associated to Dickeya solani susceptibility. Transcript levels of a UBX-domain protein was negatively associated to yield and a GLABRA2 expression modulator was negatively associated to growth rate.Conclusion: In our study, we identify 100’s of transcripts, putatively linked based on expression with 17 traits of potato, representing both well-known and novel associations. This approach can be used to link the transcriptome to traits. We explore the possibility of associating the level of transcript expression from controlled, optimal environments to traits in a progeny population with different methods introducing the application of DUO for the first time on transcriptome data. We verify the expression pattern for five of the putative transcript markers in another progeny population.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Seed coat transcriptomic profiling of 5-593, a genotype important for genetic studies of seed coat color and patterning in common bean ( Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) market classes have distinct seed coat colors, which are directly related to the diverse flavonoids found in the mature seed coat. To understand and elucidate the molecular mechanisms underlying the regulation of seed coat color, RNA-Seq data was collected from the black bean 5-593 and used for a differential gene expression and enrichment analysis from four different seed coat color development stages. 5-593 carries dominant alleles for 10 of the 11 major genes that control seed coat color and expression and has historically been used to develop introgression lines used for seed coat genetic analysis. Pairwise comparison among the four stages identified 6,294 differentially expressed genes (DEGs) varying from 508 to 5,780 DEGs depending on the compared stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and plant hormone signal transduction comprised the principal pathways expressed during bean seed coat pigment development. Transcriptome analysis suggested that most structural genes for flavonoid biosynthesis and some potential regulatory genes were significantly differentially expressed. Further studies detected 29 DEGs as important candidate genes governing the key enzymatic flavonoid biosynthetic pathways for common bean seed coat color development. Additionally, four gene models, Pv5-593.02G016100, 593.02G078700, Pv5-593.02G090900, and Pv5-593.06G121300, encode MYB-like transcription factor family protein were identified as strong candidate regulatory genes in anthocyanin biosynthesis which could regulate the expression levels of some important structural genes in flavonoid biosynthesis pathway. These findings provide a framework to draw new insights into the molecular networks underlying common bean seed coat pigment development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Optimal production of Phanerochaete chrysosporium manganese peroxidases and Trametes sp. C30 laccase hybrid Lac131 in Aspergillus niger for lignin bioconversion

Background Incorporating the production of related ligninolytic enzymes into industrial filamentous fungus Aspergillus niger will enhance the bioconversion of lignocelluloses to various chemical products. Results In this study, transgenic expression of Phanerochaete chrysosporium manganese peroxidases (mnps) and Trametes sp. C30 laccase hybrid Lac131 (lac131) were examined and optimized in A. niger 11414 prtT∆ strain. Five mnps (mnp1, mnp2, mnp3, mnp4, and mnp5) and lac131 genes were expressed separately or in combination. The transgenic strain containing the entire mnp2 genomic coding sequence (gmnp2) exhibited the highest mnP activity among the five mnp over-expression strains in the modified minimal medium (mMM) with addition of 5 g/L bovine hemoglobin (bHg). We examined the effects of hemin and bHg on mnP production in the gmnp2 strain cultures and found that at least 1 g/L bHg was required, while hemin was not. Culture conditions for mnP production were further optimized for the gmnp2 strain and the highest mnP activities were detected in the cultures grown at 25 °C and 200 rpm with an initial pH of 4.5. Effects of soy protein, skim milk, and bovine serum albumin on mnP production were investigated; 5 g/L of soy proteins or skim milk had comparable effects to 2.5 g/L bHg, while cultures with bovine serum albumin had diminished mnP activity. Disruption of both prtT and vsm1 substantially augmented the mnP production and its activity reached 575 U/L. Trametes sp. C30 laccase hybrid lac131 was strongly expressed in either A. niger gmnp2 (1975 U/L) or 11414prtT∆ (3895 U/L) strain. Both mnP and laccase in the culture supernatants effectively decolorized selected phenolic compounds (dyes) and cleaved tagged model lignin dimers. Conclusion The mnP was successfully produced in A. niger by optimizing the culture conditions and host strain. Co-expression of all four mnp genes in the same expression host by multiplex CRISPR will lead to the mnP production reaching levels comparable to P. chrysosporium, while only requiring 36 h at 25 °C. The Lac131 activity in transgenic A. niger strain is 4- to 7-times higher than that in previous studies. Co-production of mnP and laccase in A. niger will enhance the lignin bioconversion efficiency.

Aspergillus niger↗
DOE OSTI2020

Evaluation of Thermolytic Hydrogen Generation Rate Models at High-Temperature/High-Hydroxide Regimes

This report describes the results of testing performed to extend the applicable ranges of temperature and hydroxide concentration for use within the Glycolate and Global Total Organic Carbon (TOC) Hydrogen Generation Rate (HGR) expressions. Seven experimental conditions (six simulants of the 242-25H Evaporator system chosen as a D-optimal set of experiments and a single test conducted at an elevated boiling point of 170 °C) were investigated in the presence of sodium glycolate and Xiameter TM AFE-1010. Glycolate was employed to study the extension of the Glycolate Thermolytic HGR expression while Xiameter TM AFE-1010 was employed to study the extension of the Global TOC Thermolytic HGR expression. The following conclusions were derived from this testing: The Glycolate Thermolytic HGR expression may be confidently used to predict thermolytic HGRs from glycolate at temperatures as high as 170 °C and hydroxide concentrations as high as 23 M.; The hydroxide and temperature-dependence predicted by the Global TOC Thermolytic HGR expression has been confirmed at temperatures as high as 170 °C and hydroxide concentrations as high as 23 M, suggesting that the Global TOC Thermolytic HGR expression may be used at these ranges.; Methane was observed from tests with Xiameter TM AFE-1010 at production rates higher than those observed for hydrogen. These rates were observed at temperatures higher than 100 °C.; Preliminary models suggest that increasing hydroxide/temperature causes an increase in Methane Generation Rate (MGR) from Xiameter TM AFE-1010. The following recommendations are based on this testing: The existing equations for thermolytic HGR from glycolate and non-glycolate organics should be used at Concentration, Storage, and Transfer Facilities (CSTF) storage and evaporation conditions, including temperatures and hydroxide concentrations exhibited in the 242-25H Evaporator.; Further investigation should be made into the influence of methylsilanes on CSTF flammability. This investigation should include: determination of the types of methylsilanes historically added to the CSTF, determination of methane formation rates from each type of methylsilane, and determination of the extent of degradation of methylsilanes in CSTF waste.; Characterization techniques should be developed by Savannah River National Laboratory (SRNL) to assist in the speciation of methylsilane-containing waste in the CSTF.; Additional testing with radioactive waste should be performed to determine the MGRs possible in radioactive waste and better inform model predictions made from testing with simulants.

08 HYDROGEN↗
DOE OSTI2022

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗