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Structural basis for $\mathrm{APE1}$ processing $\mathrm{DNA}$ damage in the nucleosome
Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3-6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.
Universally Accessible Structural Data on Macromolecular Conformation, Assembly, and Dynamics by Small Angle X-Ray Scattering for DNA Repair Insights.
Structures provide a critical breakthrough step for biological analyses, and small angle X-ray scattering (SAXS) is a powerful structural technique to study dynamic DNA repair proteins. As toxic and mutagenic repair intermediates need to be prevented from inadvertently harming the cell, DNA repair proteins often chaperone these intermediates through dynamic conformations, coordinated assemblies, and allosteric regulation. By measuring structural conformations in solution for both proteins, DNA, RNA, and their complexes, SAXS provides insight into initial DNA damage recognition, mechanisms for validation of their substrate, and pathway regulation. Here, we describe exemplary SAXS analyses of a DNA damage response protein spanning from what can be derived directly from the data to obtaining super resolution through the use of SAXS selection of atomic models. We outline strategies and tactics for practical SAXS data collection and analysis. Making these structural experiments in reach of any basic and clinical researchers who have protein, SAXS data can readily be collected at government-funded synchrotrons, typically at no cost for academic researchers. In addition to discussing how SAXS complements and enhances cryo-electron microscopy, X-ray crystallography, NMR, and computational modeling, we furthermore discuss taking advantage of recent advances in protein structure prediction in combination with SAXS analysis.
xGFabric: Coupling Sensor Networks and HPC Facilities with Private 5G Wireless Networks for Real-Time Digital Agriculture
Advanced scientific applications require coupling distributed sensor networks with centralized high-performance computing facilities. Citrus Under Protective Screening (CUPS) exemplifies this need in digital agriculture, where citrus research facilities are instrumented with numerous sensors monitoring environmental conditions and detecting protective screening damage. CUPS demands access to computational fluid dynamics codes for modeling environmental conditions and guiding real-time interventions like water application or robotic repairs. These computing domains have contrasting properties: sensor networks provide low-performance, limited-capacity, unreliable data access, while high-performance facilities offer enormous computing power through high-latency batch processing. Private 5G networks present novel capabilities addressing this challenge by providing low latency, high throughput, and reliability necessary for near-real-time coupling of edge sensor networks with HPC simulations. This work presents xGFabric, an end-to-end system coupling sensor networks with HPC facilities through Private 5G networks. The prototype connects remote sensors via 5G network slicing to HPC systems, enabling real-time digital agriculture simulation.
AI-Enabled Robots for Automated Nondestructive Evaluation and Repair of Power Plant Boilers. Final Report
Boiler failure could cause loss of life and safety issues, cost hundreds of thousands of dollars in equipment repairs, property damage and production losses, and drive up the cost of electric power. Boiler maintenance is challenging and risky for inspectors working on scaffolding in confined hazardous spaces inside of a boiler and sometimes the space is hard to access. The operation is also time-consuming due to the large area of vertical structures for inspection and the tremendous effort needed for scaffolding. Recently, the use of robotics (e.g., drones and crawlers) in power plants for maintenance is growing rapidly. However, the existing robotics solutions show two notable technological gaps: no live repair capability, and no Artificial Intelligence (AI) for smart autonomy. The objective of this project is to develop an integrated autonomous robotic platform that is equipped with compact non-destructive evaluation (NDE) sensors to perform live inspection, operates onboard repair devices to perform live repair, and uses AI for intelligent data fusion and predictive analysis for automated and smart spatiotemporal inspection, analysis and repair of the furnace walls in coal-fired boilers. The approach to achieve the objective includes developing NDE sensors with signal processing techniques, designing and evaluating repair devices for robots based on fusion and solid-state technologies, and an autonomous robotic platform that can attach to and navigate on boiler furnace walls using magnetic drive tracks. The robot is also powered by AI to automate data gathering (e.g., 3D mapping and damage localization) and predictive analysis. This project has advanced the state-of-the-art by providing technological breakthroughs including compact NDE and repair tools for robots, AI capabilities for smart autonomy, and a robotic platform for automated boiler maintenance. This project has great potential to result in significant benefits including limiting or eliminating the need to send operators to assess difficult-to-access or hazardous areas, enabling automated live inspection and repair, avoiding time consuming scaffolding (especially for partial maintenance during unplanned outage), collecting comprehensive and well-organized data smartly, and avoiding or limiting the need for onsite or remote piloting technicians. The impacts can be tremendous in terms of the time and cost savings, reducing the risk for human operators, and increasing boiler reliability, usability, and efficiency. In addition, by developing the new technologies on the autonomous inspection and repair robot, by involving multiple undergraduate and graduate students working together with the faculty members on this project, and by generating knowledge and building up collaborations with industrial partners, this effort will significantly update the education capabilities, support long-term fundamental research, and maintain the leadership of Colorado School of Mines and Michigan State University in energy fields.
Time as a microbial resource
Microbes need resources for energy and cellular building material. They also need access to clement conditions with liquid water and a cellular damage rate that is lower than repair. When deprived of resources and clement conditions, microbes often enter some form of dormancy (e.g., by ceasing cell division, slowing metabolic rate, or forming an endospore) until they can grow again. For example, at night, phototrophs wait for the sun to return. In winter, soil microbes wait for warmer temperatures. Microbes that cause diseases like tuberculosis can stay dormant for years, waiting for the cessation of antibiotic or immune system bombardment. But what about longer timescales? Unlike multicellular life, microbes survive in an extremely broad range of conditions and can access an amazing variety of resources to maintain cellular functions in the absence of cell division. This means that they have the potential to be dormant for much longer than a few months or years. There is no theoretical reason microbes cannot survive on maintenance energy for hundreds or thousands of years, or longer, with little to no cell proliferation. Here, given this lack of theoretical constraints on the length of microbial dormancy intervals, two questions arise, (i) is there evidence for the existence of organisms experiencing very long dormancies? And (ii) what could be the advantages of such long wait times?
Front shielding of the 25-kW beam absorber in Linac-to-Booster transfer line
The 25-kW beam absorber in the Linac-to-Booster transfer line is supposed to take in the beam from PIP-II 800-MeV Linac occasionally, on the average for one hour once a week. Such an operational scenario suggests that residual dose of the absorber front part can be relatively low. In order to simplify access to the front part of the absorber and beam window in case of repair or maintenance, using a slide-in shielding plug is considered as a preferred option.
Bivalent molecular mimicry by ADP protects metal redox state and promotes coenzyme B 12 repair
Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5′-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5′-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5′-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.
Non-Resource Conservation and Recovery Act Corrective Action Unit (CAU) Post-Closure Inspection Report, Nevada National Security Site, Nevada: For Calendar Year 2023
This report includes visual inspection results, descriptions of maintenance and repair activities, and recommendations for CY 2023 for URs at CASs located on the NNSS and on the NTTR that are accessed through the NNSS Main Gate. This document is formatted by project activity (i.e., Industrial Sites, Soils, and DTRA]; then by CAU; and then by CAS.
Post-Closure Inspection Report for the Tonopah Test Range and Nevada Test and Training Range, Nevada, For Calendar Year 2019, Revision 0
This report provides the results of the annual post-closure use restriction (UR) inspections at the closed corrective action sites (CASs) located on the Tonopah Test Range (TTR) and Nevada Test and Training Range (NTTR) that are accessed through the TTR/NTTR main gate. This report covers post-closure UR inspections for calendar year 2019, and includes visual inspections and repair activities completed at the following corrective action units (CAUs): • CAU 400, Bomblet Pit and Five Points Landfill (TTR) • CAU 407, Roller Coaster RadSafe Area (TTR) • CAU 424, Area 3 Landfill Complexes (TTR) • CAU 453, Area 9 UXO Landfill (TTR) • CAU 487, Thunderwell Site (TTR)
Cathodic Protection Modeling for Hanford Underground Double-Shell Tank Farms
Hanford stores millions of gallons of radioactive and chemically hazardous waste from the production of weapon materials in tank farms consisting of underground carbon-steel storage tanks surrounded by reinforced concrete. Six of these Hanford tank farms use double-shell storage tanks (DSTs). The DST farms were constructed from 1968 to 1986 with a planned 40–50 year design life, so some are already operating beyond their initial life expectancy. Ultrasonic testing (UT) has indicated significant thinning on the bottom of the secondary (outer) liner of these tanks, believed to arise from groundwater intrusion driving concrete side corrosion. There is no direct access to the steel/concrete interface between the tank and the concrete pad, making it difficult to apply a chemical-based mitigation strategy or to conduct repairs, but cathodic protection (CP) is a possible method to inhibit further concrete-side corrosion. Hanford already uses CP to protect below grade steel piping within the tank farms and connected to the tanks, but this system was not designed to protect the tank bottoms. CP design must account for the structures surrounding the DSTs, including the steel reinforcing bars (rebar) within the concrete pad and vault, various process lines, and the existing CP system. In this study, finite element analysis (FEA) modeling was carried out to simulate CP protection of 1) a single tank and CP anode to develop options for modeling the rebar and to compare to a simpler circuit model and 2) the entire Hanford AN tank farm as a representative example consisting of seven tanks, associated piping, and both existing and new CP anodes. Both circuit and FEA models predict that significant protective current could be delivered to the bottoms of the tanks with the addition of tank-protection anodes below the depth of the tanks. Simulations with only the existing pipe-protection anodes active confirmed that only a very small current to the tank bottoms is predicted under present conditions. Multiple simplified representations of the dome and wall rebar were tested to reduce the computational complexity of the tank-farm simulations, resulting in modeling the rebar as edge elements with a prescribed effective circumference that matches the real rebar surface area. The geometry of the rebar is also simplified into horizontal hoops around the tank walls and radial rebar over the dome with increased effective circumference to retain the target surface area. This simplification was found to greatly reduce the complexity and solution time of the models without large changes in current distributions, especially to the tank bottom. A range of values were tested for model parameters such as soil and concrete resistivities and polarization resistance to investigate their impact on the current and electric potential distributions. Depending on the parameters used, FEA simulations predict some risk of overprotection, particularly on the piping system; since overprotection can also lead to surface damage associated with hydrogen gas generation at the interface (e.g. hydrogen embrittlement or damage to coatings), this needs to be considered when refining the design of the new CP system. Comparison between the FEA models and the circuit model representation demonstrated that the circuit model could not match the predicted FEA current distribution, even when using the exact same surface areas. This discrepancy appeared to be at least partly attributable to the impact of the relative positions of the tank components and anodes to each other and to the ground surface. The FEA model accounts for the relative positions since it solves the governing equations in three dimensions, but the circuit model cannot account for the positioning. In particular, the circuit model underpredicts the current to the tank bottom and overpredicts the current to the dome compared to FEA for the baseline geometry. The FEA models omitted the electrically isolated rebar in the bottom concrete slab. However, a circuit based stray current model estimated that only 2.1% of the total current through the slab would stray into the rebar, corresponding to ~0.21 A for a target current density of 2 mA/ft2 to the tank bottom. The estimated corrosion driven by this amount of stray current is predicted to yield a lifetime of >400 years for the minimum rebar diameter, assuming an acceptable cross-section area loss of 10%.
Development of an Innovative Inspection Tool for Superheater Tubes in Fossil Fuel Power Plants
Fossil fuel power plants are complex systems containing multiple components that require periodic health monitoring. Failures in these systems can lead to increased downtime for the plant, reduction of power, and significant cost for repairs. Inspections of the plant’s superheater tubes are typically manual, laborious, and extremely time-consuming. This is due to their small diameter size (between 1.3 and 7.6 cm) and the coiled structure of the tubing. In addition, the tubes are often stacked close to each other, limiting access for external inspection. This paper presents the development and testing of an electrically powered pipe crawler that can navigate inside 5 cm diameter tubes and provide an assessment of their health. The crawler utilizes peristaltic motion within the tubes via interconnected modules for gripping and extending. The modular nature of the system allows it to traverse through straight sections and multiple 90° and 180° bends. Additional modules in the system include an ultrasonic sensor for tube thickness measurements, as well as environmental sensors, a light detecting and ranging (LiDAR) sensor, and camera. Furthermore, these modules utilize a gear system that allows for 360° rotation and provides a means to inspect the entire internal circumference of the tubes.
Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites
Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.
Non-Resource Conservation and Recovery Act Corrective Action Unit (CAU) Post-Closure Inspection Report, Nevada National Security Site, Nevada For Calendar Year 2024
This report includes visual inspection results, descriptions of maintenance and repair activities, and recommendations for calendar year 2024 for use restrictions at corrective action sites located on the Nevada National Security Site (NNSS) and on the Nevada Test and Training Range that are accessed through the NNSS Main Gate.
Non-Resource Conservation and Recovery Act Corrective Action Unit (CAU) Post-Closure Inspection Report, Nevada National Security Site, Nevada For Calendar Year 2025
This report includes visual inspection results, descriptions of maintenance and repair activities, and recommendations for CY 2025 for use restrictions (URs) at corrective action sites (CASs) located on the Nevada National Security Site and on the Nevada Test and Training Range (NTTR) that are accessed through the NNSS Main Gate. This document is arranged by project activity (i.e., Industrial Sites, Soils, and Defense Threat Reduction Agency [DTRA]); then by corrective action unit (CAU); and then by CAS. Post-closure UR inspections are performed on an annual basis, unless as noted in text. Post-closure UR inspections verify the condition of physical controls required by each UR, which may include the following activities: • Visual verification of the legibility of signs • Visual inspection of fencing, signs, monuments, and/or markers for indications of wear • Visual inspection of soil covers for indications of subsidence, erosion, or unauthorized use Deficiencies in UR requirements that were identified during CY 2024 inspections and not addressed in the CY 2024 report are addressed in this CY 2025 report.
Non-Resource Conservation and Recovery Act Corrective Action Unit (CAU) Post-Closure Inspection Report, Nevada National Security Site, Nevada (CY2021, Revision 0)
This report includes visual inspection results, maintenance and repair activities, and recommendations for calendar year (CY) 2021 for use restrictions (URs) at corrective action sites (CASs) located on the Nevada National Security Site (NNSS) and on the Nevada Test and Training Range (NTTR) that are accessed through the NNSS Main Gate. This document is formatted by project activity (i.e., Industrial Sites, Soils, and Defense Threat Reduction Agency [DTRA]); then by corrective action unit (CAU); and then by CAS.
Non-Resource Conservation and Recovery Act Corrective Action Unit (CAU) Post-Closure Inspection Report, Nevada National Security Site, Nevada For Calendar Year 2022
This report includes visual inspection results, descriptions of maintenance and repair activities, and recommendations for calendar year (CY) 2022 for use restrictions (URs) at corrective action sites (CASs) located on the Nevada National Security Site (NNSS) and on the Nevada Test and Training Range (NTTR) that are accessed through the NNSS Main Gate. This document is formatted by project activity (i.e., Industrial Sites, Soils, and Defense Threat Reduction Agency [DTRA]); then by corrective action unit (CAU); and then by CAS.
Virtual and Augmented Realities to Support Development, Training, Deployment, and Operations of Remote Systems - 20516
Throughout the U.S. Department of Energy complex, there is a need to deploy new, unique equipment and systems for cleanup activities, waste processing, infrastructure repair, and decommissioning and demolition in radioactive and hazardous environments. The new equipment often consists of custom systems created by integrating and modifying commercial technologies. In addition, the systems are deployed in unique environments due to limited access, waste properties, and the presence of previously installed equipment or infrastructure that has been left in place. The deployment of custom systems that are 'turnkey' ready for first-time installation and application has its risks. Use of test platforms and mockups for cold (i.e., non-radioactive) testing is one method to mitigate that risk: however, full-scale test mockups for operations such as waste retrieval and tank inspection can be expensive and must be modified to incorporate the uniqueness of each application. An alternative to full-scale mockups is to use three-dimensional (3D) virtual environments and virtual reality (VR) technology to develop interactive virtual test beds. These can be used throughout the down-selection, development, system integration, operational planning, operator training, and mission execution phases of a task. Virtual test beds are readily reconfigured, can be used for multiple tasks, support multiple users in parallel, and can accommodate enhancements and changes not originally planned or conceived in the initial design. Virtual test beds also provide a platform for developing and testing augmented reality (AR) tools for improving operator efficiency. A VR experience has been generated for waste retrieval from a Hanford waste tank to present the potential applications of a virtual test bed. Features of the VR experience are presented, potential applications of VR and AR for remote applications are discussed, and development areas for improving future application are considered. (authors)