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All sourcesNASA NTRSDOE OSTI
NASA NTRS2017

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System

Exposure to space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized exposure to ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-week old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 mega electron volts per nucleon) or high-LET (sup 56) Fe ions (600 mega electron volts per nucleon) using either low (5 or 10 centigrays) or high (50 or 200 centigrays) doses at NASAs Space Radiation Lab at Brookhaven National Lab (NSRL/BNL). Tissues were harvested 5 weeks or 1 year after irradiation and bones were analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed for select groups by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) during the proliferative phase or the mineralizing phase, and differentiation was analyzed by imaging mineralized nodules (percentage surface area). Representative genes were selected for expression analyses, including cell proliferation (PCNA, Cdk2, p21, p53), differentiation (Runx2, Alpl, Bglap), oxidative metabolism (Catalase, GPX, MnSOD, CuZnSOD, iNos, Foxo1), DNA-damage repair (Gadd45), or apoptosis (Caspase 3). As expected, a high dose (200 centigrays), but not low doses, of either (sup 56) Fe or protons caused a loss of cancellous bone volume per total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; (sup 56) Fe (200 centigrays) inhibited median nodule area by more than 90 percent at 5 weeks and 1 year post-irradiation, compared to controls. At 5 weeks post exposure, irradiation with protons or (sup 56) Fe caused few changes in gene expression levels during osteoblastogenesis, although a high dose of (sup 56) Fe (200 centigrays) increased levels of Catalase and Gadd45. In addition, supplementing cell culture media with SOD protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET ((sup 137) Cs gamma) if irradiated in vitro, but had limited protective effects on high-LET (sup 56) Fe-exposed cells. In sum, exposure of mice to either protons or (sup 56) Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET (sup 56) Fe increased expression of redox-related genes and inhibited osteoblastogenesis, albeit to a limited extent. We conclude that high-LET irradiation impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

ionizing radiation↗
NASA NTRS2022

Merging of the Case 2 Regional Coast Colour and Maximum‑Peak Height Chlorophyll‑a Algorithms: Validation and Demonstration of Satellite‑derived Retrievals Across US Lakes

Water quality monitoring is relevant for protecting the designated, or beneficial uses, of water such as drinking, aquatic life, recreation, irrigation, and food supply that support the economy, human well-being, and aquatic ecosystem health. Managing finite water resources to support these designated uses requires information on water quality so that managers can make sustainable decisions. Chlorophyll-a (chl-a, µg L−1) concentration can serve as a proxy for phytoplankton biomass and may be used as an indicator of increased anthropogenic nutrient stress. Satellite remote sensing may present a complement to in situ measures for assessments of water quality through the retrieval of chl-a with in-water algorithms. Validation of chl-a algorithms across US lakes improves algorithm maturity relevant for monitoring applications. This study compares performance of the Case 2 Regional Coast Colour (C2RCC) chl-a retrieval algorithm, a revised version of the Maximum-Peak Height (MPH(P)) algorithm, and three scenarios merging these two approaches. Satellite data were retrieved from the MEdium Resolution Imaging Spectrometer (MERIS) and the Ocean and Land Colour Instrument (OLCI), while field observations were obtained from 181 lakes matched with U.S. Water Quality Portal chl-a data. The best performance based on mean absolute multiplicative error (MAEmult) was demonstrated by the merged algorithm referred to as C15−M10 (MAEmult = 1.8, biasmult = 0.97, n = 836). In the C15−M10 algorithm, the MPH(P) chl-a value was retained if it was > 10 µg L−1; if the MPH(P) value was ≤ 10 µg L−1, the C2RCC value was selected, as long as that value was < 15 µg L−1. Time-series and lake-wide gradients compared against independent assessments from Lake Champlain and long-term ecological research stations in Wisconsin were used as complementary examples supporting water quality reporting requirements. Trophic state assessments for Wisconsin lakes provided examples in support of inland water quality monitoring applications. This study presents and assesses merged adaptations of chl-a algorithms previously reported independently. Additionally, it contributes to the transition of chl-a algorithm maturity by quantifying error statistics for a number of locations and times.

Satellite↗
DOE OSTI2026

Smart Planning for Radioactive Source Transport Advanced Tools for Increased Safety and Efficiency

End-of-life (EOL) management of high-activity radioactive sources is made uniquely challenging by the inherent risks associated with storage and transportation of these sources, the complex logistics involved, and the strict requirements for regulatory compliance. Traditional methods lack comprehensive tools for accurate site assessments and precision planning for the transportation of radioactive sources. They also frequently fail to provide the adaptability required to consider diverse operational environments, resulting in inefficiencies and potential safety concerns. This paper introduces a novel software solution developed to address these issues by integrating advanced technologies such as light detection and ranging (LiDAR)-based 3D environment modeling, smart dynamic route planning, and customizable measurement functionalities. This software enables detailed terrain visualizations, facilitating thorough environmental assessments and enabling users to virtually navigate, analyze, and plan site-specific operations. Among the key features are a user-centric interface for virtual navigation, precise site measurement tools for site evaluations, interactive visualizations that highlight potential operational hazards, dynamic route planning capabilities, and real-time collision detection to promote safe workflows. By demonstrating the effectiveness of this tool through real-world application, the present work underscores the tool’s potential to revolutionize radioactive source EOL management by improving operational efficiencies, minimizing risk, and advancing the state of practice to achieve suitable and secure radioactive material handling.

99 - GENERAL AND MISCELLANEOUS↗
DOE OSTI2025

Evaluation of a high-throughput method for processing sponge-stick samples to detect viable, non-spore-forming biothreat agents

After a bioterrorism incident, surface sampling is often used to determine the extent of contamination and exposure, guiding decontamination efforts and decisions for re-occupancy of affected sites. The sponge-stick (SS) is a preferred and commonly used device for sample collection to detect both spore-forming and non-spore-forming biothreat agents from non-porous surfaces. Here, in this study, a recently developed high-throughput method (HTM) for processing SS samples to detect viable Bacillus anthracis spores was adapted for detection of non-spore-forming biothreat agents, Yersinia pestis and Francisella tularensis. The scalable HTM was used to process up to 20 SS samples simultaneously, compared to the current stomacher-based method which processes one SS at a time. Comparisons of the HTM and the stomacher-based method were statistically indistinguishable for most experiments (P > 0.05) with HTM recoveries of 37–60 % for Y. pestis inoculated at 102–103 cells/SS and held 48 h at 4 °C to mimic sample transport/storage. The HTM was integrated with Rapid Viability-Polymerase Chain Reaction (RV-PCR) analysis to detect viable Y. pestis in the presence of particulate contamination (Arizona Test Dust, ATD). This approach detected Y. pestis inoculated at 20 cells/SS and ATD did not impact detection (P > 0.05). F. tularensis showed significantly lower recoveries between no-hold time and 48-h hold time (4 °C, P < 0.05) using the HTM, which further testing showed could be due to toxicity of the neutralizing buffer used for SS pre-wetting. With modifications, this method could enhance throughput capacity while maintaining similar recovery efficiencies to current methods for other non-spore-forming bacterial pathogens.

Biological and medical sciences↗
DOE OSTI2025

Outlook towards deployable continual learning for particle accelerators

Particle accelerators are high power complex machines. To ensure uninterrupted operation of these machines, thousands of pieces of equipment need to be synchronized, which requires addressing many challenges including design, optimization and control, anomaly detection and machine protection. With recent advancements, machine learning (ML) holds promise to assist in more advance prognostics, optimization, and control. While ML based solutions have been developed for several applications in particle accelerators, only few have reached deployment and even fewer to long term usage, due to particle accelerator data distribution drifts caused by changes in both measurable and non-measurable parameters. In this paper, we identify some of the key areas within particle accelerators where continual learning can allow maintenance of ML model performance with distribution drifts. Particularly, we first discuss existing applications of ML in particle accelerators, and their limitations due to distribution drift. Next, we review existing continual learning techniques and investigate their potential applications to address data distribution drifts in accelerators. By identifying the opportunities and challenges in applying continual learning, this paper seeks to open up the new field and inspire more research efforts towards deployable continual learning for particle accelerators.

43 PARTICLE ACCELERATORS↗
DOE OSTI2026

FY 2026 Midyear Report: Seismic Monitoring of Underground Vibration Sources Using Distributed Acoustic Sensing and Seismometers

Safeguards-relevant temporal changes in underground facilities can be observed using geophysical monitoring techniques. Seismic waves, in particular, provide valuable insights into subsurface activities and can serve as an important tool for detecting anomalous events that may indicate containment breaches at geological repositories. This midyear report summarizes ongoing efforts to automatically and rapidly detect and locate anomalous vibration signals that could be indicative of potential containment breaches. Previous work during FY25 focused on compiling continuous seismic datasets from two underground sites and developing a database of continuous waveforms and ground-truth event data derived from multiple sensing modalities. Building on this foundation, we are adapting anomaly detection and geolocation algorithms to explore methods for monitoring underground activities using two relatively low-maintenance sensing technologies: a dense surface geophone array deployed at the Pleasant Gap mine in Pennsylvania, and a three-dimensional fiber-optic cable array for distributed acoustic sensing (DAS) installed in the subsurface at the Sanford Underground Research Facility (SURF) in South Dakota. This report summarizes work conducted during the first two quarters of FY26, during which we refined a dynamic power spectral density (PSD)-based detector, applied it independently to each geophone station, and then combined the per‑station detections with density-based spatial clustering of applications with noise (DBSCAN) to cluster events and produce spatial maps over a nine‑day interval. In addition, we outline plans for a field trial at the Waste Isolation Pilot Plant (WIPP) in New Mexico to compare traditional seismic monitoring approaches with DAS techniques and to evaluate the benefits of combined data analysis. Activities during the past two quarters have included the preparation and submission of a Field Test Plan to WIPP for approval, as well as submission to headquarters for review and feedback.

58 GEOSCIENCES↗
DOE OSTI2020

Feedback on Forty-year Long Clean-up Operations of a Contaminated Soil for Environmental Purpose - 20026

One of the CEA facilities in France is the place of miscellaneous mapping and clean-up operations since the 1980's. The final purpose of the conducted work deals with the environmental remediation of the building. In France, the absence of a regulatory framework for the management of sites polluted by radioactive substances, especially as the lack of release thresholds, has led to develop different approaches of soil radiological characterization. The geostatistical approach is part of them. Preliminary investigations are an unavoidable step prior to clearance and remediation. First, historical analysis enables to define site perimeters to investigate as well as to precisely locate expected contaminated parts. For radioactive pollutants, the media (water, soil, air) and transfer routes participate in the definition of the investigation perimeter and thus also need to be identified through a geological study. At the same time, functional analysis leads to choose the best measurement means which could provide useful information when combined with visual inspection. An equipment well adapted to easy-to-measure radionuclides as gamma emitters, such as gamma probe or on-site gamma spectrometry, is usually selected as a non-destructive assay. Coupled with spatial positions, processed on-site radiological data can be mapped. Data processing consists in mapping and kriging before a geostatistical analysis in order to identify the zones of interest to be targeted. Selected zones are not only linked to high level count rates but also to low level ones so that a pollution-free reference is known in the area. This data processing also enables to categorize waste types according to their origin and contamination levels. An expected volume of different waste then follows as well as the waste characterization equipments. In-depth investigations, e.g. core drillings and samplings, naturally ensue to aim at remediation optimization through the assessment of environmental impact. After clean-up operations, non-destructive assays as well as destructive samplings enable to check the effective clearance. Following this methodology in the present remediation work, results of the first non-destructive assay campaign highlighted a long-lived transuranic radionuclides contamination in the concrete flagstone. Successive non-destructive assays were then performed in the defined zones of interest. Count rates measured with a surface probe were first mapped. The best localization of destructive assays, i.e. core drillings at a 1 meter depth, came from this surface mapping followed by a geostatistical analysis. The concrete flagstone was totally removed. Then, the depth of soil to remove under the flagstone was optimized from the results of radiological activities measured in core drillings samples. A new surface mapping was then drawn after this partial soil excavation. The sand and demolition rubble samples, homogeneously constituted from the flagstone fragments, were analyzed by gamma spectrometry. Meanwhile, waste drums and other containers were also measured by gamma spectrometry and passive neutron measurement devices. All these results have brought miscellaneous pieces of information. In this context, major issues appear in terms of physical constraints such as the premises tininess as well as of soil sampling techniques and in terms of measurement performances assessment and the representativeness of homogeneous samples. The choice of radiological soil and waste characterization devices is described in the light of enhanced performances by keeping in mind radiation protection requirements and a willing to always optimize waste categories (very low level waste) and volume. The improvement of the characterization methods appears through this forty-year long work while sharing the feedback of encountered difficulties. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2023

Quantification of LEU Holdup using gamma ray imaging and inverse transport solver

Holdup is the residual amount of special nuclear material (SNM) remaining in a processing facility after the bulk materials have been cleaned out. In commercial uranium processing facilities, quantification of holdup is a major challenge because of the highly variable shapes and sizes of the deposits. Any method that attempts to generalize and calibrate deposit shapes in order to quantify holdup will be prone to high uncertainties. Uncertainties on the order of ±50% are typical in holdup results. In international safeguards applications, a ±50% uncertainty can result in a large amount of material unaccounted for (MUF) thereby increasing the difficulty of detecting material diversion and facility misuse. An imaging-based methodology has been developed with the objective of significantly reducing this uncertainty by using the true deposit shape, instead of relying on oversimplified geometric assumptions. The project is a collaboration between ORNL, Y-12, and the University of Tennessee, Knoxville, TN. Uranium sources of known masses were measured using the Germanium Gamma-ray Imager (GeGI), a high-resolution imaging spectrometer, creating a pixelated map for each spectral bin. Two different gamma imaging methods are employed in this work: coded aperture imaging and Compton imaging. A validated MonteCarlo model of the detector has been developed using the GEANT4 code for determining the intrinsic response of the detector, its enclosure, and the coded aperture mask. An inverse transport solver based on the Markov Chain Monte-Carlo approach known as Differential Evolution Adaptive Metropolis (DREAM) is employed to use the measurement data from the image pixels (coded aperture or Compton) to solve for the mass of 235 U in the deposit. A reliable method based on the DREAM solver has been developed to flag the infinite thickness condition of a uranium deposit. The project team is working towards improving the image reconstruction for Compton imaging so that a better localization of the source can be achieved. Besides treating the coded aperture and Compton imaging methods independently, the project is also evaluating a combined method that uses the Compton scatter data from a coded aperture measurement. GEANT4 simulations are being performed to evaluate the combined approach. The impact on the DREAM optimization as the source thickness progressively approaches infinite thickness is being evaluated. A number of uranium sources available at ORNL have been measured, and the DREAM results have been tested and validated for the coded aperture imaging. A similar effort will be carried out to validate the Compton based method once the development of algorithms for better localization are complete. The imaging based quantification is very amenable to unattended monitoring of holdup accumulation at key measurement points. A proof of concept measurement has been completed to demonstrate this capability The current work used the high energy resolution imager GeGI. However, the approach and methodologies are applicable to other imagers such as the cadmium zin telluride (CZT) based imager manufactured by H3D, Inc.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2020

Economic Model for Estimation of GDP Losses in the MACCS Offsite Consequence Analysis Code

The MACCS (MELCOR Accident Consequence Code System) code is the U.S. Nuclear Regulatory Commission (NRC) tool used to perform probabilistic health and economic consequence assessments for atmospheric releases of radionuclides. It is also used by international organizations, both reactor owners and regulators. It is intended and most commonly used for hypothetical accidents that could potentially occur in the future rather than to evaluate past accidents or to provide emergency response during an ongoing accident. It is designed to support probabilistic risk and consequence analyses and is used by the NRC, U.S. nuclear licensees, the Department of Energy, and international vendors, licensees, and regulators. This report describes the modeling framework, implementation, verification, and benchmarking of a GDP-based model for economic losses that has recently been developed as an alternative to the original cost-based economic loss model in MACCS. The GDP-based model has its roots in a code developed by Sandia National Laboratories for the Department of Homeland Security to estimate short-term losses from natural and manmade accidents, called the Regional Economic Accounting analysis tool (REAcct). This model was adapted and modified for MACCS and is now called the Regional Disruption Economic Impact Model (RDEIM). It is based on input-output theory, which is widely used in economic modeling. It accounts for direct losses to a disrupted region affected by an accident, indirect losses to the national economy due to disruption of the supply chain, and induced losses from reduced spending by displaced workers. RDEIM differs from REAcct in its treatment and estimation of indirect loss multipliers, elimination of double counting associated with inter-industry trade in the affected area, and that it is designed to be used to estimate impacts for extended periods that can occur from a major nuclear reactor accident, such as the one that occurred at the Fukushima Daiichi site in Japan. Most input-output models do not account for economic adaptation and recovery, and in this regard RDEIM differs from its parent, REAcct, because it allows for a user-definable national recovery period. Implementation of a recovery period was one of several recommendations made by an independent peer review panel to ensure that RDEIM is state-of-practice. For this and several other reasons, RDEIM differs from REAcct. Both the original and the RDEIM economic loss models account for costs from evacuation and relocation, decontamination, depreciation, and condemnation. Where the original model accounts for an expected rate of return, based on the value of property, that is lost during interdiction, the RDEIM model instead accounts for losses of GDP based on the industrial sectors located within a county. The original model includes costs for disposal of crops and milk that the RDEIM model currently does not, but these costs tend to contribute insignificantly to the overall losses. This document discusses three verification exercises to demonstrate that the RDEIM model is implemented correctly in MACCS. It also describes a benchmark study at five nuclear power plants chosen to represent the spectrum of U.S. commercial sites. The benchmarks provide perspective on the expected differences between the RDEIM and the original cost-based economic loss models. The RDEIM model is shown to consistently predict larger losses than the original model, probably in part because it accounts for national losses by including indirect and induced losses; whereas, the original model only accounts for regional losses. Nonetheless, the RDEIM model predicts losses that are remarkably consistent with the original cost-based model, differing by 16% at most for the five sites combined with three source terms considered in this benchmark.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
NASA NTRS2016

Pilot Field Test: Use of a Compression Garment During a Stand Test After Long-Duration Space Flight

Orthostatic intolerance (OI) is a concern for astronauts returning from long-duration space flight. One countermeasure that has been used to protect against OI after short-duration bed rest and space flight is the use of lower body and abdominal compression garments. However, since the end of the Space Shuttle era we have not been able to test crewmembers during the first 24 hours after landing on Earth. NASA's Pilot Field Test provided us the opportunity to test cardiovascular responses of crewmembers wearing the Russian Kentavr compression garment during a stand test at multiple time points throughout the first 24 hours after landing. HYPOTHESIS We hypothesized that the Kentavr compression garment would prevent an increase in heart rate (HR) >15 bpm during a 3.5-min stand test. METHODS: The Pilot Field Test was conducted up to 3 times during the first 24 hours after crewmembers returned to Earth: (1) either in a tent adjacent to the Soyuz landing site in Kazakhstan (approx.1 hr) or after transportation to the Karaganda airport (approx. 4 hr); (2) during a refueling stop in Scotland (approx.12 hr); and (3) upon return to NASA Johnson Space Center (JSC) (approx.24 hr). We measured HR and arterial pressure (finger photoplethysmography) for 2 min while the crewmember was prone and throughout 3.5 min of quiet standing. Eleven crewmembers consented to participate; however, 2 felt too ill to start the test and 1 stopped 30 sec into the stand portion of the test. Of the remaining 8 crewmembers, 2 did not wear the Russian Kentavr compression garment. Because of inclement weather at the landing site, 5 crewmembers were flown by helicopter to the Karaganda airport before initial testing and received intravenous saline before completing the stand test. One of these crewmembers wore only the portion of the Russian Kentavr compression garment that covered the lower leg and thus lacked thigh and abdominal compression. All crewmembers continued wearing the Russian Kentavr compression garment during the second testing session in Scotland, but none wore it during testing at JSC. RESULTS: The mean Delta HR from the supine to standing position in the 8 crewmembers measured pre-flight or 60 days after return from long-duration space flight was 9.8 bpm. During the first few hours after landing from long-duration space flight, the mean Delta HR of the 6 crewmembers who wore the Russian Kentavr compression garment in Kazakhstan or Karaganda was +14 bpm and the change in mean arterial pressure (Delta MAP) was +0.8 mmHg, while the 2 crewmembers who did not wear the Russian Kentavr compression garment had a Delta HR of +38 bpm and a Delta MAP of +1.1 mmHg. In Scotland, 4 crewmembers wore the Russian Kentavr compression garment and had a Delta HR of +7.4 bpm while the 3 crewmembers who did not wear it had a Delta HR of +25.0 bpm. Seven crewmembers were tested upon return to JSC approx. 24 hr after landing, but none wore the Russian Kentavr compression garment and their Delta HR was 16.0 bpm. CONCLUSIONS: These are the first stand-test data to be collected from long-duration crewmembers during the first 24 hr of re-adaptation to gravity on Earth. The Delta HR measured in crewmembers who completed the stand-test while wearing Kentavr within the first approx.4 hours after returning to Earth was only slightly elevated from pre-flight Delta HR, while the few subjects who did not wear the Russian Kentavr compression garment had a much larger increase in HR in order to maintain arterial pressure throughout 3.5-min of standing. These data demonstrate the effectiveness of a compression garment in preventing large increases in HR during a 3.5 min stand test after long-duration space flight. However, the fact that three crewmembers were too ill to complete the test or was not able to complete 3.5 min of standing despite wearing the Russian Kentavr compression garment indicates that wearing a compression garment does not resolve all problems crewmembers face during the period of re-adaptation immediately after return to Earth's gravity.

Laurie, S. S.↗
DOE OSTI2023

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗