Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Repair”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58
All sourcesNASA NTRSDOE OSTI
NASA NTRSDate not supplied

Investigating Laser Beam Welding as an In-Space Joining Technique via Thermal Vacuum and Microgravity and Vacuum Experiments

In-space joining technologies are crucial for stimulating an in-space economy and for enabling sustained space exploration by in-space manufacturing and repair of metallic structures. Compared to brazing or soldering, in-space welding (ISW) can provide highly hermetic, strong, and complex joints, potentially without introducing additional material. However, the influence of extreme temperatures, reduced pressure, and reduced gravity on ISW is not yet fully elucidated. Several efforts at NASA are investigating laser beam welding (LBW) as a joining and repair method in both thermal vacuum (TVAC) and combined vacuum & reduced gravity environments. NASA Marshall Space Flight Center (MSFC) shepherded several ISW projects in its past, including the 1973 electron beam welding on Skylab, the 1989 low-power LBW on parabolic flights, and the unflown 1990s-era In-Space Welding Experiment. Recent parabolic flights and 3 degree-of-freedom ground testing build upon this heritage. A collaboration with the Ohio State University using NASA Langley Research Center (LaRC) hardware retrofitted for LBW achieved the first high-powered laser welds under vacuum and low gravity and developed a workforce capable developing such experimental hardware. A ground testing campaign at the MSFC Flat Floor simulated fit-up and welding representative of ISW in 3 degrees of freedom to emulate microgravity effects on inertial systems. One ongoing effort is a NASA Early Career Initiative project – Lunar Assembly and Servicing by Autonomous Robotics (LASAR). Ruggedized LBW components were developed by an external partner for use in TVAC. A TVAC-rated robotic arm was procured by MSFC and used in the first known robotic laser weld where all components save the laser generator were under vacuum. NASA Johnson Space Center (JSC) is advancing supervised autonomy of ISW. NASA LaRC continues to adapt their unique snowflake joint geometry, suitable for connecting segments in trusses and other structures, to LBW. Upcoming TVAC campaigns will focus on testing extreme temperatures, proving out autonomous operations, and demonstrating weld repair. Weld inspection will occur via a non-contact nondestructive evaluation (NDE) technique – electromagnetic acoustic transduction (EMAT). Another ongoing effort based at MSFC is the DISCMAN -- DIsk-Shaped Configurable and Modular vAcuum uNit – which seeks to development a compact, modular payload that can provide a vacuum environment while in a reduced gravity condition. This payload could support multiple in-space manufacturing developmental efforts, with the first demonstration technology being LBW. Currently, the design is targeting operations in the pressurized volume of a space station, but the payload could readily be adapted to other flight platforms such as parabolic or even suborbital vehicles. LASAR elucidates the effects of temperature and vacuum on LBW while DISCMAN probes those of vacuum and gravity. Through these complementary efforts, NASA is addressing the primary challenges of ISW across the space environment while simultaneously developing and maturing technologies including robotic systems and inspection methodologies for future practical implementation on the Moon and beyond. This approach is timely, as upcoming missions requiring sustained human presence in space will depend on reliable ISW capabilities to create robust metallic joints currently unproven in the space environment and to perform repairs in situ .

hypogravity↗
DOE OSTI2017

Estimating a Service-Life Distribution Based on Production Counts and a Failure Database

A manufacturer wanted to compare the service-life distributions of two similar products. These concern product lifetimes after installation (not manufacture). For each product, there were available production counts and an imperfect database providing information on failing units. In the real case, these units were expensive repairable units warrantied against repairs. Failure (of interest here) was relatively rare and driven by a different mode/mechanism than ordinary repair events (not of interest here). Approach: Data models for the service life based on a standard parametric lifetime distribution and a related limited failure population were developed. These models were used to develop expressions for the likelihood of the available data that properly accounts for information missing in the failure database. Results: A Bayesian approach was employed to obtain estimates of model parameters (with associated uncertainty) in order to investigate characteristics of the service-life distribution. Custom software was developed and is included as Supplemental Material to this case study. One part of a responsible approach to the original case was a simulation experiment used to validate the correctness of the software and the behavior of the statistical methodology before using its results in the application, and an example of such an experiment is included here. Because of confidentiality issues that prevent use of the original data, simulated data with characteristics like the manufacturer’s proprietary data are used to illustrate some aspects of our real analyses. Lastly, we also note that, although this case focuses on rare and complete product failure, the statistical methodology provided is directly applicable to more standard warranty data problems involving typically much larger warranty databases where entries are warranty claims (often for repairs) rather than reports of complete failures.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2018

Evaluating Energy Impacts and Costs from PV Component Failures: Preprint

The PV Reliability Performance Model (PV-RPM) tool is used to simulate the cost and energy impacts of component faults and failures for a hypothetical PV system. This model, originally developed by Sandia National Laboratories, is a new feature in the National Renewable Energy Laboratory's System Advisor Model (SAM), which performs stochastic analysis sampling of probability distributions for faults, failures, and repairs. One PV system was created for this analysis to be representative of a portfolio of maintenance data maintained by Sandia National Laboratories. Fault, failure, and repair distributions from this system are varied from current baseline conditions to simulate different reliability and repair scenarios and resulting energy and cost impacts. Results highlight ways to express the uncertainty around PV system performance when considering the probabilistic behavior of components in the system, and ways the PV-RPM model can be used to explore different failure and repair scenarios.

14 SOLAR ENERGY↗
DOE OSTI2021

Smart Methane Emission Detection System Development (Final Report)

Working with the Department of Energy’s National Energy Technology Laboratory, Southwest Research Institute® (SwRI®) developed a system to identify methane leaks reliably, accurately, and autonomously at critical midstream sections of the natural gas distribution network in real- time for the purpose of mitigating methane emissions using Optical Gas Imaging (OGI) cameras. SwRI’s Smart Leak Detection – Methane (SLED/M) adds a high degree of automation to the process of methane leak detection to minimize sources of human error, minimize response time to a leak event, and maximize midstream visibility. Furthermore, SwRI has been working towards integrating Quantitative OGI (QOGI) capabilities into this existing technology. By leveraging Deep Learning, SwRI now has the capability to estimate fugitive emission leak rates quickly and reliably, which allows operators to detect emissions, quantify leak rate, prioritize repairs, and validate the repairs in a single instrument. The next generation QOGI technology leverages the same cameras used in Leak Detection and Repair (LDAR) programs, with improvements in safety and speed for traditional quantification-based repairs, ultimately leading to less overhead cost for the operators. The goals for this research were to develop two types of models with the following goals: 1. Run in real-time on the edge (≥ 12 Hz) 2. Classification: Achieve less than 5% false positive detection 3. Classification: Achieve ≥ 95% methane plume detection rate 4. Regression: achieve ≤ 10 standard cubic feet per hour (scfh) prediction > 70% of the time In order to achieve these results, multiple infrared (IR) and other sensors were investigated in tandem with the midwave IR (MWIR) OGI to provide additional information to train the underlying models. Information on atmospheric conditions including humidity, temperature, pressure, and solar radiation was provided by a weather station. Several machine learning and deep learning architectures and methods, including looking at quantized classification networks and regressions networks, were explored. As further data was collected, curated, and labeled, it allowed for more refined regressive networks to be adequately trained, leading to better insight into the true flow rates being observed. An important valuable deliverable of this research effort was the development of an advanced network which underwent multiple iterations capable of giving a continuous output. The current network has a predicted mean average percentage error (MAPE) of 12.3% just outside our target goal of 10.00%, but an accuracy of 97.78% at ±50 scfh, well within the overall goal for the Department of Energy (DOE) program. Upon closer inspection, it was observed that more than 10% of datapoints contributing to the MAPE predictions were the result of low flow rate predictions and are beyond the sensitivity of instrument measurement as a result of normal operational variation and noise.

03 NATURAL GAS↗
DOE OSTI2014

Structure and specificity of FEN-1 from Methanopyrus kandleri

DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Ultimately, we found both to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Xylem embolism refilling and resilience against drought-induced mortality in woody plants: processes and trade-offs

Understanding which species are able to recover from drought, under what conditions, and the mechanistic processes involved, will facilitate predictions of plant mortality in response to global change. In response to drought, some species die because of embolism-induced hydraulic failure, whilst others are able to avoid mortality and recover, following rehydration. Several tree species have evolved strategies to avoid embolism, whereas others tolerate high embolism rates but can recover their hydraulic functioning upon drought relief. Here, we focus on structures and processes that might allow some plants to recover from drought stress via embolism reversal. We provide insights into how embolism repair may have evolved, anatomical and physiological features that facilitate this process, and describe possible trade-offs and related costs. Recent controversies on methods used for estimating embolism formation/repair are also discussed, providing some methodological suggestions. Although controversial, embolism repair processes are apparently based on the activity of phloem and ray/axial parenchyma. The mechanism is energetically demanding, and the costs to plants include metabolism and transport of soluble sugars, water and inorganic ions. We propose that embolism repair should be considered as a possible component of a ‘hydraulic efficiency-safety’ spectrum. We also advance a framework for vegetation models, describing how vulnerability curves may change in hydrodynamic model formulations for plants that recover from embolism.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2015

Regulation of Fanconi anemia protein FANCD2 monoubiquitination by miR-302

Fanconi anemia (FA) is a recessively inherited multigene disease characterized by congenital defects, progressive bone marrow failure, and heightened cancer susceptibility. Monoubiquitination of the FA pathway member FANCD2 contributes to the repair of replication stalling DNA lesions. However, cellular regulation of FANCD2 monoubiquitination remains poorly understood. In the present study, we identified the miR-302 cluster as a potential regulator of FANCD2 by bioinformatics analysis. MicroRNAs (miRNAs) are the major posttranscriptional regulators of a wide variety of biological processes, and have been implicated in a number of diseases. Expression of the exogenous miR-302 cluster (without miR-367) reduced FANCD2 monoubiquitination and nuclear foci formation. Furthermore, miR-302 cells showed extensive chromosomal breakage upon MMC treatment when compared to mock control cells. Taken together, our results suggest that overexpression of miR-302 plays a critical role in the regulation of FANCD2 monoubiquitination, resulting in characteristic defects in DNA repair within cells. - Highlights: • miR-302 binds to the 3′UTR promoter of the FANCD2 gene to regulate gene expression. • miR-302 cluster down-regulates FANCD2 protein expression. • miR-302 cluster reduces FANCD2 monoubiquitination and nuclear foci formation. • miR-302 exhibits the characteristic defects in DNA repair in cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers

Deinococcus radiodurans can survive extreme doses of ionizing radiation due to the very efficient DNA repair mechanisms that are able to cope even with hundreds of double-strand breaks. RecA, the critical protein of homologous recombination in bacteria, is one of the key components of the DNA-repair system. Repair of double-strand breaks requires RecA binding to DNA and assembly of the RecA nucleoprotein helical filaments. The Escherichia coli RecA protein (EcRecA) and its interactions with DNA have been extensively studied using various approaches including single-molecule techniques, while the D. radiodurans RecA (DrRecA) remains much less characterized. However, DrRecA shows some remarkable differences from E. coli homolog. Here we combine microfluidics and single-molecule DNA manipulation with optical tweezers to follow the binding of DrRecA to long double-stranded DNA molecules and probe the mechanical properties of DrRecA nucleoprotein filaments at physiological pH. Our data provide a direct comparison of DrRecA and EcRecA binding to double-stranded DNA under identical conditions. We report a significantly faster filaments assembly as well as lower values of persistence length and contour length for DrRecA nucleoprotein filaments compared to EcRecA. Our results support the existing model of DrRecA forming more frequent and less continuous filaments relative to those of EcRecA. - Highlights: • We investigate Deinococcus radiodurans RecA interactions with long double-stranded DNA at the single-molecule level. • At physiological pH D. radiodurans RecA forms nucleoprotein filaments significantly faster relative to Escherichia coli RecA. • D. radiodurans RecA-dsDNA nucleoprotein filaments are more flexible and slightly shorter compared to those of E. coli RecA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

DNA damage preceding dopamine neuron degeneration in A53T human α-synuclein transgenic mice

Defective DNA repair has been linked with age-associated neurodegenerative disorders. Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by genetic and environmental factors. Whether damages to nuclear DNA contribute to neurodegeneration of PD still remain obscure. in this study we aim to explore whether nuclear DNA damage induce dopamine neuron degeneration in A53T human α-Synuclein over expressed mouse model. We investigated the effects of X-ray irradiation on A53T-α-Syn MEFs and A53T-α-Syn transgene mice. Our results indicate that A53T-α-Syn MEFs show a prolonged DNA damage repair process and senescense phenotype. DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice and decrease the number of nigrostriatal dopaminergic neurons. Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages. - Highlights: • This study explore contribution of DNA damage to neurodegeneration in Parkinson's disease mice. • A53T-α-Syn MEF cells show a prolonged DNA damage repair process and senescense phenotype. • DNA damage preceded onset of motor phenotype in A53T-α-Syn transgenic mice. • DNA damage decrease the number of nigrostriatal dopaminergic neurons. • Neurons of A53T-α-Syn transgenic mice are more fragile to DNA damages.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

The combination of A-966492 and Topotecan for effective radiosensitization on glioblastoma spheroids

Radiotherapy is one of the modalities in the treatment of glioblastoma patients, but glioma tumors are resistant to radiation and also chemotherapy drugs. Thus, researchers are investigating drugs which have radiosensitization capabilities in order to improve radiotherapy. PARP enzymes and topoisomerase I enzymes have a critical role in repairing DNA damage in tumor cells. Thus, inhibiting activity of these enzymes helps stop DNA damage repair and increase DSB lethal damages. In the current study, we investigated the combination of TPT as a topoisomerase I inhibitor, and A-966492 as a novel PARP inhibitor for further radiosensitization. U87MG cells (a human glioblastoma cell line) were cultured in Poly-Hema coated flasks to reach 300 μm-diameter spheroids. Treatments were accomplished by using non-toxic concentrations of A-966492 and Topotecan. The surviving fraction of treated cells was determined by clonogenic assay after treatment with drugs and 6 MV X-ray. The γ-H2AX expression was measured by an immunofluorescence staining method to examine the influence of A-966492, TPT and radiation on the induction of double stranded DNA breaks. Treatments using the A-966492 drug were conducted in concentration of 1 μM. Combining A-966492 and TPT with radiation yielded enhanced cell killing, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER{sub 50}) 1.39 and 1.16 respectively. Radio- and chemo-sensitization was further enhanced when A-966492 was combined with both X-ray and TPT, with SER{sub 50} of 1.53. Also γ-H2AX expression was higher in the group treated with a combination of drugs and radiation. A-966492 is an effective PARP inhibitor and has significant radio-sensitivity on U87MG spheroids. By accumulating cells in the S phase and by inhibiting the DNA damage repair, TPT enhanced radio-sensitivity. A-966492 combined with TPT as a topoisomerase I inhibitor had additive radio-sensitizing effects. As a result, applying PARP and topoisomerase I inhibitors can be a suitable strategy for improving radiotherapy in clinics. - Highlights: • A-966492 is an effective drug with significant radiosensitivity on U87MG spheroids. • Combination of A-966492 and TPT had synergistic radiosensitizing effect. • Applying PARP and topoisomerase I inhibitors can improve radiotherapy treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RecBCD (Exonuclease V) is inhibited by DNA adducts produced by cisplatin and ultraviolet light

Highlights: • DNA adducts inhibited the recombinational repair enzyme RecBCD. • UV-induced DNA adducts inhibited RecBCD. • Cisplatin-induced DNA adducts inhibited RecBCD. The presence of adducts on the DNA double-helix can have major consequences for the efficient functioning of DNA repair enzymes. E. coli RecBCD (exonuclease V) is involved in recombinational repair of double-strand breaks that are caused by defective DNA replication, DNA damaging agents and other factors. The holoenzyme possesses a bipolar helicase activity which helps unwind DNA from both 3′- and 5′-directions and is coupled with a potent exonuclease activity that is also capable of digesting DNA from both 3′- and 5′-ends. In this study, DNA sequences were damaged with cisplatin or UV followed by RecBCD treatment. DNA damaging agents such as cisplatin and UV induce the formation of intrastrand adducts in the DNA template. It was demonstrated that RecBCD degradation was inhibited by either cisplatin-damaged or UV-damaged DNA sequences. This is the first occasion that RecBCD has been demonstrated to be inhibited by DNA adducts induced by cisplatin or UV. In addition, we quantified the amounts of DNA remaining after RecBCD treatment and observed that the level of inhibition was concentration and dose dependent. A DNA-targeted 9-aminoacridinecarboxamide cisplatin analogue was also found to inhibit RecBCD activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Cell-type specific role of the RNA-binding protein, NONO, in the DNA double-strand break response in the mouse testes

Here, the tandem RNA recognition motif protein, NONO, was previously identified as a candidate DNA double-strand break (DSB) repair factor in a biochemical screen for proteins with end-joining stimulatory activity. Subsequent work showed that NONO and its binding partner, SFPQ, have many of the properties expected for bona fide repair factors in cell-based assays. Their contribution to the DNA damage response in intact tissue in vivo has not, however, been demonstrated. Here we compare DNA damage sensitivity in the testes of wild-type mice versus mice bearing a null allele of the NONO homologue (Nono gt ). In wild-type mice, NONO protein was present in Sertoli, peritubular myoid, and interstitial cells, with an increase in expression following induction of DNA damage. As expected for the product of an X-linked gene, NONO was not detected in germ cells. The Nono gt/0 mice had at most a mild testis developmental phenotype in the absence of genotoxic stress. However, following irradiation at sublethal, 2–4 Gy doses, Nono gt/0 mice displayed a number of indicators of radiosensitivity as compared to their wild-type counterparts. These included higher levels of persistent DSB repair foci, increased numbers of apoptotic cells in the seminiferous tubules, and partial degeneration of the blood-testis barrier. There was also an almost complete loss of germ cells at later times following irradiation, evidently arising as an indirect effect reflecting loss of stromal support. Results demonstrate a role for NONO protein in protection against direct and indirect biological effects of ionizing radiation in the whole animal.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Neutron diffraction illustrates residual stress behavior of welded alloys used as radioactive confinement boundary

Corrosion-resistant welded alloys are frequently used as a leak-tight boundary in critical applications that require confinement of hazardous and/or radioactive substances, including an increasing population of spent nuclear fuel (SNF) canisters. The behavior of residual stresses generated as a result of irregular elastic–plastic deformation during processes such as welding is one of today's key issues to a full understanding of the aging mechanisms that may compromise the confinement boundary. Whether such processes and any subsequent weld repairs, not subjected to post-weld heat treatment, would negatively affect the initial material by introducing through-thickness tensile stresses remains an open question. Here we report the first residual stress measurements using neutron diffraction on the welded joints of a SNF canister. We found significant tensile residual stresses in the as welded sample, indicating that initiation and through-thickness growth of cracks may be possible. Following repair, we observed a stress redistribution and introduction of beneficial compressive stresses. We anticipate our results will improve understanding of confinement susceptibility to aging and guide improvements in repair techniques.

36 MATERIALS SCIENCE↗
DOE OSTI2014

Targeting Nucleophosmin 1 Represents a Rational Strategy for Radiation Sensitization

Purpose: To test the hypothesis that small molecule targeting of nucleophosmin 1 (NPM1) represents a rational approach for radiosensitization. Methods and Materials: Wilde-type and NPM1-deficient mouse embryo fibroblasts (MEFs) were used to determine whether radiosensitization produced by the small molecule YTR107 was NPM1 dependent. The stress response to ionizing radiation was assessed by quantifying pNPM1, γH2AX, and Rad51 foci, neutral comet tail moment, and colony formation. NPM1 levels in a human-derived non-small-cell lung cancer (NSCLC) tissue microarray (TMA) were determined by immunohistochemistry. YTR107-mediated radiosensitization was assessed in NSCLC cell lines and xenografts. Results: Use of NPM1-null MEFs demonstrated that NPM1 is critical for DNA double- strand break (DSB) repair, that loss of NPM1 increases radiation sensitivity, and that YTR107-mediated radiosensitization is NPM1 dependent. YTR107 was shown to inhibit NPM1 oligomerization and impair formation of pNPM1 irradiation-induced foci that colocalized with γH2AX foci. Analysis of the TMA demonstrated that NPM1 is overexpressed in subsets of NSCLC. YTR107 inhibited DNA DSB repair and radiosensitized NSCLC lines and xenografts. Conclusions: These data demonstrate that YTR107-mediated targeting of NPM1 impairs DNA DSB repair, an event that increases radiation sensitivity.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2015

Survival Fraction at 2 Gy and γH2AX Expression Kinetics in Peripheral Blood Lymphocytes From Cancer Patients: Relationship With Acute Radiation-Induced Toxicities

Purpose: Predictive assays for acute radiation toxicities would be clinically relevant in radiation oncology. We prospectively examined the predictive role of the survival fraction at 2 Gy (SF2) and of γH2AX (double-strand break [DSB] DNA marker) expression kinetics in peripheral blood mononuclear cells (PBMCs) from cancer patients before radiation therapy. Methods and Materials: SF2 was measured with Trypan Blue assay in the PBMCs from 89 cancer patients undergoing radiation therapy at 4 hours (SF2{sub [4h]}) and 24 hours (SF2{sub [24h]}) after ex vivo irradiation. Using Western blot analysis and band densitometry, we further assessed the expression of γH2AX in PBMC DNA at 0 hours, 30 minutes, and 4 hours (33 patients) and 0 hour, 4 hours, and 24 hours (56 patients), following ex vivo irradiation with 2 Gy. Appropriate ratios were used to characterize each patient, and these were retrospectively correlated with early radiation therapy toxicity grade. Results: The SF2{sub (4h)} was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio{sub (30min)} (band density of irradiated/non-irradiated cells at 30 minutes) revealed, similarly, a significant inverse association (P=.0001). The DSB DNA repair rate from 30 minutes to 4 hours, calculated as the relative RγH2AX-ratio (γH2AX-ratio{sub (4h)}/γH2AX-ratio{sub (30min)}) showed a significant direct association with high toxicity grade (P=.01). Conclusions: Our results suggest that SF2 is a significant radiation sensitivity index for patients undergoing radiation therapy. γH2AX Western blot densitometry analysis provided 2 important markers of normal tissue radiation sensitivity. Low γH2AX expression at 30 minutes was linked with high toxicity grade, suggesting that poor γH2AX repair activity within a time frame of 30 minutes after irradiation predicts for poor radiation tolerance. On the other hand, rapid γH2AX content restoration at 4 hours after irradiation, compatible with efficient DSB repair ability, predicts for increased radiation tolerance.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2016

MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Results: Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. Conclusions: These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2016

Influence of Nucleoshuttling of the ATM Protein in the Healthy Tissues Response to Radiation Therapy: Toward a Molecular Classification of Human Radiosensitivity

Purpose: Whereas post–radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases. Methods and Materials: Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers of micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group. Results: OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. Conclusions: Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III).

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2016

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells

Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

62 RADIOLOGY AND NUCLEAR MEDICINE↗