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DOE OSTI2021

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Multimeric antibodies from antigen-specific human IgM+ memory B cells restrict Plasmodium parasites

Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Magnetoquantum oscillations in the specific heat of a topological Kondo insulator

Surprisingly, magnetoquantum oscillations (MQOs) characteristic of a metal with a Fermi surface have been observed in measurements of the topological Kondo insulator SmB 6 . As these MQO have only been observed in measurements of magnetic torque (dHvA) and not in measurements of magnetoresistance (SdH), a debate has arisen as to whether the MQO are an extrinsic effect arising from rare-earth impurities, defects, and/or aluminum inclusions or an intrinsic effect revealing the existence of charge-neutral excitations. In this work, we report the first observation of MQO in the low-temperature specific heat of SmB 6 . The observed frequencies and their angular dependence for these flux-grown samples are consistent with previous results based on magnetic torque for SmB 6 but the inferred effective masses are significantly larger than previously reported. Such oscillations can only be observed if the MQO are of bulk thermodynamic origin; the measured magnetic-field dependent oscillation amplitude and effective mass allow us to rule out suggestions of an extrinsic, aluminum inclusion-based origin for the MQO.

topological materials↗

DOE OSTI2025

Development of a military-specific mesh-type computational phantom library and its application to internal dosimetry and in-field radiological triage screening

Estimates of organ-absorbed and committed doses to individuals exposed to radioactive materials via acute inhalation often rely on internal dose coefficients and detector responses from reference human computational models. To achieve more accurate dose assessments to United States Armed Forces service members exposed in-field, computational models with varying morphometric parameters representative of this population are necessary. The International Commission on Radiological Protection (ICRP) Publication 145 provides detailed mesh reference computational phantoms (MRCPs) for adult males and females, with morphometric parameters matched to the 50th percentile. Previously, these phantoms were 2D and 3D scaled to match desired height, mass, and secondary anthropomorphic parameters in the creation of the University of Florida / Memorial Sloan Kettering (UF/MSK) computational phantom library. To achieve body fat percentage targets required for accession into the US Armed Forces, muscle and fat volumes were adjusted accordingly, thus, creating the UF/Department of Defence computational phantom library presented in this study. A comprehensive library of mesh-type computational human phantoms was created, including 57 adult males and 49 adult females with morphometric parameters aligned with United States Armed Forces service members. Phantoms were restricted to a body mass index between 19 and 27.5, with body fat percentages below 26% for males and 36% for females. Specific absorbed fractions were computed for selected source and target combinations, demonstrating how variations in height and body mass influence energy absorption in target regions relative to the ICRP MRCPs. Radiation detector responses were also computed, revealing that higher body masses resulted in decreased registered counts in the detection volume. These findings highlight the importance of incorporating morphometric variability in computational phantoms to achieve more accurate dose assessments and radiation detection responses for United States Armed Forces service members who inhale radioactive materials in-field.

computational phantoms↗

DOE OSTI2023

The magnificent ACT of flavor-specific neutrino self-interaction

Here we revisit the cosmology of neutrino self-interaction and use the latest cosmic microwave background data from the Atacama Cosmology Telescope (ACT) and the Planck experiment to constrain the interaction strength. In both flavor-universal and nonuniversal coupling scenarios, we find that the ACT data prefers strong neutrino self-interaction that delays neutrino free streaming until just before the matter-radiation equality. When combined with the Planck 2018 data, the preference for strong interaction decreases due to the Planck polarization data. For the combined dataset, the flavor-specific interaction still provides a better fit to the CMB data than ΛCDM. This trend persists even when neutrino mass is taken into account and extra radiation is added. We also study the prospect of constraining such strong interaction by future terrestrial and space telescopes, and find that the upcoming CMB-S4 experiment will improve the upper limit on neutrino self-interaction by about a factor of three.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2023

Rotor and wake aerodynamic analysis of the Hybrid-Lambda concept - an offshore low-specific-rating rotor concept

The low-specific-rating rotor concept Hybrid-Lambda introduces a blade design with nonuniform distribution of design variables (design tip speed ratio and axial induction) along the blade span to alleviate loads of the outboard section in strong winds. In this paper, we validate aerodynamic design calculations, which were carried out with the blade element momentum theory by comparing the results to free vortex wake investigations (FVW). Furthermore, we investigate the wake behaviour with FVW and large-eddy simulations. The results show good agreements between the blade element momentum theory and FVW for integrated rotor quantities (power and thrust). Small deviations are present when the gradients of axial induction along the span are large. The wake of the Hybrid-Lambda Rotor shows advantages in the near-wake region (up to 4 diameters [D] downstream), especially in the outer wake annulus and in low turbulence scenarios. For further downstream positions, the wake is comparable to that of the reference turbine.

17 WIND ENERGY↗

DOE OSTI2023

Specifications and pre-production of n + -in-p large-format strip sensors fabricated in 6-inch silicon wafers, ATLAS18, for the Inner Tracker of the ATLAS Detector for High-Luminosity Large Hadron Collider

Abstract The ATLAS experiment is constructing new all-silicon inner tracking system for HL-LHC. The strip detectors cover the radial extent of 40 to 100 cm. A new approach is adopted to use p-type silicon material, making the readout in n + -strips, so-called n + -in-p sensors. This allows for enhanced radiation tolerance against an order of magnitude higher particle fluence compared to the LHC. To cope with varying hit rates and occupancies as a function of radial distance, there are two barrel sensor types, the short strips (SS) for the inner 2 and the long strips (LS) for the outer 2 barrel cylinders, respectively. The barrel sensors exhibit a square, 9.8 × 9.8 cm 2 , geometry, the largest possible sensor area from a 6-inch wafer. The strips are laid out in parallel with a strip pitch of 75.5 μm and 4 or 2 rows of strip segments. The strips are AC-coupled and biased via polysilicon resistors. The endcap sensors employ a “stereo-annulus” geometry exhibiting a skewed-trapezoid shapes with circular edges. They are designed in 6 unique shapes, R0 to R5, corresponding to progressively increasing radial extents and which allows them to fit within the petal geometry and the 6-inch wafer maximally. The strips are in fan-out geometry with an in-built rotation angle, with a mean pitch of approximately 75 μm and 4 or 2 rows of strip segments. The eight sensor types are labeled as ATLAS18xx where xx stands for SS, LS, and R0 to R5. According to the mechanical and electrical specifications, CAD files for wafer processing were laid out, following the successful designs of prototype barrel and endcap sensors, together with a number of optimizations. A pre-production was carried out prior to the full production of the wafers. The quality of the sensors is reviewed and judged excellent through the test results carried out by vendor. These sensors are used for establishing acceptance procedures and to evaluate their performance in the ATLAS collaboration, and subsequently for pre-production of strip modules and stave and petal structures.

Instruments & Instrumentation↗

DOE OSTI2021

Reduction in total and major cause-specific mortality from tobacco smoking cessation: a pooled analysis of 16 population-based cohort studies in Asia

Little is known about the time course of mortality reduction following smoking cessation in Asians who have smoking behaviours distinct from their Western counterparts. We evaluated the level of reduction in all-cause, cardiovascular disease (CVD) and lung cancer mortality by years since quitting smoking, in Asia. Using Cox regression, we analysed individual participant data (n = 709 151) from 16 prospective cohorts conducted in China, Japan, Korea/Singapore, and India/Bangladesh, separately by cohorts. Cohort-specific hazard ratios (HRs) were combined using a random-effects meta-analysis. During a mean follow-up of 12.0 years, 108 287 deaths were ascertained—35 658 from CVD and 7546 from lung cancer. Among Asian men, a dose-response relationship of risk reduction in deaths from all causes, CVD and lung cancer was observed with an increase in years after smoking cessation. Compared with never smokers, however, all-cause and CVD mortality among former smokers remained elevated 10–14 years after quitting [multivariable-adjusted HR (95% confidence interval (CI) = 1.25 (1.13–1.37) and 1.20 (1.02–1.41), respectively]. Lung cancer mortality stayed almost 2-fold higher than among never smokers 15–19 years after smoking cessation [1.97 (1.41–2.73)], particularly among former heavy smokers [2.62 (1.71–4.00)]. Women who quitted for ≥5 years retained a significantly elevated mortality from all causes, CVD and lung cancer. Overall patterns of the cessation-mortality associations were similar across countries. Our findings suggest that adverse effects of tobacco smoking persist for an extended time period, even for more than two decades, which is beyond the time windows defined in current clinical guidelines for risk assessment of lung cancer and CVD.

Public, Environmental & Occupational Health↗

DOE OSTI2020

Genotype-Specific Neutralization of Norovirus Is Mediated by Antibodies Against the Protruding Domain of the Major Capsid Protein

Abstract Human noroviruses are the most common viral agents of acute gastroenteritis. Recently, human intestinal enteroids were shown to be permissive for norovirus infection. We tested their suitability as a system to study norovirus neutralization. Hyperimmune sera raised against virus-like particles (VLPs) representing different genotypes showed highly specific neutralization activity against GII.4 and GII.6 noroviruses. Carbohydrate blocking assays and neutralization exhibited similar patterns in antibody responses. Notably, sera produced against chimeric VLPs that presented swapped structural shell and protruding (P) domains, from different genotypes showed that neutralization is primarily mediated by antibodies mapping to the P domain of the norovirus capsid protein. This study provides empirical information on the antigenic differences among genotypes as measured by neutralization, which could guide vaccine design.

Ford-Siltz, Lauren A.↗

DOE OSTI2025

Thermophilic site-specific recombination system for rapid insertion of heterologous DNA into the Clostridium thermocellum chromosome

Clostridium thermocellum is an anaerobic thermophile capable of producing ethanol and other commodity chemicals from lignocellulosic biomass. The insertion of heterologous DNA into the C. thermocellum chromosome is currently achieved via a time-consuming homologous recombination process, where a single stable insertion can take 2–4 weeks or more to construct. In this work, we developed a thermostable version of the Serine recombinase Assisted Genome Engineering (tSAGE) approach for gene insertion in C. thermocellum utilizing a site-specific recombinase from Geobacillus sp. Y412MC61, enabling quick and easy insertion of DNA into the chromosome for accelerated genetic tool screening and heterologous gene expression. Using tSAGE, chromosomal insertion of plasmid DNA occurred at a maximum transformation efficiency of 5 × 10 3 CFU/µg, which is comparable to the transformation efficiency of a replicating control plasmid in C. thermocellum. Using tSAGE, we chromosomally integrated and characterized 17 reporter genes, 15 homologous and 31 heterologous constitutive promoters of varying strengths, 4 inducible promoters, and 5 riboswitches in C. thermocellum. We also determined that a 6–7 nucleotide gap between the ribosome binding site (RBS) and the start codon is optimal for high expression by employing a library of superfolder green fluorescent protein expression constructs driven by our strongest tested promoter (P clo1313_1194 ) with different distances between the RBS and start codon. The tools developed here will aid in accelerating C. thermocellum strain engineering for producing sustainable fuels and chemicals directly from plant biomass.

Biofuels↗

DOE OSTI2025

Habitat-specific seasonal densities of sympatric raccoons and opossums in the southeastern United States

Raccoon and opossum densities have implications for rabies management, but estimates of seasonal densities of both species are lacking for rural nonagricultural habitats of the southeastern United States, a core portion of their geographic range. Consequently, it remains unclear whether the densities of 1 species limit the other, which is possible considering their substantial niche overlap. We carried out a mark–recapture study of raccoons and opossums in 4 rural nonagricultural habitats (bottomland hardwood forest, riparian forest, upland pine forest, and isolated wetlands) in South Carolina, United States (2020 to 2022), and combined this with previous data from the same habitats (2017 to 2019) to estimate habitat-specific spring and fall densities. Raccoon densities ranged from 5.17 ± 0.96 animals/km 2 (bottomland hardwood fall) to 1.63 ± 0.83 animals/km 2 (upland pine spring) and were on average 19% higher in fall compared to spring. Opossum densities ranged from 10.35 ± 1.98 animals/km 2 (bottomland hardwood fall) to 1.11 ± 1.55 animals/km 2 (upland pine spring) with divergent seasonal patterns among habitats. These low densities across all habitats compared to other studies are likely the result of low resource availability, consistent with other habitats that have minimal anthropogenic influence. We observed a positive association between raccoon and opossum densities across trapping grids, suggesting that raccoons do not suppress opossum densities, but that densities of both species increase with increasing resource availability. Furthermore, our results can be used to inform oral rabies vaccination efforts such as refining bait densities and timing of bait distribution in these habitats.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Developmentally-specific physiological and metabolic responses support drought resilience in switchgrass and constrains biofuel yield

Switchgrass (Panicum virgatum) is a promising bioenergy crop due in part to its resilience to drought stress. However, the significance of drought timing remains poorly understood, both from a plant biology perspective and its impact on downstream biofuel production. This study determines the developmental stage-specific physiological and metabolic responses of switchgrass to drought stress and its implications for biofuel production using a custom-built programmable irrigation system. Vegetative, flowering, and senescence-stage drought significantly reduced carbon dioxide assimilation, and stomatal conductance without affecting biomass yield. Metabolic profiling revealed significant accumulation of glucose, fructose, quinic acid, shikimate and GABA during vegetative-stage drought, while flowering and senescence stages exhibited limited metabolic changes. Similarly, specialized metabolites also displayed distinct developmental patterns, with vegetative-stage drought driving the most pronounced metabolic alterations. Thermochemically-treated and hydrolyzed switchgrass biomass from vegetative-stage drought showed elevated lignocellulose-derived compounds and saponins with the latter most positively correlating with fermentation lag times. Conversely, senescence-stage drought enhanced ethanol yields while lowering saponin levels in the hydrolysates. While vegetative-stage drought enhanced physiological resilience, it compromises downstream biofuel production by introducing fermentation inhibitors, particularly saponins.

biofuel↗

DOE OSTI2020

Non-LTE modelling of cyanoacetylene: evidence for isomer-specific excitation

ABSTRACT Cyanoacetylene molecules are widespread in the interstellar medium (ISM) and several of its isomers have been detected in cold molecular clouds and circumstellar gas. Accurate estimates of the abundance ratio of cyanoacetylene isomers may provide deep insight into their environment. Such knowledge requires rigorous modelling of the emission spectra based on non-local thermodynamic equilibrium (LTE) radiative transfer calculations. To this end, we computed excitation cross-sections of HC2NC and HNC3 induced by collision with para- and ortho-H2, using a quantum mechanical close-coupling method. Then, by thermally averaging these data, we derived rate coefficients for the first 31 low-lying rotational levels of each isomer for temperatures up to 80 K. For the para-H2 collider, the propensity rules are in favour of rotational transitions involving Δj1 = 2 for both isomers, while for the ortho-H2 collider, Δj1 = 2 and Δj1 = 1 rotational transitions are favoured for HC2NC and HNC3, respectively. A comparison of rate coefficients for the HC3N isomers shows differences up to an order of magnitude, especially at low temperatures. Finally, we performed non-LTE radiative transfer calculations to assess the impact of such variations in the analysis of observations. Our simulation suggests that the lack of collisional data specific to each isomer could lead to errors up to a factor of 2–3 in the excitation temperatures. We expect that these data could help in better understanding the cyanoacetylene chemistry and constraining the nitrogen chemistry in the ISM.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2021

Structural insights into the substrate specificity of the endonuclease activity of the influenza virus cap-snatching mechanism

Abstract The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN) of the influenza RNA dependent RNA polymerase, and we report here complexes of PAN with RNA and DNA oligonucleotides to understand its specificity and the structural basis of baloxavir resistance mutations. The RNA and DNA oligonucleotides bind within the substrate binding groove of PAN in a similar fashion, explaining the ability of the enzyme to cleave both substrates. The individual nucleotides occupy adjacent conserved pockets that flank the two-metal active site. However, the 2′ OH of the RNA ribose moieties engage in additional interactions that appear to optimize the binding and cleavage efficiency for the natural substrate. The major baloxavir resistance mutation at position 38 is at the core of the substrate binding site, but structural studies and modeling suggest that it maintains the necessary virus fitness via compensating interactions with RNA. These studies will facilitate the development of new influenza therapeutics that spatially match the substrate and are less likely to elicit resistance mutations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Prediction of condition-specific regulatory genes using machine learning

Recent advances in genomic technologies have generated data on large-scale protein–DNA interactions and open chromatin regions for many eukaryotic species. How to identify condition-specific functions of transcription factors using these data has become a major challenge in genomic research. To solve this problem, we have developed a method called ConSReg, which provides a novel approach to integrate regulatory genomic data into predictive machine learning models of key regulatory genes. Using Arabidopsis as a model system, we tested our approach to identify regulatory genes in data sets from single cell gene expression and from abiotic stress treatments. Our results showed that ConSReg accurately predicted transcription factors that regulate differentially expressed genes with an average auROC of 0.84, which is 23.5–25% better than enrichment-based approaches. To further validate the performance of ConSReg, we analyzed an independent data set related to plant nitrogen responses. ConSReg provided better rankings of the correct transcription factors in 61.7% of cases, which is three times better than other plant tools. We applied ConSReg to Arabidopsis single cell RNA-seq data, successfully identifying candidate regulatory genes that control cell wall formation. Our methods provide a new approach to define candidate regulatory genes using integrated genomic data in plants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP’s enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP’s role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Improved age- and gender-specific radiation risk models applied on cohorts of Swedish patients

The aim of this study is to implement lifetime attributable risk (LAR) predictions for radiation induced cancers for Swedish cohorts of patients of various age and sex, undergoing diagnostic investigations by nuclear medicine methods. Methods: Calculations are performed on Swedish groups of patients with Paget's disease and with bone metastases from prostatic cancer and diagnosed with bone scintigraphy with an administration of 500 MBq 99m Tc-phosphonate. Results: The inclusion of patient survival rates into the calculations lowers the induced radiation cancer risk, as it takes into account that cohorts of patients have shorter predicted survival times than the general population. Conclusion: LAR estimations could be valuable for referring physicians, nuclear medicine physicians, nurses, medical physicists, radiologists, and oncologists and as well as ethical committees for risk estimates for specific subgroups of patients. Caution is however advised with respect to application of LAR predictions to individuals (because of individual sensitivities, circumstances, etc.).

61 RADIATION PROTECTION AND DOSIMETRY↗