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DOE OSTI2021

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Property Measurements of the NaCl-PuCl 3 Molten Salt System

Thermal properties of several compositions of the binary NaCl-PuCl 3 salt were measured to confirm the eutectic composition and provide high quality data for use by MSR developers. The PuCl 3 was generated by reducing PuO 2 to plutonium metal in the presence of calcium metal and then chlorinating the metallic plutonium with NH 4 Cl. The resulting PuCl 3 was used to make seven binary NaCl-PuCl 3 compositions with between 59.9 mol % PuCl 3 (Salt 1) and 20.0 mol % PuCl 3 (Salt 7). The eutectic temperature was measured to be 457 ± 4 °C by onset determination in DSC analyses of the seven mixtures, which is consistent with the values in the literature. Additional transitions were observed at approximately 332 and 363 °C in analyses of the PuCl 3 -rich compositions (Salts 1-3) at about 376 and 439 °C in analyses the Na-rich compositions (Salts 4-7). The solid state heat capacity decreased with increasing PuCl 3 content. The liquid state heat capacity was measured for Salt 3 (37.4 mol % PuCl 3 ) and Salt 4 (38.3 mol % PuCl 3 ). A higher heat capacity was measured for Salt 4 and measurements with both salts showed a positive correlation with temperature between 520 and 730 °C. Cells fabricated from nickel and molybdenum for use in DSC measurements at high temperatures could not be adequately sealed because these materials are not sufficiently malleable, even after high temperature annealing. It is recommended that future development focus on corrosion-resistant materials that have mechanical and thermal properties similar to the commercially available gold cells, such as Pt-Rh (80–20) and pure platinum. Those materials are chemically inert, soft, and malleable like gold, but have melting temperatures higher than 1000 °C (Rakhtsaum, 2013). Use of the Pt-Rh alloy should be evaluated first based on superior machinability for making precision parts and its current use in commercial DSC high temperature crucibles. The commercial Pt-Rh cells are not hermetically sealable, but it is expected that cells made from thinner stock can be sealed.

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

DOE OSTI2015

Cleaning up the Legacy of the Cold War: Plutonium Oxides and the Role of Synchrotron Radiation Research

The deceptively simple binary formula of AnO 2 belies an incredibly complex structural nature, and propensity to form mixed-valent, nonstoichiometric phases of composition AnO 2±x . For plutonium, the very formation of PuO 2+x has challenged a long-established dogma, and raised fundamental questions for long-term storage and environmental migration. This presentation covers two aspects of Los Alamos synchrotron radiation studies of plutonium oxides: (1) the structural chemistry of laboratory-prepared AnO 2+x systems (An = U, Pu; 0 ≤ x ≤ 0.25) determined through a combination of x-ray absorption fine structure spectroscopy (XAFS) and x-ray scattering of laboratory prepared samples; and (2) the application of synchrotron radiation towards the decontamination and decommissioning of the Rocky Flats Environmental Technology Site. Making the case for particle transport mechanisms as the basis of plutonium and americium mobility, rather than aqueous sorption-desorption processes, established a successful scientific basis for the dominance of physical transport processes by wind and water. The scientific basis was successful because it was in agreement with general theory on insolubility of PuO 2 in oxidation state IV, results of ultrafiltration analyses of field water/sediment samples, XAFS analyses of soil, sediment, and concrete samples, and was also in general agreement with on-site monitoring data. This understanding allowed Site contractors to rapidly move to application of soil erosion and sediment transport models as the means of predicting plutonium and americium transport, which led to design and application of site-wide soil erosion control technology to help control downstream concentrations of plutonium and americium in streamflow.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

DOE OSTI2016

Alloying effect of Nb and Sn on the Zirconium alloy fuel claddings behavior at high temperature oxidation in steam - 6.1 STP-2016-0072

As the material of fuel claddings for water cooled reactors most commonly used binary and multicomponent zirconium alloys Zr-Nb (E110,M5), Zr-Sn-Fe (Zircaloy-2,-4) and Zr-Nb-Sn-Fe (E635, Zirlo) are used, which improvement extends, in particular, by varying their composition by Nb, Sn and Fe. At the same time, the importance is given to ensuring the safe operation of the fuel rods not only in normal conditions but also in emergency situations such as LOCA. In this paper the researches results of the studies of influence of alloying elements Nb, Sn (and Fe) on corrosion resistance and embrittlement at high temperature steam oxidation of fuel claddings based on alloys types Zr-xNb (x=1.0/2.5) and Zr-xNb-ySn-zFe (x=0.6/2.4; y=0.24/1.1; z=0.18/0.34) made with using zirconium sponge are presented. High temperature steam oxidation tests were carried out at temperatures of 1000, 1100 and 1200 deg. C with continuous measurement of the weight gain during the experiment and subsequent cooling in steam with rate ∼ 20 deg. C/s. The studies of kinetics of high-temperature oxidation in steam, structural-phase state changes, alloying elements distribution, absorbed hydrogen content and residual ductility after rapid cooling of the oxidized specimens were made. The difference in the oxidation kinetics of the materials studied was revealed, it decreases with the oxidation temperature increase. At the same time, an increase of the Sn content in the alloy influences on specimens 'breakaway' oxidation intensification at 1000 deg. C. It is shown that the mechanical properties of fuel claddings oxidized under the same conditions depend on their alloying composition, the hydrogen fraction absorbed by the specimen and formed by the oxidation ZrO2, a-Zr(O) and 'ex-β' layers structure, which in turn varies depending on the alloy composition. The increase of Nb, Sn and Fe content in the zirconium alloy leads to decrease in residual ductility of fuel claddings after high temperature steam oxidation. According to obtained results the most resistant to high-temperature steam oxidation and embrittlement at temperatures of 1000-1200 deg. C among the materials studied is Zr-1Nb alloy. (authors)

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

DOE OSTI2025

mzPeak: Designing a Scalable, Interoperable, and Future-Ready Mass Spectrometry Data Format

Advances in mass spectrometry (MS) instrumentation, such as higher resolution, faster scan speeds, and improved sensitivity, have significantly increased the volume and complexity of data. The growing adoption of imaging and ion mobility further amplifies these challenges across MS-based omics fields, including proteomics, metabolomics, and lipidomics. While these technologies unlock new possibilities, they also present significant challenges in data management, storage, and accessibility. Existing open formats, such as the XML-based community standards mzML and imzML, struggle to meet the demands of modern MS workflows due to their large file sizes, slow data access, and limited metadata support. Vendor-specific formats, while optimized for proprietary instruments, lack interoperability, comprehensive metadata support and long-term archival reliability. This white paper lays the groundwork for mzPeak, a next-generation community data format designed to address these challenges and support high-throughput, multi-dimensional MS workflows. By adopting a hybrid model that combines efficient binary storage for numerical data and both human and machine-readable metadata storage, mzPeak will reduce file sizes, accelerate data access, and offer a scalable, adaptable solution for evolving MS technologies. For researchers, mzPeak will enable enhanced interoperability across platforms, seamless support for complex workflows including ion mobility and MS imaging, and faster data access compared to existing community formats such as mzML. Its design will ensure data is managed in compliance with regulatory standards, essential for applications such as precision medicine and chemical safety, where long-term data integrity and accessibility are critical. For vendors, mzPeak provides a streamlined, open alternative to proprietary formats, reducing the burden of regulatory compliance while aligning with the industry's push for transparency and standardization. By offering a high-performance, interoperable solution, mzPeak positions vendors to meet customer demands for sustainable data management tools which will be able to handle emerging and future data types and workflows. mzPeak aspires to become the cornerstone of MS data management, empowering researchers, vendors, and developers to innovate and collaborate more effectively.

data formats↗

DOE OSTI2026

Custom surface reflectance, shade mask, and equivalent water thickness maps for the Colorado Headwaters Ecological Spectroscopy Study (2025)

This dataset contains land surface reflectance estimates and additional derived products generated from NEON Imaging Spectrometer (NIS) data collected in the Upper Gunnison river basin during June and July of 2025. Data was collected over three domains: the Upper East River (CRBU), Almont Triangle (ALMO), and the Upper Taylor Basin (UPTA). These products were derived from radiance and LiDAR data collected by the NEON Airborne Observation Platform (AOP) campaign funded by the Colorado Headwaters Ecological Spectroscopy Study (CHESS) (doi:10.15485/3017965). Products include per-pixel surface reflectance (rfl) and reflectance uncertainty (rfl_unc), observational data (obs), canopy equivalent water thickness (ewt), and shade masks. Atmospheric correction was performed per flightline using the ISOFIT (Imaging Spectrometer Optimal FITting) optimal estimation framework to estimate surface reflectance and the associated per-band reflectance uncertainty. Reflectance retrievals achieved a mean absolute error of 1.5% across diverse validation surfaces (see validation report.pdf). Equivalent water thickness was calculated from surface reflectance using the Beer–Lambert absorption of liquid water. Shade masks were generated based on the geometry between the sun angle, ground surface, and sensor at the time of flight. Data products are provided per-flightline and as mosaics for each domain. Flightline data products are provided as ENVI-formatted binary files (rfl, rfl_unc, ewt) and GeoTIFFs (shade). Reflectance and uncertainty mosaics are provided as tiled NetCDFs, while all other mosaicked products are provided as cloud-optimized GeoTIFFs. These formats are supported by common geospatial software (e.g., QGIS, ArcGIS, ENVI) and programmatic libraries in Python (e.g., rasterio, xarray, spectral, netCDF4) and R (e.g., terra, ncdf4). Processing workflows were designed to be equivalent to those used to generate the 2018 CHESS campaign airborne imaging spectroscopy data products (doi:10.15485/3013527). All outputs were co-registered to a common spatial grid to support time series analyses. CHESS Project Description: The Colorado Headwaters Ecological Spectroscopy Study (CHESS) comprised a multi-week airborne remote sensing and field observation campaign in the Upper Gunnison Basin, Colorado, conducted in June and July of 2025. Airborne remote sensing was conducted by the National Ecological Observatory Network Airborne Observation Platform (NEON AOP), concurrent with a field campaign run by the Rocky Mountain Biological Laboratory (RMBL), the Lawrence Berkeley National Laboratory (LBNL) and SLAC National Accelerator Laboratory Watershed Function Science Focus Area (SFA), and NASA-JPL (Jet Propulsion Laboratory) Earth Surface Mineral Dust Source Investigation (EMIT) program. Between June 10 and July 18, 2025, the NEON AOP flight team collected high-resolution aerial imaging spectroscopy and Light Detection and Ranging (LiDAR) data over three domains: the Upper East River (CRBU), Almont Triangle (ALMO), and the Upper Taylor Basin (UPTA). In coordination with the flights, a field campaign acquired ground-truth observations, including observations of vegetation composition, foliar traits, forest demography, and subsurface properties in 18 core sampling areas within the domains. Additional surface water observations were taken at over 380 point locations. All CHESS campaign datasets can be found within the CHESS ESS-DIVE data portal: https://data.ess-dive.lbl.gov/portals/chess. Funding Acknowledgment: Data acquisition was performed under a grant from the National Aeronautics and Space Administration (80NSSC24K1005). Computational research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004) and was funded by EMIT Extended Mission Phase E Science.

2018 NEON and 2025 CHESS Campaigns↗