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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 487 records · Page 27
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid-mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. In this work, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species ( Aliivibrio fisheri ) and two important human pathogens ( Vibrio cholerae and Vibrio vulnificus ). We found all three proteins, including one in a superintegron ( V. cholerae ), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis

Abstract Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular‐like “rosette” state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide‐binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high‐resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG‐3 PDZ domains from M. brevicollis . Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG‐3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity.

Gao, Melody↗
DOE OSTI2023

Sequential Image Recovery Using Joint Hierarchical Bayesian Learning

Abstract Recovering temporal image sequences (videos) based on indirect, noisy, or incomplete data is an essential yet challenging task. We specifically consider the case where each data set is missing vital information, which prevents the accurate recovery of the individual images. Although some recent (variational) methods have demonstrated high-resolution image recovery based on jointly recovering sequential images, there remain robustness issues due to parameter tuning and restrictions on the type of sequential images. Here, we present a method based on hierarchical Bayesian learning for the joint recovery of sequential images that incorporates prior intra- and inter-image information. Our method restores the missing information in each image by “borrowing” it from the other images. More precisely, we couple sequential images by penalizing their pixel-wise difference. The corresponding penalty terms (one for each pixel and pair of subsequent images) are treated as weakly-informative random variables that favor small pixel-wise differences but allow occasional outliers. As a result, all of the individual reconstructions yield improved accuracy. Our method can be used for various data acquisitions and allows for uncertainty quantification. Some preliminary results indicate its potential use for sequential deblurring and magnetic resonance imaging.

Xiao, Yao↗
DOE OSTI2021

Trajectory of Growth of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants in Houston, Texas, January through May 2021, Based on 12,476 Genome Sequences

Certain genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of substantial concern because they may be more transmissible or detrimentally alter the pandemic course and disease features in individual patients. SARS-CoV-2 genome sequences from 12,476 patients in the Houston Methodist health care system diagnosed from January 1 through May 31, 2021 are reported here. Prevalence of the B.1.1.7 (Alpha) variant increased rapidly and caused 63% to 90% of new cases in the latter half of May. Eleven B.1.1.7 genomes had an E484K replacement in spike protein, a change also identified in other SARS-CoV-2 lineages. Compared with non–B.1.1.7-infected patients, individuals with B.1.1.7 had a significantly lower cycle threshold (a proxy for higher virus load) and significantly higher hospitalization rate. Other variants [eg, B.1.429 and B.1.427 (Epsilon), P.1 (Gamma), P.2 (Zeta), and R.1] also increased rapidly, although the magnitude was less than that in B.1.1.7. Twenty-two patients infected with B.1.617.1 (Kappa) or B.1.617.2 (Delta) variants had a high rate of hospitalization. Breakthrough cases (n = 207) in fully vaccinated patients were caused by a heterogeneous array of virus genotypes, including many not currently designated variants of interest or concern. Finally, in the aggregate, this study delineates the trajectory of SARS-CoV-2 variants circulating in a major metropolitan area, documents B.1.1.7 as the major cause of new cases in Houston, TX, and heralds the arrival of B.1.617 variants in the metroplex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

{sup 1}H NMR spectroscopic studies establish that heparanase is a retaining glycosidase

Highlights: •{sup 1}H and {sup 13}C NMR chemical shifts of fondaparinux were fully assigned by 1D and 2D NMR techniques. •Hydrolysis of fondaparinux by heparanase was monitored by {sup 1}H NMR spectroscopy. •Heparanase is established to be a retaining glycosidase. -- Abstract: Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains of proteoglycans in basement membranes and the extracellular matrix (ECM). Heparanase is implicated in several diverse pathological processes associated with ECM degradation such as metastasis, inflammation and angiogenesis and is thus an important target for anti-cancer and anti-inflammatory drug discovery. Heparanase has been classed as belonging to the clan A glycoside hydrolase family 79 based on sequence analysis, secondary structure predictions and mutagenic analysis, and thus it has been inferred that it is a retaining glycosidase. However, there has been no direct experimental evidence to support this conclusion. Herein we describe {sup 1}H NMR spectroscopic studies of the hydrolysis of the pentasaccharide substrate fondaparinux by heparanase, and provide conclusive evidence that heparanase hydrolyses its substrate with retention of configuration and is thus established as a retaining glycosidase. Knowledge of the mechanism of hydrolysis may have implications for future design of inhibitors for this important drug target.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli

Highlights: • Mycobacterial ABC transporter Rv1272c involved in fatty acid transport. • First report on Rv1272c showing that it enhances long-chain fatty acid import. • Metabolic incorporation of fatty acids into lipids enhanced by Rv1272c. Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies

Highlights: • We constructed a novel scFv display library based on lentivirus vector. • Stable transduced cell lines were obtained. • Characterization of mutants showed approximately upto three fold increase in antibody affinity. Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10{sup 6}. Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The FGF2 aptamer inhibits the growth of FGF2-FGFR pathway driven lung cancer cells

Cancers, including lung cancer, are a leading cause of death worldwide. To overcome this deadly disease, multiple modality inhibitors have been developed. These include cytotoxic agents, molecular targeted small molecules, such as tyrosine kinase inhibitors, and neutralizing antibodies. An aptamer is a short single-stranded nucleic acid molecule that is selected in vitro from a large random sequence library based on its high and specific affinity to a target molecule. Aptamers can be applied to therapeutics of various types of diseases, including cancer, due to their strong and specific neutralizing activities. However, the efficacy of aptamer-based therapy for cancer cells is not well characterized. In this study, we aimed to show that the FGF2 aptamer is effective for the treatment of FGF2 dependent lung cancer cells. We previously developed PC9GR lung cancer cells, whose proliferation is dependent on EGFR and FGF2-FGFR pathways in a cell autonomous manner. Using PC9GR cells, we demonstrate that the addition of the FGF2 aptamer induces more significant inhibition of PC9GR cell proliferation than does the addition of EGFR inhibitor alone. Furthermore, the addition of the FGF2 aptamer more significantly inhibits the downstream signals and induces apoptosis to a higher extent than does the addition of EGFR inhibitor alone. Our results show that the FGF2 aptamer inhibits the growth of FGF2-FGFR pathway-dependent lung cancer cells. The findings provide preclinical evidence that aptamers can be useful for cancer treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

The crystal structure of HIV CRF07 B′/C gp41 reveals a hyper-mutant site in the middle of HR2 heptad repeat

HIV CRF07 B′/C is a strain circulating mainly in northwest region of China. The gp41 region of CRF07 is derived from a clade C virus. In order to compare the difference of CRF07 gp41 with that of typical clade B virus, we solved the crystal structure of the core region of CRF07 gp41. Compared with clade B gp41, CRF07 gp41 evolved more basic and hydrophilic residues on its helix bundle surface. Based on sequence alignment, a hyper-mutant cluster located in the middle of HR2 heptads repeat was identified. The mutational study of these residues revealed that this site is important in HIV mediated cell–cell fusion and plays critical roles in conformational changes during viral invasion. - Highlights: • We solved the crystal structure of HIV CRF07 gp41 core region. • A hyper-mutant cluster in the middle of HR2 heptads repeat was identified. • The hyper-mutant site is important in HIV-cell fusion. • The model will help to understand the HIV fusion process.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926 T ) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926 T ) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Discovery of novel plant interaction determinants from the genomes of 163 root nodule bacteria

Root nodule bacteria (RNB) or “rhizobia” are a type of plant growth promoting bacteria, typified by their ability to fix nitrogen for their plant host, fixing nearly 65% of the nitrogen currently utilized in sustainable agricultural production of legume crops and pastures. In this study, we sequenced the genomes of 110 RNB from diverse hosts and biogeographical regions, and undertook a global exploration of all available RNB genera with the aim of identifying novel genetic determinants of symbiotic association and plant growth promotion. Specifically, we performed a subtractive comparative analysis with non-RNB genomes, employed relevant transcriptomic data, and leveraged phylogenetic distribution patterns and sequence signatures based on known precepts of symbioticand host-microbe interactions. A total of 184 protein families were delineated, including known factors for nodulation and nitrogen fixation, and candidates with previously unexplored functions, for which a role in host-interaction, -regulation, biocontrol, and more, could be posited. Lastly, these analyses expand our knowledge of the RNB purview and provide novel targets for strain improvement in the ultimate quest to enhance plant productivity and agricultural sustainability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Temporal dynamics of hot desert microbial communities reveal structural and functional responses to water input

The temporal dynamics of desert soil microbial communities are poorly understood. Given the implications for ecosystem functioning under a global change scenario, a better understanding of desert microbial community stability is crucial. Here, we sampled soils in the central Namib Desert on sixteen different occasions over a one-year period. Using Illumina-based amplicon sequencing of the 16S rRNA gene, we found that α-diversity (richness) was more variable at a given sampling date (spatial variability) than over the course of one year (temporal variability). Community composition remained essentially unchanged across the first 10 months, indicating that spatial sampling might be more important than temporal sampling when assessing β-diversity patterns in desert soils. However, a major shift in microbial community composition was found following a single precipitation event. This shift in composition was associated with a rapid increase in CO2 respiration and productivity, supporting the view that desert soil microbial communities respond rapidly to re-wetting and that this response may be the result of both taxon-specific selection and changes in the availability or accessibility of organic substrates. Recovery to quasi pre-disturbance community composition was achieved within one month after rainfall.

desert soil↗
DOE OSTI2014

Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

Here we describe an ultrasensitive electrochemical nucleic acids assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL) related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation, and used to label and amplify target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The effect of DP and HRP loading of the CNT-based labels on its signal-to-noise ratio of electrochemical detection was studied systematically for the first time. Under optimized conditions, the signal-amplified assay achieved a detection limit of 83 fM targets oligonuecleotides and a 4-order wide dynamic range of target concentration. The resulting assay allowed a robust discrimination between the perfect match and a three-base mismatch sequence. When subjected to full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of approximately 33 pg of the target gene. The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60. The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, point-of-care and early disease diagnostic applications.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2015

PRESTO polarization transfer to quadrupolar nuclei: Implications for dynamic nuclear polarization

In this study, we show both experimentally and numerically on a series of model systems that in experiments involving transfer of magnetization from 1 H to the quadrupolar nuclei under magic-angle-spinning (MAS), the PRESTO technique consistently outperforms traditionally used cross polarization (CP), affording more quantitative intensities, improved lineshapes, better overall sensitivity, and straightforward optimization. This advantage derives from the fact that PRESTO circumvents the convoluted and uncooperative spin dynamics during the CP transfer under MAS, by replacing the spin-locking of quadrupolar nuclei with a single central transition selective 90° pulse and using a symmetry-based recoupling sequence in the 1 H channel. This is important in the context of dynamic nuclear polarization (DNP) NMR of quadrupolar nuclei, where the efficient transfer of enhanced 1 H polarization is desired to obtain the highest sensitivity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗