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

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At least 829 records · Page 46
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding

While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle

We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3C LT , used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3C LT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2013

Altered cardiac muscle mTOR regulation during the progression of cancer cachexia in the ApcMin/+ mouse

Cancer cachexia is a muscle wasting condition that occurs in response to a malignant growth in the body. The mechanisms regulating cardiac muscle mass with cachexia are not well understood. Using the ApcMin/+ mouse model of colorectal cancer, we investigated how cachexia affects the regulation of 5'-adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling in the heart. Compared to age-matched C57BL/6 (BL6) mice, ApcMin/+ body mass and heart mass were lower at 12 (11±5 and 8±3%, respectively) and 20 weeks (26±3 and 6±4%, respectively) of age (P<0.05). Diminished heart mass in the 20-week-old ApcMin/+ mice coincided with a decreased rate of myofibrillar protein synthesis and increased AMPKα phosphorylation. Cachexia decreased mTOR phosphorylation and the phosphorylation of the mTOR substrates, S6 ribosomal protein and 4EBP1 independent of Akt activation. These changes in mTOR-related protein signaling were accompanied by modest increases in the amount of Beclin1 but not protein ubiquitination or cardiomyocyte apoptosis. Taken together, these data suggest that loss of cardiac mass during cachexia progression in the ApcMin/+ mouse is associated with an Akt-independent suppression of anabolic signaling and evidence of increased autophagy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Lipids accumulation in nitrogen and phosphorus-limited yeast is caused by less growth-related dilution

Oleaginous yeasts are used commercially to produce oleochemicals and hold potential also for biodiesel production. In response to nitrogen or phosphorous limitation, oleaginous yeast accumulate triacylglycerol. Previous work has investigated potential mechanisms by which nutrient limitation induces lipid biosynthesis without verifying whether lipid biosynthesis flux is actually enhanced. Here we show, using 13C-glucose tracing, that in nitrogen or phosphorous limitation, lipid accumulation occurs without consistent increases in biosynthetic flux. Instead, the main driver of increased lipid pools is decreased growth-related dilution. This conclusion holds across two divergent oleaginous yeasts: Rhodotorula toruloides and Yarrowia lipolytica. Quantitative proteomics shows a substantial proteome reallocation in response to nitrogen and phosphorous limitation, with ribosomal proteins strongly down regulated, while lipid biosynthetic enzymes are preserved but not consistently upregulated in absolute quantity. Thus, nutrient limitation, rather than triggering greatly enhanced lipid synthesis, results in roughly sustained lipid enzyme levels and biosynthetic flux. Due to slower lipid dilution by cell division, this suffices to drive marked lipid accumulation.

Keber, Felix [Lewis Sigler Institute for Integrati↗
DOE OSTI2016

Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biases in genome reconstruction from metagenomic data

Background Advances in sequencing, assembly, and assortment of contigs into species-specific bins has enabled the reconstruction of genomes from metagenomic data (MAGs). Though a powerful technique, it is difficult to determine whether assembly and binning techniques are accurate when applied to environmental metagenomes due to a lack of complete reference genome sequences against which to check the resulting MAGs. Methods We compared MAGs derived from an enrichment culture containing ~20 organisms to complete genome sequences of 10 organisms isolated from the enrichment culture. Factors commonly considered in binning software—nucleotide composition and sequence repetitiveness—were calculated for both the correctly binned and not-binned regions. This direct comparison revealed biases in sequence characteristics and gene content in the not-binned regions. Additionally, the composition of three public data sets representing MAGs reconstructed from the Tara Oceans metagenomic data was compared to a set of representative genomes available through NCBI RefSeq to verify that the biases identified were observable in more complex data sets and using three contemporary binning software packages. Results Repeat sequences were frequently not binned in the genome reconstruction processes, as were sequence regions with variant nucleotide composition. Genes encoded on the not-binned regions were strongly biased towards ribosomal RNAs, transfer RNAs, mobile element functions and genes of unknown function. Our results support genome reconstruction as a robust process and suggest that reconstructions determined to be >90% complete are likely to effectively represent organismal function; however, population-level genotypic heterogeneity in natural populations, such as uneven distribution of plasmids, can lead to incorrect inferences.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2015

Characterization of the salivary microbiome in patients with pancreatic cancer

Clinical manifestations of pancreatic cancer often do not occur until the cancer has undergone metastasis, resulting in a very low survival rate. In this study, we investigated whether salivary bacterial profiles might provide useful biomarkers for early detection of pancreatic cancer. Using high-throughput sequencing of bacterial small subunit ribosomal RNA (16S rRNA) gene, we characterized the salivary microbiota of patients with pancreatic cancer and compared them to healthy patients and patients with other diseases, including pancreatic disease, non-pancreatic digestive disease/cancer and non-digestive disease/cancer. A total of 146 patients were enrolled at the UCSD Moores Cancer Center where saliva and demographic data were collected from each patient. Of these, we analyzed the salivary microbiome of 108 patients: 8 had been diagnosed with pancreatic cancer, 78 with other diseases and 22 were classified as non-diseased (healthy) controls. Bacterial 16S rRNA sequences were amplified directly from salivary DNA extractions and subjected to high-throughput sequencing (HTS). Several bacterial genera differed in abundance in patients with pancreatic cancer. We found a significantly higher ratio of Leptotrichia to Porphyromonas in the saliva of patients with pancreatic cancer than in the saliva of healthy patients or those with other disease (Kruskal–Wallis Test; P < 0.001). Leptotrichia abundances were confirmed using real-time qPCR with Leptotrichia specific primers. Similar to previous studies, we found lower relative abundances of Neisseria and Aggregatibacter in the saliva of pancreatic cancer patients, though these results were not significant at the P < 0.05 level (K–W Test; P = 0.07 and P = 0.09 respectively). However, the relative abundances of other previously identified bacterial biomarkers, e.g., Streptococcus mitis and Granulicatella adiacens , were not significantly different in the saliva of pancreatic cancer patients. Overall, this study supports the hypothesis that bacteria abundance profiles in saliva are useful biomarkers for pancreatic cancer though much larger patient studies are needed to verify their predictive utility.

Science & Technology - Other Topics↗
DOE OSTI2014

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Large scale surveys in mammalian tissue culture cells suggest that the protein expressed at the median abundance is present at 8000–16 000 molecules per cell and that differences in mRNA expression between genes explain only 10–40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances and the relative importance of transcription. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170 000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher. We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression. We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 = 0.13). Based on this, our second strategy suggests that mRNA levels explain ~81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimate that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the absence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the ~40% of genes that are non-expressed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

DISCo-microbe: design of an identifiable synthetic community of microbes

Microbiomes are extremely important for their host organisms, providing many vital functions and extending their hosts’ phenotypes. Natural studies of host-associated microbiomes can be difficult to interpret due to the high complexity of microbial communities, which hinders our ability to track and identify individual members along with the many factors that structure or perturb those communities. For this reason, researchers have turned to synthetic or constructed communities in which the identities of all members are known. However, due to the lack of tracking methods and the difficulty of creating a more diverse and identifiable community that can be distinguished through next-generation sequencing, most such in vivo studies have used only a few strains. To address this issue, we developed DISCo-microbe, a program for the design of an identifiable synthetic community of microbes for use in in vivo experimentation. The program is composed of two modules; (1) create, which allows the user to generate a highly diverse community list from an input DNA sequence alignment using a custom nucleotide distance algorithm, and (2) subsample, which subsamples the community list to either represent a number of grouping variables, including taxonomic proportions, or to reach a user-specified maximum number of community members. As an example, we demonstrate the generation of a synthetic microbial community that can be distinguished through amplicon sequencing. The synthetic microbial community in this example consisted of 2,122 members from a starting DNA sequence alignment of 10,000 16S rRNA sequences from the Ribosomal Database Project. We generated simulated Illumina sequencing data from the constructed community and demonstrate that DISCo-microbe is capable of designing diverse communities with members distinguishable by amplicon sequencing. Using the simulated data we were able to recover sequences from between 97–100% of community members using two different post-processing workflows. Furthermore, 97–99% of sequences were assigned to a community member with zero sequences being misidentified. We then subsampled the community list using taxonomic proportions to mimic a natural plant host–associated microbiome, ultimately yielding a diverse community of 784 members. DISCo-microbe can create a highly diverse community list of microbes that can be distinguished through 16S rRNA gene sequencing, and has the ability to subsample (i.e., design) the community for the desired number of members and taxonomic proportions. Although developed for bacteria, the program allows for any alignment input from any taxonomic group, making it broadly applicable. The software and data are freely available from GitHub and Python Package Index (PYPI).

16S rRNA↗
NASA NTRS1971

The possible participation of esters as well as amides in prebiotic polymers.

Demonstration that alpha-hydroxy acids may have participated in the formation of prebiological polymers in a manner similar to the participation of alpha-amino acids. Ex periments are described which indicate that the system for forming peptide bonds in present-day biological organisms is equally competent in forming ester and polyester bonds. In particular, the experiments described are directed toward answering questions regarding the action of peptidyl transferase in ester formation. Also, an attempt is made to determine whether a complete protein synthetic system can operate with transfer RNA molecules which have alpha-hydroxyl acids attached to them instead of alpha-amino acids, using both synthetic and natural mRNA. The ability of ribosomal peptidyl transferase to catalyze the formation of an ester bond as well as its normal product, the peptide bond, is demonstrated.

Rich, A.↗
NASA NTRS1970

Origins of life; Proceedings of the First Conference, Princeton, N.J., May 21-24, 1967.

The dialogs on the origin of life commence with the fossil record. They deal with the age of the earth; the primitive atmosphere; precambrian microfossils; the Fig Tree series of rocks, which is part of the Swaziland system; the Bitter Springs Formation in Australia; and simulated Precambrian microfossils. The syntheses of biologically important monomers, amino acids, and nucleic acid derivatives are discussed. Polymerization mechanisms; the thermal origin of amino acid polymers; nonnucleic acid information carriers; the relationship between amino acid and nucleic acid polymers; the origin of the triple code; early reciprocating systems; and the evolution of the ribosomal system receive attention. Some brief comments on extraterrestrial life are presented. An appendix deals with the geological evidence for the thermal origin of living systems.

Margulis, L.↗
NASA NTRS1972

Zinc in Entamoeba invadens.

Atomic absorption spectroscopy, electron microprobe analysis, and dithizone staining of trophozoites and cysts of Entamoeba invadens demonstrate that these cells have a high concentration of zinc (approximately one picogram per cell or 1% of their dry weight). In the cysts of this organism, the zinc is confined to the chromatoid bodies, which previous work has shown to contain crystals of ribosomes. The chemical state and function of this zinc are unknown.

Morgan, R. S.↗
NASA NTRS1973

Macromolecular fractionation and determination

Biological materials were recovered from other materials ultracentrifugation techniques. Data cover fatty acid synthetase, acetyl-CoA synthetase, ribosomes, DNA, RNA, and AMP.

Chao, F.↗