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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 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Upregulation of eIF6 inhibits cardiac hypertrophy induced by phenylephrine

Cardiac hypertrophy is determined by an increase of cell size in cardiomyocytes (CMCs). Among the cellular processes regulating the growth of cell size, the increase of protein synthesis rate represents a critical event. Most of translational factors promoting protein synthesis stimulate cardiac hypertrophy. In contrast, activity of translational repressor factors, in cardiac hypertrophy, is not fully determined yet. Here we report the effect of a translational modulator, eIF6/p27{sup BBP} in the hypertrophy of neonatal rat CMCs. The increase of eIF6 levels surprisingly prevent the growth of cell size induced by phenylephrine, through a block of protein synthesis without affecting skeletal rearrangement and ANF mRNA expression. Thus, this work uncovers a new translational cardiac regulator independent by other well-known factors such as mTOR signalling or eIF2β.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Corticosteroid therapy and airflow obstruction influence the bronchial microbiome, which is distinct from that of bronchoalveolar lavage in asthmatic airways

The lung has a diverse microbiome that is modest in biomass. This microbiome differs in asthmatic patients compared with control subjects, but the effects of clinical characteristics on the microbial community composition and structure are not clear. OBJECTIVES: We examined whether the composition and structure of the lower airway microbiome correlated with clinical characteristics of chronic persistent asthma, including airflow obstruction, use of corticosteroid medications, and presence of airway eosinophilia. METHODS: DNA was extracted from endobronchial brushings and bronchoalveolar lavage fluid collected from 39 asthmatic patients and 19 control subjects, along with negative control samples. 16S rRNA V4 amplicon sequencing was used to compare the relative abundance of bacterial genera with clinical characteristics. RESULTS: Differential feature selection analysis revealed significant differences in microbial diversity between brush and lavage samples from asthmatic patients and control subjects. Lactobacillus, Pseudomonas, and Rickettsia species were significantly enriched in samples from asthmatic patients, whereas Prevotella, Streptococcus, and Veillonella species were enriched in brush samples from control subjects. Generalized linear models on brush samples demonstrated oral corticosteroid use as an important factor affecting the relative abundance of the taxa that were significantly enriched in asthmatic patients. In addition, bacterial α-diversity in brush samples from asthmatic patients was correlated with FEV1 and the proportion of lavage eosinophils. CONCLUSION: The diversity and composition of the bronchial airway microbiome of asthmatic patients is distinct from that of nonasthmatic control subjects and influenced by worsening airflow obstruction and corticosteroid use. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

16S ribosomal RNA↗
DOE OSTI2011

Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Discrimination of haptens from prohaptens using the metabolically deficient Cpr{sup low/low} mouse

The murine local lymph node assay (LLNA) is a validated, well accepted method for identification of chemical contact allergens. Both direct acting haptens and prohaptens (requiring metabolic activation) can be identified, but not differentiated by this assay. This study was used to assess the utility of a pan microsomal metabolic deficient mouse to distinguish between direct acting haptens and prohaptens in the LLNA. Hapten and prohapten induced cell proliferation was compared in C57BL/6J (B6) wild type (WT) versus homozygous (HO) knockout mice with a hypomorphic NADPH-Cytochrome P450 Reductase (CPR) gene (termed Cpr{sup low/low}) resulting in low CPR enzyme activity. Mice were dosed with known prohaptens; benzo(a)pyrene (BaP), carvone oxime (COx) and paracetamol (PCM) and haptens; oxazolone (OX), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (EtOX), and N-acetylbenzoquinoneimine (NABQI) in this study. Skin microsomes from the WT, HO and heterozygous (HT) Cpr{sup low/low} mice were compared and evaluated for CPR activity. Lymphocyte proliferative responses to BaP, COx and PCM were significantly abrogated by 36.4%, 45.2% and 50.8%, respectively; in Cpr{sup low/low} knock out (KO) mice versus WT mice; while the lymphocyte proliferative responses to the direct acting haptens OX, EtOX and NABQI were comparable. CPR activity, determined as Units/mg protein, was determined to be significantly lower in the Cpr{sup low/low} mice compared to the WT. Results of the present study suggest potential utility of the Cpr{sup low/low} mice in the LLNA to differentiate prohaptens from direct acting haptens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100 nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2 nM [{sup 3}H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2{alpha}. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50 days of feeding 3 {mu}g of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50 days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3 days and remained elevated for up to 28 days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50 days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14 days of feeding both PAHs that was not significant after 50 days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28 days. There was no clear association between AHR2{alpha} affinity and BaP and BeP-induced oxidative stress. - Highlights: > No direct association between aryl hydrocarbon receptor affinity and polyaromatic hydrocarbon induced oxidative stress. > There was a strong correlation between cytochrome P4501A activity and oxidative stress as measured with the comet assay. > There was no correlation between cytochrome P4501A protein and oxidative stress as measured with the comet assay.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

Metabolism of aflatoxin B{sub 1} in Turkey liver microsomes: The relative roles of cytochromes P450 1A5 and 3A37

The extreme sensitivity of turkeys to aflatoxin B{sub 1} (AFB{sub 1}) is associated with efficient epoxidation by hepatic cytochromes P450 (P450) 1A5 and 3A37 to exo-aflatoxin B{sub 1}-8,9-epoxide (exo-AFBO). The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB{sub 1} to the exo-AFBO and to detoxification products aflatoxin M{sub 1} (AFM{sub 1}) and aflatoxin Q{sub 1} (AFQ{sub 1}), respectively, complicates the kinetic analysis of AFB{sub 1} in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB{sub 1} in TLMs. In control TLMs, AFB{sub 1} was converted to exo-AFBO in addition to AFM{sub 1} and AFQ{sub 1} confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar ({approx} 0.1 {mu}M), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (> 50 {mu}M) AFB{sub 1} concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM{sub 1}, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ{sub 1} following the kinetic Hill equation. At 0.1 {mu}M AFB{sub 1}, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM{sub 1}) compared to 3A37 (0.15: 1, exo-AFBO: AFQ{sub 1}), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB{sub 1} bioactivation and metabolism at environmentally-relevant AFB{sub 1} concentrations in turkey liver. - Graphical abstract: Display Omitted Highlights: > Efficient bioactivation by P450s 1A5 and 3A4 associated with extreme aflatoxin B{sub 1} sensitivity in turkeys. > These P450s exhibit different metabolite profiles and enzyme kinetic models toward AFB{sub 1}. > Study conducted to determine which P450 is primary bioactivator in turkey liver. > Immunoinhibition studies show 1A5 predominates at low, environmentally-relevant AFB{sub 1} concentrations. > 3A37 predominates at only at very high AFB{sub 1} concentrations, not relevant to liver in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Deficiency in the nuclear factor E2-related factor 2 renders pancreatic β-cells vulnerable to arsenic-induced cell damage

Chronic human exposure to inorganic arsenic (iAs), a potent environmental oxidative stressor, is associated with increased prevalence of type 2 diabetes, where impairment of pancreatic β-cell function is a key pathogenic factor. Nuclear factor E2-related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. However, persistent activation of Nrf2 in response to chronic oxidative stress, including inorganic arsenite (iAs{sup 3+}) exposure, blunts glucose-triggered reactive oxygen species (ROS) signaling and impairs glucose-stimulated insulin secretion (GSIS). In the current study, we found that MIN6 pancreatic β-cells with stable knockdown of Nrf2 (Nrf2-KD) by lentiviral shRNA and pancreatic islets isolated from Nrf2-knockout (Nrf2−/−) mice exhibited reduced expression of several antioxidant and detoxification enzymes in response to acute iAs{sup 3+} exposure. As a result, Nrf2-KD MIN6 cells and Nrf2−/− islets were more susceptible to iAs{sup 3+} and monomethylarsonous acid (MMA{sup 3+})-induced cell damage, as measured by decreased cell viability, augmented apoptosis and morphological change. Pretreatment of MIN6 cells with Nrf2 activator tert-butylhydroquinone protected the cells from iAs{sup 3+}-induced cell damage in an Nrf2-dependent fashion. In contrast, antioxidant N‐acetyl cysteine protected Nrf2-KD MIN6 cells against acute cytotoxicity of iAs{sup 3+}. The present study demonstrates that Nrf2-mediated antioxidant response is critical in the pancreatic β-cell defense mechanism against acute cytotoxicity by arsenic. The findings here, combined with our previous results on the inhibitory effect of antioxidants on ROS signaling and GSIS, suggest that Nrf2 plays paradoxical roles in pancreatic β-cell dysfunction induced by environmental arsenic exposure. -- Highlights: ► Lack of Nrf2 reduced expression of antioxidant genes induced by iAs{sup 3+} in β-cells. ► Deficiency of Nrf2 in β-cells sensitized to iAs{sup 3+} and MMA{sup 3+}-induced cytotoxicity. ► Nrf2 activation protected β-cells from acute iAs{sup 3+} cytotoxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

The JmjC-domain protein NO66/RIOX-1 affects the balance between proliferation and maturation in acute myeloid leukemia

Highlights: • NO66/RIOX1 is not expressed in the AML cell line KG1a regarded as a “stem cell-like” model system. • NO66/RIOX1 is not expressed in primary HSC-enriched CD34{sup +}/CD38{sup -} cells of healthy donors. • Ectopic expression of NO66/RIOX1 in KG1a cells inhibits proliferation and rDNA transcription. • NO66/RIOX1 impacts on the transition from stem cells to progenitors by regulation of gene expression. As epigenetic regulators are frequently dysregulated in acute myeloid leukemia (AML) we determined expression levels of the JmjC-protein NO66 in AML cell lines and sub fractions of healthy human hematopoietic cells. NO66 is absent in the AML cell lines KG1/KG1a which consist of cells with the immature CD34{sup +}/CD38{sup -} phenotype and is regarded as a “stem cell-like” model system. Similarly, NO66 is not detectable in CD34{sup +}/CD38{sup -} cells purified from healthy donors but is clearly expressed in the more committed CD34{sup +}/CD38{sup +} cell population. Loss of NO66 expression in KG1/KG1a cells is due to hyper-methylation of its promoter and is released by DNA-methyltransferase inhibitors. In KG1a cells stably expressing exogenous wild type (KG1a66wt) or enzymatically inactive mutant (KG1a66mut) NO66, respectively, the wild type protein inhibited proliferation and rDNA transcription. Gene expression profiling revealed that the expression of NO66 induces a transcriptional program enriched for genes with roles in proliferation and maturation (e.g.EPDR1, FCER1A, CD247, MYCN, SNORD13). Genes important for the maintenance of stem cell properties are downregulated (e.g. SIRPA, Lin28B, JAML). Our results indicate that NO66 induces lineage commitment towards myeloid progenitor cell fate and suggest that NO66 contributes to loss of stem cell properties.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Structural Analysis of 23S rRNA Methylating Enzyme Cfr Reveals RNA-Binding Determinants for Methylation Regiospecificity and Antibiotic Resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli, and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPS A phenotype). Cfr catalyzes methylation of the C8 position of the A2503 base in 23S rRNA, the recognition site of the above antibiotic classes. Along with the RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfr’s dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb PhLOPS A resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in cellulo 23S rRNA methylated species, and in cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multidrug resistance activity. In particular, we found that Cfr’s Q329 residue is important for C8- specific methylation. These data provide a framework for further studies of the biochemistry, structure, and inhibition of this important resistance determinant.

AlphaFold↗
DOE OSTI2014

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. In this paper we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Finally, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Widespread polycistronic gene expression in green algae

Significance Historically, it has been understood that for gene expression in eukaryotes, each messenger RNA encodes a single protein. With the recent development of technologies to sequence full-length transcripts en masse, we have discovered hundreds of examples in two species of green algae where two, three, or more proteins are translated from a single transcript. These “polycistronic” transcripts are found in diverse species throughout the green algal lineage, which highlights their biological importance. We have leveraged these findings to coexpress pairs of genes on polycistronic transcripts in vitro, which should facilitate efforts to engineer algae for research and industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota)

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

59 BASIC BIOLOGICAL SCIENCES↗