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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 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

A Versatile Remediation Module for Remote Repair of Spent Nuclear Fuel and High-Level Waste Storage Containers

Oak Ridge National Laboratory (ORNL) successfully demonstrated the Versatile Remediation Module (VRM), a prototype module designed and built by ORNL for on-site remote repair of welded stainless steel storage containers for spent nuclear fuel and high-level radioactive waste. This paper describes the VRM prototype and its design features and components to support continued long-term storage or off-site transportation of spent nuclear fuel and high-level radioactive waste currently stored in storage containers. A remote (100 ft away from the simulated radiative environment) demonstration of the VRM was successfully performed on a full-scale mock-up welded stainless steel canister. The VRM is designed with features to accommodate remediation techniques beyond those currently selected and described in this paper. Therefore, many of the VRM’s features may benefit other remote nuclear or nonnuclear applications. The VRM is envisioned to serve as a development center to facilitate and enhance further development of new remediation technologies.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2015

WE-EF-BRA-08: Cell Survival in Modulated Radiation Fields and Altered DNA-Repair at Field Edges

Purpose: Tissue damage prognoses in radiotherapy are based on clonogenic assays that provide dose dependent cell survival rates. However, recent work has shown that apart from dose, systemic reactions and cell-cell communication crucially influence the radiation response. These effects are probably a key in understanding treatment approaches such as microbeam radiation therapy (MRT). In this study we tried to quantify the effects on a cellular level in spatially modulated radiation fields. Methods: Pancreas carcinoma cells were cultured, plated and irradiated by spatially modulated radiation fields with an X-ray tube and at a synchrotron. During and after treatment cells were able to communicate via the intercellular medium. Afterwards we stained for DNA and DNA damage and imaged with a fluorescence microscope. Results: Intriguingly we found that DNA damage does not strictly increase with dose. Two cell entities appear that have either a high or a low amount of DNA lesions, indicating that DNA damage is also a cell stress reaction. Close to radiation boundaries damage-levels became alike; they were higher than expected at low and lower than expected at high doses. Neighbouring cells reacted similarly. 6 hours after exposure around 40% of the cells resembled in their reactions neighbouring cells more than randomly chosen cells that received the same dose. We also observed that close to radiation boundaries the radiation induced cell-cycle arrest disappeared and the size of DNA repair-centres increased. Conclusion: Cell communication plays an important role in the radiation response of tissues and may be both, protective and destructive. These effects may not only have the potential to affect conventional radiotherapy but may also be exploited to spare organs at risk by intelligently designing irradiation geometries. To that end intensive work is required to shed light on the still obscure processes in cell-signalling and radiation biology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in Δnei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of Δlhr cells by mutant lhr alleles depended on Lhr’s ATPase/helicase activities and its tetrameric quaternary structure. The lhr–nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

X-ray crystal structure of the Escherichia coli RadD DNA repair protein bound to ADP reveals a novel zinc ribbon domain

Genome maintenance is an essential process in all cells. In prokaryotes, the RadD protein is important for survival under conditions that include DNA-damaging radiation. Precisely how RadD participates in genome maintenance remains unclear. Here we present a high-resolution X-ray crystal structure of ADP-bound Escherichia coli RadD, revealing a zinc-ribbon element that was not modelled in a previous RadD crystal structure. Insights into the mode of nucleotide binding and additional structure refinement afforded by the new RadD model will help to drive investigations into the activity of RadD as a genome stability and repair factor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Residential Home Repairs, Energy Efficiency, and Electrification in the Hill District of Pittsburgh [Slides]

Through Communities LEAP, a community coalition focused on the Hill District neighborhood of Pittsburgh is working with a technical assistance provider network led by the National Renewable Energy Laboratory (NREL). The coalition includes community organizations, nonprofits, the city government, and the utility. Technical assistance provides analysis and information to support Hill District stakeholders in their goals to create informed residential energy efficiency and renewable energy transition strategies. This report summarizes the insights from stakeholder engagement with organizations that provide home repair services, weatherization, and residential energy efficiency in the Hill District as well as from a building analysis to characterize Hill District housing stock and the potential costs and benefits of energy efficiency and electrification measures.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2021

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A goldilocks computational protocol for inhibitor discovery targeting DNA damage responses including replication-repair functions

While many researchers can design knockdown and knockout methodologies to remove a gene product, this is mainly untrue for new chemical inhibitor designs that empower multifunctional DNA Damage Response (DDR) networks. Here, we present a robust Goldilocks (GL) computational discovery protocol to efficiently innovate inhibitor tools and preclinical drug candidates for cellular and structural biologists without requiring extensive virtual screen (VS) and chemical synthesis expertise. By computationally targeting DDR replication and repair proteins, we exemplify the identification of DDR target sites and compounds to probe cancer biology. Our GL pipeline integrates experimental and predicted structures to efficiently discover leads, allowing early-structure and early-testing (ESET) experiments by many laboratories. By employing an efficient VS protocol to examine protein-protein interfaces (PPIs) and allosteric interactions, we identify ligand binding sites beyond active sites, leveraging in silico advances for molecular docking and modeling to screen PPIs and multiple targets. A diverse 3,174 compound ESET library combines Diamond Light Source DSI-poised, Protein Data Bank fragments, and FDA-approved drugs to span relevant chemotypes and facilitate downstream hit evaluation efficiency for academic laboratories. Two VS per library and multiple ranked ligand binding poses enable target testing for several DDR targets. This GL library and protocol can thus strategically probe multiple DDR network targets and identify readily available compounds for early structural and activity testing to overcome bottlenecks that can limit timely breakthrough drug discoveries. By testing accessible compounds to dissect multi-functional DDRs and suggesting inhibitor mechanisms from initial docking, the GL approach may enable more groups to help accelerate discovery, suggest new sites and compounds for challenging targets including emerging biothreats and advance cancer biology for future precision medicine clinical trials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1964

Self repair techniques

Computer simulation program for self-repairing system design - reliability improvement as compared to use of redundant logic circuits

SELF-REPAIRING SYSTEM↗
NASA NTRS2003

Quantifying clustered DNA damage induction and repair by gel electrophoresis, electronic imaging and number average length analysis

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

NASA Program Biological Systems Research↗
NASA NTRS1997

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Non-NASA Center↗
NASA NTRS1995

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

NASA Discipline Radiation Health↗