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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

NASA Discipline Plant Biology↗
NASA NTRS1999

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

Non-NASA Center↗
DOE OSTI2020

Chemical changes in thirdhand smoke associated with remediation using an ozone generator

Ozonation is a common remediation approach to eliminate odors from mold, tobacco and fire damage in buildings. Little information exists to: 1) assess its effectiveness; 2) provide guidance on operation conditions; and 3) identify potential risks associated with the presence of indoor ozone and ozonation byproducts. The goal of this study is to evaluate chemical changes in thirdhand smoke (THS) aerosols induced by high levels of ozone, in comparison with THS aerosols aged under similar conditions in the absence of ozone. Samples representing different stages of smoke aging in the absence of ozone, including freshly emitted secondhand smoke (SHS) and THS, were collected inside an 18-m 3 room-sized chamber over a period of 42 h after six cigarettes were consumed. The experiments involved collection and analysis of gas phase species including volatile organic compounds (VOCs), volatile carbonyls, semivolatile organic compounds (SVOCs), and particulate matter. VOC analysis was carried out by gas chromatography/mass spectrometry with a thermal desorption inlet (TD-GC/MS), and volatile carbonyls were analyzed by on-line derivatization with dinitrophenylhydrazine (DNPH), followed by liquid chromatography with UV/VIS detection. SVOCs were extracted from XAD-coated denuders and Teflon-coated fiberglass filters in the absence of ozone. In those extracts, tobacco-specific nitrosamines (TSNAs) and other SVOCs were analyzed by gas chromatography with positive chemical ionization-triple quadrupole mass spectrometric detection (GC/PCI-QQQ-MS), and polycyclic aromatic hydrocarbons (PAHs) were quantified by gas chromatography with ion trap mass spectrometric detection (GC/IT-MS) in selected ion monitoring mode. Particulate matter concentration was determined gravimetrically. In a second experiment, a 300 mg h -1 commercial ozone generator was operated during 1 h, one day after smoke was generated, to evaluate the remediation of THS by ozonation. VOCs and volatile carbonyls were analyzed before and after ozonation. Extracts from fabrics that were exposed in the chamber before and after ozonation as surrogates for indoor furnishings were analyzed by GC/IT-MS, and aerosol size distribution was studied with a scanning mobility particle sizer. Ozone concentration was measured with a photometric detector. An estimated 175 mg ozone reacted with THS after 1 h of treatment, corresponding to 58% of the total O 3 released during that period. Fabric-bound nicotine was depleted after ozonation, and the surface concentration of PAHs adsorbed to fabric specimens decreased by an order of magnitude due to reaction with ozone, reaching pre-smoking levels. These results suggest that ozonation has the potential to remove harmful THS chemicals from indoor surfaces. However, gas phase concentrations of volatile carbonyls, including formaldehyde, acetaldehyde and acetone were higher immediately after ozonation. Ultrafine particles (UFP, in most cases with size <60 nm) were a major ozonation byproduct. UFP number concentrations peaked shortly after ozonation ended, and remained at higher-than background levels for several hours. Based on these results, minimum re-entry times after ozone treatment were predicted for different indoor scenarios. Clearly defining re-entry times can serve as a practical measure to prevent acute exposures to ozone and harmful ozonation byproducts after treatment. This study evaluated potential benefits and risks associated with THS remediation using ozone, providing insights into this technology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Uncovering novel mutational signatures by de novo extraction with SigProfilerExtractor

Mutational signature analysis is commonly performed in cancer genomic studies. Here, we present SigProfilerExtractor, an automated tool for de novo extraction of mutational signatures, and benchmark it against another 13 bioinformatics tools by using 34 scenarios encompassing 2,500 simulated signatures found in 60,000 synthetic genomes and 20,000 synthetic exomes. For simulations with 5% noise, reflecting high-quality datasets, SigProfilerExtractor outperforms other approaches by elucidating between 20% and 50% more true-positive signatures while yielding 5-fold less false-positive signatures. Applying SigProfilerExtractor to 4,643 whole-genome- and 19,184 whole-exome-sequenced cancers reveals four novel signatures. Two of the signatures are confirmed in independent cohorts, and one of these signatures is associated with tobacco smoking. In summary, this report provides a reference tool for analysis of mutational signatures, a comprehensive benchmarking of bioinformatics tools for extracting signatures, and several novel mutational signatures, including one putatively attributed to direct tobacco smoking mutagenesis in bladder tissues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Nicotine Monitoring with a Wearable Sweat Band

The tobacco epidemic is a public health threat that has taken a heavy toll of lives around the globe each year. Smoking affects both the smokers and those who are exposed to secondhand smoke, and careful tracking of exposure can be key to mitigating the potential hazards. For smokers, the variation of chemical compositions between commercial cigarettes has led to ambiguity in estimating the health risks, both for active smokers and others involuntarily exposed to tobacco smoke and byproducts. In this regard, sweat possesses an attractive opportunity to monitor smoke exposure due to sweat's abundance in biomolecules and its great accessibility. In this paper, we present a wearable sweat band to monitor nicotine, a prominent ingredient in cigarettes, as a viable way to quantitatively assess a wearer's exposure to smoking. Both smokers and normal subjects are tested to demonstrate the use of this device for smoke-related health monitoring. Our results exhibit confirmable and elevated nicotine levels in sweat for subjects inhaling cigarette smoke. This continuous and personalized sweat sensing device is leverage to monitor smoke pollution for a potentially broad population.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Triacylglycerol stability limits futile cycles and inhibition of carbon capture in oil-accumulating leaves

Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g. oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered tobacco (Nicotiana tabacum) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14 CO 2 tracking describe metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEAFY COTYLEDON 2 (LEC2) line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested interference of the glyoxylate cycle with photorespiration that limited CO 2 assimilation by 50%. In contrast, inclusion of the LEC2 transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO 2 assimilation and plant growth comparable to wild type but with much higher lipid levels in leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Transposon Signatures of Allopolyploid Genome Evolution

Hybridization brings together chromosome sets from two or more distinct progenitor species. Genome duplication associated with hybridization, or allopolyploidy, allows these chromosome sets to persist as distinct subgenomes during subsequent meioses. Here, we present a general method for identifying the subgenomes of a polyploid based on shared ancestry as revealed by the genomic distribution of repetitive elements that were active in the progenitors. This subgenome-enriched transposable element signal is intrinsic to the polyploid, allowing broader applicability than other approaches that depend on the availability of sequenced diploid relatives. We develop the statistical basis of the method, demonstrate its applicability in the well-studied cases of tobacco, cotton, and Brassica napus, and apply it to several cases: allotetraploid cyprinids, allohexaploid false flax, and allooctoploid strawberry. These analyses provide insight into the origins of these polyploids, revise the subgenome identities of strawberry, and provide perspective on subgenome dominance in higher polyploids.

Genomics↗
DOE OSTI2022

Functional analysis of Pogostemon cablin farnesyl pyrophosphate synthase gene and its binding transcription factor PcWRKY44 in regulating biosynthesis of patchouli alcohol

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin . In this study, the full-length cDNA of the FPP synthase gene from P. cablin ( PcFPPS ) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene ( PcPTS ), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.

Wang, Xiaobing↗
NASA NTRS1992

Time threshold for second positive phototropism is decreased by a preirradiation with red light

A second positive phototropic response is exhibited by a plant after the time of irradiation has exceeded a time threshold. The time threshold of dark-grown seedlings is about 15 minutes for Arabidopsis thaliana. This threshold is decreased to about 4 minutes by a 669-nanometer preirradiation. Tobacco (Nicotiana tabacum) seedlings show a similar response. The time threshold of dark-grown seedlings is about 60 minutes for tobacco, and is decreased to about 15 minutes after a preirradiation with either 450- or 669- nanometer light. The existence of a time threshold for second positive phototropism and the dependence of this threshold on the irradiation history of the seedling contribute to the complexity of the fluence response relationship for phototropism.

NASA Discipline Number 40-50↗
NASA NTRS1989

Polyamine binding to proteins in oat and Petunia protoplasts

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

NASA Discipline Plant Biology↗
NASA NTRS1989

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Non-NASA Center↗
NASA NTRS2000

Plant nuclei can contain extensive grooves and invaginations

Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Non-NASA Center↗
NASA NTRS2000

An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

Non-NASA Center↗
DOE OSTI2023

An efficient virus–induced gene silencing (VIGS) system for gene functional studies in Miscanthus

Virus-induced gene silencing (VIGS) is a powerful tool for transient gene functional analysis in plants, especially for monocot species (e.g., grasses) that are recalcitrant to transformation. Despite various VIGS systems that have been developed in different plant species, none was previously available for the bioenergy crop Miscanthus. Here, we report the establishment of an efficient and robust VIGS system mediated by Tobacco Rattle Virus (TRV) in Miscanthus. We first investigated the impact of various factors that may affect gene silencing efficiency using the Miscanthus sinensis Phytoene Desaturase (MsPDS) gene as a visual indicator of photobleaching. Then, we optimized the TRV-elicited VIGS procedure using an orthogonal experimental design with four factors (sprout size, Agrobacterium concentration, vacuum infiltration time, and co-incubation time) each at three levels. The following led to the highest silencing efficiency (~76%): inoculation of germinating seedlings (1.0–2.0 mm), Agrobacterium tumefaciens culture grown to optical density at 600 nm (OD 600 ) of 0.4, vacuum infiltration for 90 min, and co-incubation for 5 h. The VIGS system established was applicable for both M. sinensis and M. lutarioriparius, with comparable gene silencing efficiency. We verified the efficacy of the VIGS system via the functional characterization of the role of a MYB transcription factor, MsMYB112, in salt stress tolerance. Expression of MsMYB112 was successfully knocked down using the VIGS system, and this led to compromised salt tolerance in the silenced Miscanthus plants. The TRV-based VIGS system established may, therefore, substantially facilitate functional genomic studies in Miscanthus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Changes in Distal Tibial Microarchitecture During Eight Weeks of U.S. Army Basic Combat Training Differ by Sex and Race

ABSTRACT Basic combat training (BCT) is a physically rigorous period at the beginning of a soldier's career that induces bone formation in the tibia. Race and sex are determinants of bone properties in young adults but their influences on changes in bone microarchitecture during BCT are unknown. The purpose of this work was to determine the influence of sex and race on changes in bone microarchitecture during BCT. Bone microarchitecture was assessed at the distal tibia via high‐resolution peripheral quantitative computed tomography at the beginning and end of 8 weeks of BCT in a multiracial cohort of trainees (552 female, 1053 male; mean ± standard deviation [SD] age = 20.7 ± 3.7 years) of which 25.4% self‐identified as black, 19.5% as race other than black or white (other races combined), and 55.1% as white. We used linear regression models to determine whether changes in bone microarchitecture due to BCT differed by race or sex, after adjusting for age, height, weight, physical activity, and tobacco use. We found that trabecular bone density (Tb.BMD), thickness (Tb.Th), and volume (Tb.BV/TV), as well as cortical BMD (Ct.BMD) and thickness (Ct.Th) increased following BCT in both sexes and across racial groups (+0.32% to +1.87%, all p < 0.01). Compared to males, females had greater increases in Tb.BMD (+1.87% versus +1.40%; p = 0.01) and Tb.Th (+0.87% versus +0.58%; p = 0.02), but smaller increases in Ct.BMD (+0.35% versus +0.61%; p < 0.01). Compared to black trainees, white trainees had greater increases in Tb.Th (+0.82% versus +0.61%; p = 0.03). Other races combined and white trainees had greater increases in Ct.BMD than black trainees (+0.56% and + 0.55% versus +0.32%; both p ≤ 0.01). Changes in distal tibial microarchitecture, consistent with adaptive bone formation, occur in trainees of all races and sexes, with modest differences by sex and race. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hughes, Julie M.↗
DOE OSTI2023

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar, fluid supported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. Further, on the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Expression of the Trichoderma reesei expansin-like protein, swollenin, in poplar results in biomass with improved sugar release by enzymatic hydrolysis

Ethanol production from lignocellulosic biomass has been considered an alternative to corn-based ethanol and fossil fuel. However, the secondary cell wall structure prevents efficient degradation of cellulose. Expression of cell wall degrading enzymes has been suggested as one solution to generate suitable feedstocks for lignocellulosic ethanol production. Swollenin (SWO), originating from Trichoderma reesei, disrupts cellulose structure with low-level hydrolytic activity to the glucan chain. Previously, characterization of T. reesei SWO has been carried out in multiple microorganisms and tobacco. This study heterologously overexpressed T. reesei SWO in hybrid poplar (Populus tremula × alba), a promising feedstock for biofuel production, under the control of either the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter or the putative vascular tissue specific Subterranean Clover Stunt Virus S7 (SCSV) promoter. Transgenic poplar lines with high or moderate SWO expression level showed abnormal growth and reduced biomass production, along with increased glucose and reduced lignin contents. Increased cellulose crystallinity was observed in two transgenic events. This study also investigated the effect of in planta SWO expression on cellulose saccharification efficiency and found that transgenic events improved glucan conversion rates compared to wild type poplar. The results suggest SWO-overexpressing transgenic poplar lines could be a potential feedstock for ethanol production.

09 BIOMASS FUELS↗