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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 1,675 records · Page 93
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

NF-κB signaling and cell-fate decision induced by a fast-dissociating tumor necrosis factor mutant

Tumor necrosis factor (TNF) is a pluripotent inflammatory cytokine that can induce both the pro-survival nuclear factor kappa B (NF-κB) pathway and the pro-apoptotic caspase pathway. Selectively activating only one of the two pathways remains challenging. We used TNF mutants with different receptor binding kinetics to study their effects on NF-κB signaling dynamics and cell apoptosis. A TNF mutant, R1antTNF, which binds to TNFR1 with increased association and dissociation rates, induced NF-κB signaling with shorter response time and first peak duration. The short nuclear stay of NF-κB led to biased activation of downstream genes, favoring the fast response ones. At the same time, R1antTNF retains pro-apoptotic activity. At 10 ng/ml, R1antTNF selectively activated the pro-apoptotic pathway rather than the pro-survival NF-κB pathway. Our study provides a new example for the emerging evidence that ligand-receptor binding kinetics play a key role in the selective activation of downstream pathways, which deserves more attention in future drug discovery and disease studies. - Highlights: • Receptor-TNF and mutant binding kinetics changes lead to functional selectivity. • R1antTNF is a fast associating and fast dissociating TNF mutant. • R1antTNF produces shortened nuclear duration of NF-κB. • R1antTNF induces biased gene expression of early-response genes. • R1antTNF selectively activates apoptosis at low concentration.

60 APPLIED LIFE SCIENCES↗
NASA NTRSDate not supplied

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

Maria Moreno-Villanueva↗
NASA NTRS2024

Gene Expression of Peripheral Blood Mononuclear Cells of Crew Members During Long-Duration Space Missions Indicate Dysregulation of Immunological and Cell Survival Mechanisms

Lymphocytes are naturally exposed to genotoxic stresses. DNA damage occurs during the entire lymphocyte’s life span and is induced mainly by reactive oxygen species (ROS), replication fork collapse, or telomere shortening during the immune response or intense cell proliferation phases. Strong evidence for the influence of immune function on DNA repair comes from studies of SCID disease. SCID mice not only have a deficient V(D)J recombination but are also unable to repair double strand breaks, leading to increased radiation sensitivity. The leukocytes’ transcriptome of 8 ISS crew members revels a dysregulated immune function and activation of cellular survival pathways in response to space environment. We have performed PCR analysis in peripheral mononuclear cells from the same crew members. A list of 62 genes were carefully selected addressing immunological and cell survival pathways. Differentially expressed genes indicated changes in chemokine receptor activity, chemokine binding, toll-like receptors, adhesion molecules and cellular response to DNA damage.

María Moreno-Villanueva↗
DOE OSTI2020

PHOTO‐SENSITIVE LEAF ROLLING 1 encodes a polygalacturonase that modifies cell wall structure and drought tolerance in rice

Summary The biosynthesis and modification of cell wall composition and structure are controlled by hundreds of enzymes and have a direct consequence on plant growth and development. However, the majority of these enzymes has not been functionally characterised. Rice mutants with leaf‐rolling phenotypes were screened in a field. Phenotypic analysis under controlled conditions was performed for the selected mutant and the relevant gene was identified by map‐based cloning. Cell wall composition was analysed by glycome profiling assay. We identified a photo‐sensitive leaf rolling 1 ( psl1 ) mutant with ‘napping’ (midday depression of photosynthesis) phenotype and reduced growth. The PSL1 gene encodes a cell wall‐localised polygalacturonase (PG), a pectin‐degrading enzyme. psl1 with a 260‐bp deletion in its gene displayed leaf rolling in response to high light intensity and/or low humidity. Biochemical assays revealed PG activity of recombinant PSL1 protein. Significant modifications to cell wall composition in the psl1 mutant compared with the wild‐type plants were identified. Such modifications enhanced drought tolerance of the mutant plants by reducing water loss under osmotic stress and drought conditions. Taken together, PSL1 functions as a PG that modifies cell wall biosynthesis, plant development and drought tolerance in rice.

Zhang, Guangheng↗
DOE OSTI2024

Bactericidal effectors of the Stenotrophomonas maltophilia type IV secretion system: functional definition of the nuclease TfdA and structural determination of TfcB

ABSTRACT Stenotrophomonas maltophilia expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A S. maltophilia mutant lacking the gene for 20845 was impaired for killing Escherichia coli , Klebsiella pneumoniae , and Pseudomonas aeruginosa . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant E. coli being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede E. coli , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the S. maltophilia T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase. IMPORTANCE Stenotrophomonas maltophilia is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to S. maltophilia infection. In hospital water systems and various types of infections, S. maltophilia co-exists with other bacteria, including other pathogens such as Pseudomonas aeruginosa . We previously demonstrated that S. maltophilia has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, S. maltophilia currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Adenovirus armed with VGLL4 selectively kills hepatocellular carcinoma with G2/M phase arrest and apoptosis promotion

Highlights: • VGLL4 causes G2/M phase arrest in hepatocellular carcinoma cells. • The transcription level of VGLL4 gene is promoted by oncolytic adenovirus. • Oncolytic adenovirus harboring VGLL4 selectively kills hepatocellular carcinoma. • Oncolytic adenovirus harboring VGLL4 exerts G2/M arrest and apoptosis promotion. The Vestigial-Like Family Member 4 (VGLL4) functions as a native inhibitor of cell proliferation and tumor growth through multiple signaling pathways. We first discovered that VGLL4 causes G2/M phase arrest in hepatocellular carcinoma (HCC) cells. Then, we designed a novel survivin-regulated oncolytic adenovirus Ad-sp-VGLL4 carrying the VGLL4 gene. Ad-sp-VGLL4 exerted high HCC-targeting-selectivity but is less harmful to normal cells. This adenovirus construction enhanced antitumor activity due to G2/M phase arrest and enhanced apoptosis. It's also indicated that Ad-sp-VGLL4 could suppress the growth of transplanted tumor of HCC in vivo experiment. Taken together, our results suggest that Ad-sp-VGLL4 possesses strong antitumor capacity and has great potential use for HCC therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

The Brachypodium ENCODE Project – from sequence to function: Predicting physiological responses in grasses to facilitate engineering of biofuel crops

The overarching goal of this proposal was to gain deep knowledge of chromatin dynamics and gene networks related to stress responses in grasses. To accomplish this goal our Brachypodium ENCODE project (1) coupled drought-focused physiological analysis with multiple layers of ENCODE ‘omics’ profiling; (2) obtained fundamental information about the Brachypodium transcriptome, epigenome, chromatin dynamics, and gene regulatory networks; and (3) elucidated the regulatory dynamics of gene networks that can be manipulated to increase energy grass crop productivity.

09 BIOMASS FUELS↗
NASA NTRS1991

Cloned Hemoglobin Genes Enhance Growth Of Cells

Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

Khosla, Chaitan↗
DOE OSTI2017

Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins, while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell-free extracts used to carry out synthesis are generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. Here we explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Full proteome experiments identified over 1000 proteins per reaction. The complete set of proteins necessary for transcription and translation were found, demonstrating the ability to define potential metabolic capabilities of the extract. Further, MS-based techniques allowed characterization of the CFPS product and provided insight into the synthesis reaction and potential functional activity of the product. These capabilities were demonstrated using two different CFPS products, the commonly used standard green fluorescent protein (GFP, 27 kDa) and the polyketide synthase DEBS1 (394 kDa). For the large, multidomain DEBS1, substantial premature termination of protein translation was observed. Additionally, MS/MS analysis, as part of a conventional full proteomics workflow, identified post-translational modifications, including the chromophore in GFP, as well as the three phosphopantetheinylation sites in DEBS1. A hypothesis-driven approach focused on these three sites identified that all were correctly modified for DEBS1 expressed in vivo but with less complete coverage for protein expressed in CFPS reactions. These post-translational modifications are essential for functional activity, and the ability to identify them with mass spectrometry is valuable for judging the success of the CFPS reaction. Collectively, the use of MS-based proteomics will prove advantageous for advancing the application of CFPS and related techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

p.Q511L mutation of HNF1α in hepatocellular carcinoma suppresses the transcriptional activity and the anti-tumor effect of HNF1α

Highlights: • A novel somatic mutation p.Q511L of HNF1α was detected in a HCC tissue. • p.Q511L mutation suppressed the transcription activity of HNF1α in HCC cells. • p.Q511L mutation disturbed the subcellular localization of HNF1α. • p.Q511L mutation reduced the inhibitory effect of HNF1α on malignancy of HCC cells. Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that regulates many aspects of hepatocyte functions. Our previous studies have demonstrated that HNF1α has potent therapeutic effects on hepatocellular carcinoma (HCC). Mutations in HNF1α gene are frequently associated with maturity-onset diabetes of the young type 3 (MODY3) and hepatocellular adenomas. However, the association of HNF1α mutation and HCC remains elusive. In this study, the point mutation of HNF1α gene with c.A1532 > T/p.Q511L was identified in an HCC patient by exon-capture high-throughput sequencing. Mutation of c.A1532 > T/p.Q511L in HNF1α gene was only detected in the tumor tissue but not in the adjacent non-tumorous liver tissue of the patient. Luciferase reporter assay and real-time PCR revealed that mutation of p.Q511L reduced the transcriptional activity of HNF1α. Immunofluorescence staining and subcellular fraction analysis revealed that mutation of p.Q511L disturbed the intracellular localization of HNF1α in HCC cells. Moreover, the inhibitory effect of HNF1α on the proliferation, migration and invasion in HCC cells was also partially abolished by the mutation of p.Q511L. Our data suggested that the missense mutation of HNF1α (p.Q511L) may associate with the progression of HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The zinc-finger transcription factor BcMF20 and its orthologs in Cruciferae which are required for pollen development

Highlights: • BcMF20 was a three-fingered protein and close to the four-fingered proteins of petunia. • The inhibition of BcMF20 led to fewer and lower rate in pollen germination. • The inhibition of BcMF20 led to lower rate in fruit setting. • BcMF20 may work in genetic pathways to act on proliferation of tapetal cells. • BcMF20 helps keep the normal development of pollens. Brassica campestris Male Fertility 20 (BcMF20) is a typical zinc-finger transcription factor that was previously isolated from flower buds of Chinese cabbage (Brassica campestris ssp. chinensis). By applying expression pattern analysis, it can be known that BcMF20 was specifically and strongly expressed in tapetum and pollen, beginning from the uninucleate stage, and was maintained during the mature-pollen stage. As BcMF20 was highly conserved in Cruciferae, it can be indicated that this zinc-finger transcription factor is important during the growth of Cruciferae. In this study, 12 C2H2-type zinc-finger TFs which shared high homology with BcMF20 were found from NCBI via BLAST. A new molecular phylogenetic tree was constructed by the comparison between BcMF20 and these 12 C2H2-type zinc-finger TFs with NJ method. By analyzing this phylogenetic tree, the evolution of BcMF20 was discussed. Then, antisense RNA technology was applied in the transgenesis of Arabidopsis thaliana to get the deletion mutants of BcMF20, so that its function during the pollen development can be identified. The results showed: BcMF20 are in the same clade with three genes from Arabidopsis. The inhibition of BcMF20 expression led to smaller amounts of and lower rate in germination of pollen and lower rate in fruit setting in certain transgenetic plants. This also led to the complete collapse of pollen grains. By SEM and TEM, pollen morphology and anther development processes were observed. In the middle uninucleate microspore stage, a relatively thin or even no primexine was formed in microspores. This may result in the malformation of the pollen wall and finally cause the deformity of pollens. Above all, it can be indicated that BcMF20 may act as a part of regulation mechanisms of TAZ1 and MS1. Together they play a role in a genetic pathway in the tapetum to act on proliferation of tapetal cells and keep the normal development of pollens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A Post-Transcriptional Feedback Mechanism for Noise Suppression and Fate Stabilization

Diverse biological systems utilize fluctuations (“noise”) in gene expression to drive lineage-commitment decisions. However, once a commitment is made, noise becomes detrimental to reliable function, and the mechanisms enabling post-commitment noise suppression are unclear. Here, we find that architectural constraints on noise suppression are overcome to stabilize fate commitment. Using single-molecule and time-lapse imaging, we find that—after a noise-driven event—human immunodeficiency virus (HIV) strongly attenuates expression noise through a non-transcriptional negative-feedback circuit. Feedback is established through a serial cascade of post-transcriptional splicing, whereby proteins generated from spliced mRNAs auto-deplete their own precursor unspliced mRNAs. Strikingly, this auto-depletion circuitry minimizes noise to stabilize HIV’s commitment decision, and a noise-suppression molecule promotes stabilization. Lastly, this feedback mechanism for noise suppression suggests a functional role for delayed splicing in other systems and may represent a generalizable architecture of diverse homeostatic signaling circuits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Protein–protein interactions between tenascin-R and RPTPζ/phosphacan are critical to maintain the architecture of perineuronal nets

Neural plasticity, the ability to alter the structure and function of neural circuits, varies throughout the age of an individual. The end of the hyperplastic period in the central nervous system coincides with the appearance of honeycomb-like structures called perineuronal nets (PNNs) that surround a subset of neurons. PNNs are a condensed form of neural extracellular matrix that include the glycosaminoglycan hyaluronan and extracellular matrix proteins such as aggrecan and tenascin-R (TNR). PNNs are key regulators of developmental neural plasticity and cognitive functions, yet our current understanding of the molecular interactions that help assemble them remains limited. Disruption of Ptprz1, the gene encoding the receptor protein tyrosine phosphatase RPTPζ, altered the appearance of nets from a reticulated structure to puncta on the surface of cortical neuron bodies in adult mice. The structural alterations mirror those found in Tnr -/- mice, and TNR is absent from the net structures that form in dissociated cultures of Ptprz1 -/- cortical neurons. These findings raised the possibility that TNR and RPTPζ cooperate to promote the assembly of PNNs. Here, we show that TNR associates with the RPTPζ ectodomain and provide a structural basis for these interactions. Furthermore, we show that RPTPζ forms an identical complex with tenascin-C, a homolog of TNR that also regulates neural plasticity. Finally, we demonstrate that mutating residues at the RPTPζ–TNR interface impairs the formation of PNNs in dissociated neuronal cultures. Overall, this work sets the stage for analyzing the roles of protein–protein interactions that underpin the formation of nets.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Ebola virus VP30 and nucleoprotein interactions modulate viral RNA synthesis

Ebola virus (EBOV) is an enveloped negative-sense RNA virus that causes sporadic outbreaks with high case fatality rates. Ebola viral protein 30 (eVP30) plays a critical role in EBOV transcription initiation at the nucleoprotein (eNP) gene, with additional roles in the replication cycle such as viral assembly. However, the mechanistic basis for how eVP30 functions during the virus replication cycle is currently unclear. Here we define a key interaction between eVP30 and a peptide derived from eNP that is important to facilitate interactions leading to the recognition of the RNA template. We present crystal structures of the eVP30 C-terminus in complex with this eNP peptide. Functional analyses of the eVP30–eNP interface identify residues that are critical for viral RNA synthesis. Altogether, these results support a model where the eVP30–eNP interaction plays a critical role in transcription initiation and provides a novel target for the development of antiviral therapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A common resequencing‐based genetic marker data set for global maize diversity

SUMMARY Maize ( Zea mays ssp. mays ) populations exhibit vast ranges of genetic and phenotypic diversity. As sequencing costs have declined, an increasing number of projects have sought to measure genetic differences between and within maize populations using whole‐genome resequencing strategies, identifying millions of segregating single‐nucleotide polymorphisms (SNPs) and insertions/deletions (InDels). Unlike older genotyping strategies like microarrays and genotyping by sequencing, resequencing should, in principle, frequently identify and score common genetic variants. However, in practice, different projects frequently employ different analytical pipelines, often employ different reference genome assemblies and consistently filter for minor allele frequency within the study population. This constrains the potential to reuse and remix data on genetic diversity generated from different projects to address new biological questions in new ways. Here, we employ resequencing data from 1276 previously published maize samples and 239 newly resequenced maize samples to generate a single unified marker set of approximately 366 million segregating variants and approximately 46 million high‐confidence variants scored across crop wild relatives, landraces as well as tropical and temperate lines from different breeding eras. We demonstrate that the new variant set provides increased power to identify known causal flowering‐time genes using previously published trait data sets, as well as the potential to track changes in the frequency of functionally distinct alleles across the global distribution of modern maize.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structurally Guided Removal of DeISGylase Biochemical Activity from Papain-Like Protease Originating from Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs. IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.

Daczkowski, Courtney M.↗
DOE OSTI2017

Ecophysiology of Freshwater Verrucomicrobia Inferred from Metagenome-Assembled Genomes

Freshwater Verrucomicrobia spp. are cosmopolitan in lakes and rivers, and yet their roles and ecophysiology are not well understood, as cultured freshwater Verrucomicrobia spp. are restricted to one subdivision of this phylum. Here, we greatly expanded the known genomic diversity of this freshwater lineage by recovering 19 Verrucomicrobia draft genomes from 184 metagenomes collected from a eutrophic lake and a humic bog across multiple years. Most of these genomes represent the first freshwater representatives of several Verrucomicrobia subdivisions. Genomic analysis revealed Verrucomicrobia to be potential (poly)saccharide degraders and suggested their adaptation to carbon sources of different origins in the two contrasting ecosystems. We identified putative extracellular electron transfer genes and so-called “ Planctomycete -specific” cytochrome c -encoding genes and identified their distinct distribution patterns between the lakes/layers. Overall, our analysis greatly advances the understanding of the function, ecophysiology, and distribution of freshwater Verrucomicrobia , while highlighting their potential role in freshwater carbon cycling.

59 BASIC BIOLOGICAL SCIENCES↗