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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 289 records · Page 16
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

A comparison of X-ray and optical emission in Cassiopeia A

Broadband optical and narrowband Si XIII X-ray images of the young Galactic supernova remnant Cassiopeia A (Cas A) obtained over several decades are used to investigate spatial and temporal emission correlations on both large and small angular scales. The data examined consist of optical and near-infrared ground-based and Hubble Space Telescope images taken between 1951 and 2011, and of X-ray images from Einstein, ROSAT, and Chandra taken between 1979 and 2013. We find weak spatial correlations between the remnant's X-ray and optical emission features on large scales, but several cases of good optical/X-ray correlations on small scales for features which have brightened due to recent interactions with the reverse shock. We also find instances (1) where a time delay is observed between the appearance of a feature's optical and X-ray emissions, (2) of displacements of several arcseconds between a feature's X-ray and optical emission peaks, and (3) of regions showing no corresponding X-ray or optical emissions. To explain this behavior, we propose a highly inhomogeneous density model for Cas A's ejecta consisting of small, dense optically emitting knots (n ∼10{sup 2-3} cm{sup –3}) and a much lower density (n ∼0.1-1 cm{sup –3}) diffuse X-ray emitting component often spatially associated with optical emission knots. The X-ray emitting component is sometimes linked to optical clumps through shock-induced mass ablation generating trailing material leading to spatially offset X-ray/optical emissions. A range of ejecta densities can also explain the observed X-ray/optical time delays since the remnant's ≈5000 km s{sup –1} reverse shock heats dense ejecta clumps to temperatures around 3 × 10{sup 4} K relatively quickly, which then become optically bright while more diffuse ejecta become X-ray bright on longer timescales. Highly inhomogeneous ejecta as proposed here for Cas A may help explain some of the X-ray/optical emission features seen in other young core-collapse supernova remnants.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2015

UNAMBIGUOUS DETECTION OF REFLECTION IN MAGNETIC CATACLYSMIC VARIABLES: JOINT NuSTAR–XMM-NEWTON OBSERVATIONS OF THREE INTERMEDIATE POLARS

In magnetic cataclysmic variables (CVs), X-ray emission regions are located close to the white dwarf surface, which is expected to reflect a significant fraction of intrinsic X-rays above 10 keV, producing a Compton reflection hump. However, up to now, a secure detection of this effect in magnetic CVs has largely proved elusive because of the limited sensitivity of non-imaging X-ray detectors. Here we report our analysis of joint NuSTAR–XMM-Newton observations of three magnetic CVs, V709 Cas, NY Lup, and V1223 Sgr. The improved hard X-ray sensitivity of the imaging NuSTAR data has resulted in the first robust detection of Compton hump in all three objects, with amplitudes of ∼1 or greater in NY Lup, and likely <1.0 in the other two. We also confirm earlier reports of a strong spin modulation above 10 keV in V709 Cas, and we report the first detection of small spin amplitudes in the others. We interpret this as due to different height of the X-ray emitting region among these objects. A height of ∼0.2 white dwarf radii provides a plausible explanation for the low reflection amplitude of V709 Cas. Since emission regions above both poles are visible at certain spin phases, this can also explain the strong hard X-ray spin modulation. A shock height of ∼0.05 white dwarf radii can explain our results on V1223 Sgr, while the shock height in NY Lup appears negligible.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2022

Functional characterization of diverse type I-F CRISPR-associated transposons

CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR–Cas systems are classified into different classes and types. Class 1 CRISPR–Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

Elevated carbon dioxide (CO 2 ) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO 2 -induced stomatal movements by catalytic transmission of the CO 2 signal in guard cells. But, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO 2 -regulated stomatal movements remains unknown. We express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO 2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO 2 regulation of stomatal conductance. Mathematical modeling of cellular CO 2 catalysis suggests that the dynamics of the intracellular HCO 3 - concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Therefore, modeling supports the notion that the intracellular HCO 3 - concentration dynamics in guard cells are a key mechanism in mediating CO 2 -regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond

The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2018

Programmed DNA destruction by miniature CRISPR-Cas14 enzymes

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli : bioinformatic and experimental approaches

Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO 2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO 2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

The complete genome sequence of the yogurt isolate Streptococcus thermophilus ACA-DC 2

Streptococcus thermophilus ACA-DC 2 is a newly sequenced strain isolated from traditional Greek yogurt. Among the 14 fully sequenced strains of S. thermophilus currently deposited in the NCBI database, the ACA-DC 2 strain has the smallest chromosome, containing 1,731,838 bp. The annotation of its genome revealed the presence of 1,850 genes, including 1,556 protein-coding genes, 70 RNA genes and 224 potential pseudogenes. A large number of pseudogenes were identified. This was also accompanied by the absence of pathogenic features suggesting evolution of strain ACA-DC 2 through genome decay processes, most probably due to adaptation to the milk ecosystem. Analysis revealed the existence of one complete lactose-galactose operon, several proteolytic enzymes, one exopolysaccharide cluster, stress response genes and four putative antimicrobial peptides. Interestingly, one CRISPR-cas system and one orphan CRISPR, both carrying only one spacer, were predicted indicating low activity or inactivation of the cas proteins. Nevertheless, four putative restriction-modification systems were determined that may compensate any deficiencies of the CRISPR-cas system. Furthermore, whole genome phylogeny indicated three distinct clades within S. thermophilus. Comparative analysis among selected strains representative for each clade, including strain ACA-DC 2, revealed a high degree of conservation at the genomic scale, but also strain specific regions. Unique genes and genomic islands of strain ACA-DC 2 contained a number of genes potentially acquired through horizontal gene transfer events, that could be related to important technological properties for dairy starters. Our study suggests genomic traits in strain ACA-DC 2 compatible to the production of dairy fermented foods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Corrective Action Decision Document/Closure Report for Corrective Action Unit 374: Area 20 Schooner Unit Crater, Nevada National Security Site, Nevada with ROTC 1 and 2, Revision 0 with ROTCs 1,2, and 3

Corrective Action Unit 374 comprises five corrective action sites (CASs): • 18-22-05, Drum • 18-22-06, Drums (20) • 18-22-08, Drum • 18-23-01, Danny Boy Contamination Area • 20-45-03, U-20u Crater (Schooner) The purpose of this Corrective Action Decision Document/Closure Report is to provide justification and documentation supporting the recommendation that no further corrective action is needed for CAU 374 based on the implementation of corrective actions. The corrective action of closure in place with administrative controls was implemented at CASs 18-23-01 and 20-45-03, and a corrective action of removing potential source material (PSM) was conducted at CAS 20-45-03. The other CASs require no further action; however, best management practices of removing PSM and drums at CAS 18-22-06, and removing drums at CAS 18-22-08 were performed. Corrective action investigation (CAI) activities were performed from May 4 through October 6, 2010, as set forth in the Corrective Action Investigation Plan for Corrective Action Unit 374: Area 20 Schooner Unit Crater, Nevada Test Site, Nevada. The approach for the CAI was divided into two facets: investigating the primary release of radionuclides and investigating other releases (migration in washes and chemical releases). The purpose of the CAI was to fulfill data needs as defined during the data quality objective (DQO) process. The CAU 374 dataset of investigation results was evaluated based on the data quality indicator parameters. This evaluation demonstrated the dataset is acceptable for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against final action levels (FALs) established in this document. Radiological doses exceeding the FAL of 25 millirem per year were found to be present in the surface soil that was sampled. It is assumed that radionuclide levels present in subsurface media within the craters and ejecta fields (default contamination boundaries) at the Danny Boy and Schooner sites exceed the FAL. It is also assumed that PSM in the form of lead-acid batteries at Schooner exceeds the FAL. Therefore, corrective actions were undertaken that consist of removing PSM, where present, and implementing a use restriction and posting warning signs at the Danny Boy and Schooner sites. These use restrictions were recorded in the FFACO database; the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office (NNSA/NSO) Facility Information Management System; and the NNSA/NSO CAU/CAS files. Therefore, NNSA/NSO provides the following recommendations: • No further corrective actions are necessary for CAU 374. • A Notice of Completion to NNSA/NSO is requested from the Nevada Division of Environmental Protection for closure of CAU 374. • Corrective Action Unit 374 should be moved from Appendix III to Appendix IV of the FFACO.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2011

Corrective Action Decision Document/Corrective Action Plan for Corrective Action Unit 547: Miscellaneous Contaminated Waste Sites, Nevada National Security Site, Nevada, Revision 0

The purpose of this CADD/CAP is to present the corrective action alternatives (CAAs) evaluated for CAU 547, provide justification for selection of the recommended alternative, and describe the plan for implementing the selected alternative. Corrective Action Unit 547 consists of the following three corrective action sites (CASs): (1) CAS 02-37-02, Gas Sampling Assembly; (2) CAS 03-99-19, Gas Sampling Assembly; and(3) CAS 09-99-06, Gas Sampling Assembly. The gas sampling assemblies consist of inactive process piping, equipment, and instrumentation that were left in place after completion of underground safety experiments. The purpose of these safety experiments was to confirm that a nuclear explosion would not occur in the case of an accidental detonation of the high-explosive component of the device. The gas sampling assemblies allowed for the direct sampling of the gases and particulates produced by the safety experiments. Corrective Action Site 02-37-02 is located in Area 2 of the Nevada National Security Site (NNSS) and is associated with the Mullet safety experiment conducted in emplacement borehole U2ag on October 17, 1963. Corrective Action Site 03-99-19 is located in Area 3 of the NNSS and is associated with the Tejon safety experiment conducted in emplacement borehole U3cg on May 17, 1963. Corrective Action Site 09-99-06 is located in Area 9 of the NNSS and is associated with the Player safety experiment conducted in emplacement borehole U9cc on August 27, 1964. The CAU 547 CASs were investigated in accordance with the data quality objectives (DQOs) developed by representatives of the Nevada Division of Environmental Protection (NDEP) and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to determine and implement appropriate corrective actions for CAU 547. Existing radiological survey data and historical knowledge of the CASs were sufficient to meet the DQOs and evaluate CAAs without additional investigation. As a result, further investigation of the CAU 547 CASs was not required. The following CAAs were identified for the gas sampling assemblies: (1) clean closure, (2) closure in place, (3) modified closure in place, (4) no further action (with administrative controls), and (5) no further action. Based on the CAAs evaluation, the recommended corrective action for the three CASs in CAU 547 is closure in place. This corrective action will involve construction of a soil cover on top of the gas sampling assembly components and establishment of use restrictions at each site. The closure in place alternative was selected as the best and most appropriate corrective action for the CASs at CAU 547 based on the following factors: (1) Provides long-term protection of human health and the environment; (2) Minimizes short-term risk to site workers in implementing corrective action; (3) Is easily implemented using existing technology; (4) Complies with regulatory requirements; (5) Fulfills FFACO requirements for site closure; (6) Does not generate transuranic waste requiring offsite disposal; (7) Is consistent with anticipated future land use of the areas (i.e., testing and support activities); and (8) Is consistent with other NNSS site closures where contamination was left in place.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2012

Predictive Capability for Strongly Correlated Systems

Diffusion Monte Carlo methods can give highly accurate results for correlated systems, provided that well optimized trial wave functions with accurate nodal surfaces are employed. The Cornell team developed powerful methods for optimizing all the parameters within a multi-determinant Slater-Jastrow form of the wave function. These include the Jastrow parameters within a flexible electron-electron-nucleus form of the Jastrow function, the parameters multiplying the configuration state functions, the orbital parameters and the basis exponents. The method optimizes a linear combination of the energy and the variance of the local energy. The optimal parameters are found iteratively by diagonalizing the Hamiltonian matrix in the space spanned by the wave function and its first-order derivatives, making use of a strong zero-variance principle. It is highly robust, has become the method of choice for correlated wave function optimization and has been adopted by other QMC groups. This optimization method was used on the first-row atoms and homonuclear diatomic molecules, demonstrating that molecular well depths can be obtained with near chemical accuracy quite systematically at the diffusion Monte Carlo level for these systems. In addition the complete ground-state potential energy curve of the C 2 molecule up to the dissociation limit was obtained, and, size consistency and broken spin-symmetry issues in quantum Monte Carlo calculations were studied. The method was used with a eight-electrons-in-eight-orbitals complete active space CAS(8,8) wave function to study the relative energies of the monocyclic and bicyclic forms of m-benzyne. The DMC calculations show that the monocyclic structure is lower in energy than the bicyclic structure by 1.92 kcal/ mole, which is in excellent agreement with the best coupled cluster results (CCSD(T)) and in disagreement with the CCSD results. QMC methods have for the most part been used only for ground states of a given symmetry. We developed a method for calculating low-lying excited states as well and tested it on the ground and lowest three adiabatic excited states of methylene with progressively larger JastrowSlater multideterminant complete active space CAS wave functions. The highest of these states has the same symmetry, 1 A 1 , as the first excited state. The DMC excitation energies obtained using any of the CAS wave functions are in excellent agreement with experiment. In contrast, single-determinant wave functions do not yield accurate DMC energies, indicating that it is important to include in the wave function Slater determinants that describe static strong correlation.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2012

Closure Report for Corrective Action Unit 547: Miscellaneous Contaminated Waste Sites, Nevada National Security Site, Nevada

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 547, Miscellaneous Contaminated Waste Sites, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 547 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 547 consists of the following three Corrective Action Sites (CASs), located in Areas 2, 3, and 9 of the Nevada National Security Site: (1) CAS 02-37-02, Gas Sampling Assembly; (2) CAS 03-99-19, Gas Sampling Assembly; AND (3) CAS 09-99-06, Gas Sampling Assembly Closure activities began in August 2011 and were completed in June 2012. Activities were conducted according to the Corrective Action Decision Document/Corrective Action Plan (CADD/CAP) for CAU 547 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The recommended corrective action for the three CASs in CAU 547 was closure in place with administrative controls. The following closure activities were performed: (1) Open holes were filled with concrete; (2) Steel casings were placed over vertical expansion joints and filled with cement; (3) Engineered soil covers were constructed over piping and exposed sections of the gas sampling system components; (4) Fencing, monuments, Jersey barriers, radiological postings, and use restriction (UR) warning signs were installed around the perimeters of the sites; (5) Housekeeping debris was picked up from around the sites and disposed; and (6) Radiological surveys were performed to confirm final radiological postings. UR documentation is included in Appendix D. The post-closure plan was presented in detail in the CADD/CAP for CAU 547 and is included as Appendix F of this report. The requirements are summarized in Section 5.2 of this report. The proposed post-closure requirements consist of visual inspections to determine the condition of postings and radiological surveys to verify contamination has not migrated. NNSA/NSO requests the following: (1) A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 547; and (2) The transfer of CAU 547 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2015

2014 Annual Summary Report for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada National Security Site, Nye County, Nevada: Review of the Performance Assessments and Composite Analyses

This report summarizes the results and conclusions of the annual review of the performance assessments (PAs) and composite analyses (CAs) of the Area 3 and Area 5 Radioactive Waste Management Sites (RWMSs). The Area 3 and Area 5 RWMSs were issued Disposal Authorization Statements (DASs) in accordance with U.S. Department of Energy Order DOE O 435.1 “Radioactive Waste Management” (U.S. Department of Energy [DOE] 2001). The Area 3 RWMS and Area 5 RWMS DASs (DOE 1999a, 2000) require preparation of an annual summary report and a determination of the continuing adequacy of the PAs and CAs. The requirement to prepare an annual summary report is implemented in the Maintenance Plan for the PAs and CAs (National Security Technologies, LLC [NSTec] 2007a). The annual summary report is submitted to the DOE Office of Environmental Management for review and approval.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2016

Multivariate Statistical Analysis of Orthogonal Mass Spectral Data for the Identification of Chemical Attribution Signatures of 3-Methylfentanyl

Critical to many modern forensic investigations is the chemical attribution of the origin of an illegal drug. This process greatly relies on identification of compounds indicative of its clandestine or commercial production. The results of these studies can yield detailed information on method of manufacture, sophistication of the synthesis operation, starting material source, and final product. In the present work, chemical attribution signatures (CAS) associated with the synthesis of the analgesic 3- methylfentanyl, N-(3-methyl-1-phenethylpiperidin-4-yl)-N-phenylpropanamide, were investigated. Six synthesis methods were studied in an effort to identify and classify route-specific signatures. These methods were chosen to minimize the use of scheduled precursors, complicated laboratory equipment, number of overall steps, and demanding reaction conditions. Using gas and liquid chromatographies combined with mass spectrometric methods (GC-QTOF and LC-QTOF) in conjunction with inductivelycoupled plasma mass spectrometry (ICP-MS), over 240 distinct compounds and elements were monitored. As seen in our previous work with CAS of fentanyl synthesis the complexity of the resultant data matrix necessitated the use of multivariate statistical analysis. Using partial least squares discriminant analysis (PLS-DA), 62 statistically significant, route-specific CAS were identified. Statistical classification models using a variety of machine learning techniques were then developed with the ability to predict the method of 3-methylfentanyl synthesis from three blind crude samples generated by synthetic chemists without prior experience with these methods.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Developing Multi-Gene CRISPRa/I Programs to Accelerate DBTL Cycles in ABF Hosts Engineered for Chemical Production (CRADA 468)

Bacterial metabolism is comprised of large and complex gene networks that can produce valuable chemical products. Sophisticated organism engineering efforts are required to optimize production of high-value compounds from these networks. In principle, synthetic multi-gene transcriptional programs could be constructed to reengineer these networks for efficient industrial chemical production. In practice, however, our incomplete ability to understand and model the underlying networks, combined with our limited ability to predictably control the expression of multiple genes makes achieving this goal difficult. To overcome these challenges, we will combine new CRISPR-Cas multi-gene expression programs with computational modeling, machine learning, and multi-omics data to enhance the efficacy of design-build-test-learn (DBTL) cycles. For industrially promising microorganisms in early stages of development, creating technologies for rapidly engineering complex multi-gene programs could be transformative for accelerating data- and model-driven strain design. New CRISPR-Cas tools allow programmable gene activation (CRISPRa) or repression (CRISPRi) at multiple genes simultaneously, using the catalytically inactive Cas9 protein (dCas9) with guide RNAs that recognize DNA targets through predictable Watson-Crick base pairing. To enable accelerated DBTL cycles, we will combine these technologies with advanced Agile BioFoundry (ABF) capabilities for multi-omics data collection and machine learning. We will demonstrate the immediate applicability of these tools by rapidly improving the production of an industrial aromatic in multiple ABF organisms. We recently identified and optimized new transcriptional activators that can be linked to programmable CRISPR-Cas DNA binding domains to activate gene expression in E. coli. We can now use these CRISPRa tools as generalizable trans-acting regulators for combinatorial multi-gene expression tuning that can be easily transferred to new pathways and networks without additional genome engineering. We anticipate these tools will also transfer to new hosts. We have recently found that CRISPRa systems developed in E. coli can be readily ported to Pseudomonas putida, suggesting that multi-gene CRISPRa/i programs for diverse ABF organisms may be within reach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Closure Report for Corrective Action Unit 574: Neptune Nevada National Security Site, Nevada with ROTCs 1 & 2

This Closure Report presents information supporting closure of CAU 574 according to the FFACO (FFACO, 1996 [as amended March 2010]) and the Streamlined Approach for Environmental Restoration Plan for CAU 574 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The following activities were performed to support closure of CAU 574: · In situ external dose rate measurements were collected using thermoluminescent dosimeters at CAS 12-45-01, U12e.05 Crater (Blanca). · Total effective dose rates were determined at both sites by summing the internal and external dose rate components. · A use restriction (UR) was implemented at CAS 12-23-10, U12c.03 Crater (Neptune). Areas that exceed the final action level (FAL) of 25 millirems per year (mrem/yr) based on the Occasional Use Area exposure scenario is within the existing use restricted area for CAU 551. The 25-mrem/yr FAL is not exceeded outside the existing CAU 551 UR for any of the exposure scenarios (Industrial Area, Remote Work Area, and Occasional Use Area). Therefore, the existing UR for CAU 551 is sufficient to bound contamination that exceeds the FAL. · An administrative UR was implemented at CAS 12-45-01, U12e.05 Crater (Blanca) as a best management practice (BMP). The 25-mrem/yr FAL was not exceeded for the Remote Work Area or Occasional Use Area exposure scenarios; therefore, a UR is not required. However, because the 25-mrem/yr FAL was exceeded for the Industrial Area exposure scenario, an administrative UR was established as a BMP.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2017

Cyanogen Metabolism in Cassava Roots: Impact on Protein Synthesis and Root Development

Cassava (Manihot esculenta Crantz), a staple crop for millions of sub-Saharan Africans, contains high levels of cyanogenic glycosides which protect it against herbivory. However, cyanogens have also been proposed to play a role in nitrogen transport from leaves to roots. Consistent with this hypothesis, analyses of the distribution and activities of enzymes involved in cyanide metabolism provides evidence for cyanide assimilation, derived from linamarin, into amino acids in cassava roots. Both β-cyanoalanine synthase (CAS) and nitrilase (NIT), two enzymes involved in cyanide assimilation to produce asparagine, were observed to have higher activities in roots compared to leaves, consistent with their proposed role in reduced nitrogen assimilation. In addition, rhodanese activity was not detected in cassava roots, indicating that this competing means for cyanide metabolism was not a factor in cyanide detoxification. In contrast, leaves had sufficient rhodanese activity to compete with cyanide assimilation into amino acids. Using transgenic low cyanogen plants, it was shown that reducing root cyanogen levels is associated with elevated root nitrate reductase activity, presumably to compensate for the loss of reduced nitrogen from cyanogens. Finally, we overexpressed Arabidopsis CAS and NIT4 genes in cassava roots to study the feasibility of enhancing root cyanide assimilation into protein. Optimal overexpression of CAS and NIT4 resulted in up to a 50% increase in root total amino acids and a 9% increase in root protein accumulation. However, plant growth and morphology was altered in plants overexpressing these enzymes, demonstrating a complex interaction between cyanide metabolism and hormonal regulation of plant growth.

59 BASIC BIOLOGICAL SCIENCES↗