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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Base Excision Repair: Mechanisms and Impact in Biology, Disease, and Medicine

Base excision repair (BER) corrects forms of oxidative, deamination, alkylation, and abasic single-base damage that appear to have minimal effects on the helix. Since its discovery in 1974, the field has grown in several facets: mechanisms, biology and physiology, understanding deficiencies and human disease, and using BER genes as potential inhibitory targets to develop therapeutics. Within its segregation of short nucleotide (SN-) and long patch (LP-), there are currently six known global mechanisms, with emerging work in transcription- and replication-associated BER. Knockouts (KOs) of BER genes in mouse models showed that single glycosylase knockout had minimal phenotypic impact, but the effects were clearly seen in double knockouts. However, KOs of downstream enzymes showed critical impact on the health and survival of mice. BER gene deficiency contributes to cancer, inflammation, aging, and neurodegenerative disorders. Medicinal targets are being developed for single or combinatorial therapies, but only PARP and APE1 have yet to reach the clinical stage.

5′-Gap↗
DOE OSTI2024

Development and Assessment of SNP Genotyping Arrays for Citrus and Its Close Relatives

Rapid advancements in technologies provide various tools to analyze fruit crop genomes to better understand genetic diversity and relationships and aid in breeding. Genome-wide single nucleotide polymorphism (SNP) genotyping arrays offer highly multiplexed assays at a relatively low cost per data point. We report the development and validation of 1.4M SNP Axiom® Citrus HD Genotyping Array (Citrus 15AX 1 and Citrus 15AX 2) and 58K SNP Axiom® Citrus Genotyping Arrays for Citrus and close relatives. SNPs represented were chosen from a citrus variant discovery panel consisting of 41 diverse whole-genome re-sequenced accessions of Citrus and close relatives, including eight progenitor citrus species. SNPs chosen mainly target putative genic regions of the genome and are accurately called in both Citrus and its closely related genera while providing good coverage of the nuclear and chloroplast genomes. Reproducibility of the arrays was nearly 100%, with a large majority of the SNPs classified as the most stringent class of markers, “PolyHighResolution” (PHR) polymorphisms. Concordance between SNP calls in sequence data and array data average 98%. Phylogenies generated with array data were similar to those with comparable sequence data and little affected by 3 to 5% genotyping error. Both arrays are publicly available.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Candidatus Nanopusillus

The genus Candidatus Nanopusillus is comprised of small coccoid cells (~100–400 nm) that live epibiotically on the surface of archaeal hosts. The first described species, Candidatus Nanopusillus acidilobi, is an anaerobic, hyperthermophilic acidophile whose best growth is observed at 82°C, pH 3.6, cultivated from a hot spring in Yellowstone National Park. Ca. Nanopusillus acidilobi cells associate with the Crenarchaeota host organism Acidilobus sp. 7A. Archaeal flagella (archaella) have been predicted from the genome sequence and shown to be expressed in the proteome. Additionally, a second putative species, Candidatus Nanopusillus massiliensis, was recently reported from human dental plaque and associates with the methanogen Methanobrevibacter oralis. The genome consists of a single scaffold which is highly fragmented by spans of ambiguous nucleotides, with 16S rRNA gene fragments from Bacteria. Both species have small genomes (~0.6 Mb) encoding few biosynthetic genes and no apparent ATP synthase complex genes, suggesting that the nanoarchaeotes rely on their host for the production of major cellular precursors.

acidophile↗
DOE OSTI2022

RNAseq-based transcriptome assembly of Clostridium acetobutylicum for functional genome annotation and discovery

Accurate genome annotations are essential in modern biology and biotechnology, yet they are still largely based on genome sequencing and comparative analyses. We show that the Clostridium acetobutylicum genome annotation can be markedly improved by integrating bioinformatic predictions with RNA sequencing (RNAseq) data. Samples were acquired under butanol, butyrate, and unstressed treatments across various growth conditions. Analysis of an initial assembly revealed errors due to background signals and limitations of assembly algorithms. Hurdles for RNAseq transcriptome mapping include optimizing library complexity and sequencing depth, yet most studies report low sequencing depth and ignore the effect of ribosomal RNA abundance. An integrative analysis was developed to combine motif predictions, single-nucleotide resolution sequencing depth, and library complexity to resolve difficulties in assembly curation. This minimized false positive error and determined gene boundaries, in some cases, to the exact base-pair of prior studies. This will be the first strand-specific transcriptome assembly in a Clostridium organism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Introgression and persistence of cultivar alleles in wild carrot ( Daucus carota ) populations in the United States

Abstract Premise Cultivated species and their wild relatives often hybridize in the wild, and the hybrids can survive and reproduce in some environments. However, it is unclear whether cultivar alleles are permanently incorporated into the wild genomes or whether they are purged by natural selection. This question is key to accurately assessing the risk of escape and spread of cultivar genes into wild populations. Methods We used genomic data and population genomic methods to study hybridization and introgression between cultivated and wild carrot (Daucus carota) in the United States. We used single nucleotide polymorphisms (SNPs) obtained via genotyping by sequencing for 450 wild individuals from 29 wild georeferenced populations in seven states and 144 cultivars from the United States, Europe, and Asia. Results Cultivated and wild carrot formed two genetically differentiated groups, and evidence of crop–wild admixture was detected in several but not all wild carrot populations in the United States. Two regions were identified where cultivar alleles were present in wild carrots: California and Nantucket Island (Massachusetts). Surprisingly, there was no evidence of introgression in some populations with a long‐known history of sympatry with the crop, suggesting that post‐hybridization barriers might prevent introgression in some areas. Conclusions Our results provide support for the introgression and long‐term persistence of cultivar alleles in wild carrots populations. We thus anticipate that the release of genetically engineered (GE) cultivars would lead to the introduction and spread of GE alleles in wild carrot populations.

Plant Sciences↗
DOE OSTI2021

Reconfigurable Two‐Dimensional DNA Lattices: Static and Dynamic Angle Control

Abstract Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle‐enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson–Crick base pairing or non‐canonical nucleobase interactions (e.g., i‐motif and G‐quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely‐ordered three‐dimensional architectures.

Wang, Wen↗
DOE OSTI2021

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. Here in this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson–Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8-oxo -7,8-dihydropurine, and 8-bromopurine analogues within A-form duplexes of RNA

Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8-oxo-7,8-dihydropurine (common products of oxidation at the C8-position of purines), and 8-bromopurine (as probes to explore conformational changes), derivatives, namely 8-oxoG, 8-oxoI, 8-oxoA, 8-BrG, and 8-BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid-phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (Tm) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H-bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti-I:syn-A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8-oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble-pair is only stabilized by G; and (d) 8-oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Temporal Galactose‐Manganese Feeding in Fed‐Batch and Perfusion Bioreactors Modulates UDP‐Galactose Pools for Enhanced mAb Glycosylation Homogeneity

ABSTRACT Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N‐linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N‐glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N‐glycosylation and how mAb glycoform composition responds to real‐time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N‐glycosylation pathway to modulate N‐glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N‐GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real‐time for bench‐scale bioprocesses operated in both fed‐batch and perfusion modes to build an understanding of how temporal changes in mAb N‐glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose‐occupied glycoforms over time under glucose‐starved fed‐batch cultures. Comparable galactosylation profiles were also observed between fed‐batch (nutrient‐limited) and perfusion (non‐nutrient‐limited) bioprocess conditions. In summary, our results demonstrate the utility of real‐time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed‐batch and perfusion bioprocessing conditions to produce higher‐quality biologics.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Mechanisms in the Synthesis of S -Alcohols with 1,4-NADH Biomimetic Co-factor N-Benzyl-1,4-dihydronicotinamide using Horse Liver Alcohol Dehydrogenase: A Hybrid Computational Study

The enantioselective reduction of prochiral ketones catalyzed by horse liver alcohol dehydrogenase (HLADH), was investigated via a hybrid computational approach, for molecular reactions involved in chiral synthesis of S-alcohols, when the natural co-factor, 1,4-dihyronicotinamide adenine dinucleotide, 1,4-NADH, was replaced with biomimetic co-factor, N-benzyl-1,4-dihydronicotinamide, 1. We surmised that different hydride and proton transfer mechanisms were involved using co-factor, 1. An alternative mechanism, where the hydride transfer step occurred, via an η 1 -keto-S-η 2 -5,6-1,4-dihydronicotinamide-Zn(II) complex, was previously investigated with a model of the HLADH-Zn(II) catalytic site (J. Organometal. Chem. 2021, 943, 121810). Presently, we studied canonical and alternative mechanisms compared to models of the entire enzyme structure. We disproved the η 2 -Zn(II) complex, and discovered a canonical hydride transfer from biomimetic 1,4-NADH, 1, to the Zn(II) bound prochiral ketone substrate, followed by a new proton relay, consisting of a water chain connecting His51 to Ser48 that accomplished the S-alkoxy anion's protonation to yield the final S-alcohol product. The HLADH catalysis, with biomimetic co-factor, 1, that replaced the ribose group, the 5'-diphosphate groups, and the adenine nucleotide with a N-benzyl group, has provided a new paradigm for the design of other structures of 1,4-NADH biomimetic co-factors, including their economic value in biocatalysis reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Identification of quantitative trait loci for sorghum leaf blight resistance

Sorghum leaf blight and northern corn leaf blight, both caused by Exserohilum turcicum {(Pass.) K. J. Leonard and Suggs [syn. Setosphaeria turcica (Luttr.) K. J. Leonard and Suggs.]}, are major diseases of sorghum [Sorghum bicolor (L.) Moench] and maize (Zea mays L.), respectively. Examining the genetic architecture of resistance in sorghum will lead to a better understanding of the relationship between resistance in sorghum and maize, which can ultimately enhance management options in both crops. In 2018 and 2019, we evaluated two sorghum recombinant inbred line (RIL) populations for resistance to E. turcicum. The BTx623 × IS3620C and BTx623 × SC155 populations consisted of 235 and 81 RILs, respectively. Resistance in both populations was moderately to highly heritable. We identified a total of six quantitative trait loci (QTL) across the two populations. Three QTL with small- to moderate-effect sizes were identified in the BTx623 × IS3620C population. Three QTL, including a large-effect QTL on chromosome three that explained 24% of the variation, were identified in the BTx623 × SC155 population. We compared the identified QTL with the position of northern corn leaf blight candidate genes and found eight candidate resistance gene orthologs that colocalize with the sorghum leaf blight QTL. There were also several nucleotide-binding leucine-rich repeat encoding genes within the candidate intervals. Understanding host resistance in multiple species furthers our understanding of the Exserohilum turcicum patho-system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genetic mapping of sugarcane aphid resistance in sorghum line SC112-14

Sugarcane aphid [Melanaphis sacchari (Zehntner)] is a destructive pest that has had an economic effect on sorghum in North America since 2013. The identification, development, and use of resistant sorghum germplasm is the most feasible strategy to control the pest. Nevertheless, the genetic control of sugarcane aphid (SCA) resistance is unknown for most sorghum resistant lines. To identify the genetic regions that confer SCA resistance in sorghum line SC112-14, 103 recombinant inbred lines (RILs) derived by its cross with the susceptible line PI 609251 were evaluated for their SCA resistance response in Georgia during two consecutive years. The resistance response was determined based on two ratings (2 wk apart) for aphid population size (APS) and aphid-induced plant damage (APD) each year. Segregation for SCA resistance was observed for the first APS and both APD ratings, and the broad-sense heritability estimate ranged from .71 to .76, respectively. A quantitative trait locus analysis using a high-density linkage map of 3,852 single nucleotide polymorphisms (SNPs) detected an 81-kb genomic region on chromosome 6 that explained 50–55% of the phenotypic variation. Comparative mapping analysis found that the resistance locus in SC112-14 is located 8- and 10-cM upstream of the Henong 16 (RMES1) and Tx2783 resistance loci, respectively, and encloses the SNP Sbv3.1_06_2316351 associated in Haitian resistant lines. Therefore, the line SC112-14 is an additional SCA resistance source that can be combined or strategically used with other resistance sources to assure a more robust host plant resistance to the SCA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Transcriptome and metabolome integration in sugarcane through culm development

Abstract Sugarcane (Saccharum sp.) is a tropical and subtropical C4 plant with a high photosynthetic and carbon assimilation efficiency that stores sucrose. Culm biomass is also composed of bagasse fibre, a by‐product of the sugarcane industry. This high‐yielding grass, high in sucrose and lignocellulosic biomass, is considered an optimal feedstock as an alternative to fossil fuels and to produce a broad range of high‐value biomaterials. The ideal sugarcane production system would optimise the relative production of sugar and these new products. Multi‐omics correlation analysis was used to generate a global view of the essential metabolic pathways identifying critical genes involved in carbon partitioning during different stages of development. This research employed an unprecedented metabolic and transcriptomic dataset of 360 samples from a selection of 1440 culms of 24 genotypes at five different development stages. Chemical composition and metabolome analysis showed an increase through the culm development of lignin, sucrose, carbon, and amino acids such as aspartic acid, serine, alanine, methionine, threonine 3‐cyano‐L‐alanine, and citric acid. Transcriptome analysis revealed functionalities such as transcription, nucleotide transport and metabolism, and the biosynthesis of amino acids that are highly activated during the immature stage and highly down‐regulated during the most mature age.

Perlo, Virginie↗
DOE OSTI2021

Grandparent Inference from Genetic Data: The Potential for Parentage-Based Tagging Programs to Identify Offspring of Hatchery Strays

Abstract Fisheries managers routinely use hatcheries to increase angling opportunity. Many hatcheries operate as segregated programs where hatchery-origin fish are not intended to spawn with natural-origin conspecifics in order to prevent potential negative effects on the natural-origin population. Currently available techniques to monitor the frequency with which hatchery-origin strays successfully spawn in the wild rely on either genetic differentiation between the hatchery- and natural-origin fish or extensive sampling of fish on the spawning grounds. We present a method to infer grandparent–grandchild trios by using only genotypes from two putative grandparents and one putative grandchild. We developed estimators of false-positive and false-negative error rates and showed that genetic panels containing 500–700 single-nucleotide polymorphisms or 200–300 microhaplotypes are expected to allow application of this technique for monitoring segregated hatchery programs. We discuss the ease with which this technique can be implemented by pre-existing parentage-based tagging programs and provide an R package that applies the method.

Delomas, Thomas A. (ORCID:000000015154759X)↗
DOE OSTI2022

Registration of the sorghum carbon–partitioning nested association mapping (CP–NAM) population

The sorghum [ Sorghum bicolor (L.) Moench] carbon-partitioning nested association mapping (CP_NAM) (Reg. no. MP-4, NSL 542189 MAP) population was developed at Clemson University, SC, using 11 diverse, male founder accessions, each crossed with a recurrent female parent ‘Grassl’. The male parents represent all five major botanical races and the four major agronomic types: cellulosic (5), sweet (3), grain (2) and forage (1). A set of 11 recombinant inbred line (RIL) families CP_NAM01 to CP_NAM011 were maintained, which consisted of 2,484 (F 6 ) individuals. Each RIL family contained a minimum of 193 individuals (CP_NAM01) and a maximum of 287 individuals (CP_NAM06). For the development of this population, the founder lines were judiciously selected from the sorghum Bioenergy Association Panel based on carbon-partitioning phenotypes that make this population an ideal genetic resource for dissecting a wide range of agronomic and compositional traits for basic and applied research. The founder accessions of the CP_NAM were phenotypically characterized for various traits, including agronomic, biomass and related components, and additional compositional components. Each of the 11 F 6 RIL families of the CP_NAM were genotyped using genotyping-by-sequencing analysis, and 144,087 single nucleotide polymorphisms were generated for each individual. Genotypic information along with phenotypic data were used for the characterization of this population and to explore the range of phenotypes that permits the understanding of carbon-partitioning dynamics. This population is a unique resource for researchers to study a wide range of contrasting carbon-partitioning characteristics in sorghum to understand the genetic architecture underlying whole-plant carbon partitioning and allocation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Pacific Lamprey Translocations to the Snake River Boost Abundance of All Life Stages

Abstract The Pacific Lamprey Entosphenus tridentatus is an anadromous fish that has severely declined in the Snake River basin. In 2007, the Nez Perce Tribe initiated translocation of adults to this region from the main‐stem Columbia River and has continued the translocations to this day. These actions are aimed at restoring larval abundance along with holistic habitat improvements. We performed parentage and sibship analyses with 260 single‐nucleotide polymorphism loci to monitor productivity of translocated lamprey over a decade (2007–2018). These results provide the first direct evidence that translocations boosted larval abundance, increased juvenile production in the interior Columbia River (~3% in 2017 and 2018), and demonstrated successful migration to the Pacific Ocean. Per capita juvenile production from Snake River adult lamprey translocations out‐performed that of their volitionally migrating counterparts in the interior Columbia River, demonstrating that translocating adults to suitable habitats increased overall productivity. We projected that the translocations may eventually return enough adult offspring to the Columbia River to replace the annual take of adults for translocations, despite many adults likely returning to other basins. These translocations are also restoring Pacific Lamprey to historical areas where they had been nearly extirpated, which has impactful benefits both culturally and ecologically. Further, this study provided an unprecedented volume of data that has redefined key biological attributes, including timing of life stage transformation (average of 6.7‐year‐old juveniles), larval growth rates (~22 mm/year for the first 4 years), ocean duration (average of 5.1 years), dispersal (~3% of Snake River‐origin adults that return to the Columbia River will divert to the Willamette River), and life span (median age of postspawn adults was 12.9 years). We even quantified a new biological phenomenon of delayed spawning, which occurs at low rates (~9%) in nature. These data provide an opportunity to monitor ongoing translocation efforts and inform adaptive management to aid species recovery.

Hess, Jon E.↗
DOE OSTI2024

Population assignment of migratory Westslope Cutthroat Trout (WCT) in the Clark Fork–Pend Oreille River basin

Abstract Objective The Clark Fork–Pend Oreille River basin of northeastern Washington and the Idaho Panhandle historically supported a robust metapopulation of the Westslope Cutthroat Trout (WCT) Oncorhynchus lewisi, a western native salmonid of high cultural and economic value. The construction of impassible hydroelectric dams and smaller instream barriers has prevented the return of migratory WCT to spawning tributaries, leading to the fragmentation of this metapopulation over the past 100 years. One such impassible barrier is Albeni Falls Dam (AFD) near Newport, Washington, which was completed without fish passage capabilities in 1955. We sought to examine large-scale genetic patterns in the study area and determine the most likely spawning tributary of origin for migratory WCT captured below AFD. Methods We created a genetic baseline representative of populations within the Clark Fork–Pend Oreille River basin from upstream and downstream of the dam using 191 biallelic single-nucleotide polymorphism genetic markers. Our data set included 124 collections, which allowed for an examination of population structure and hatchery influence across the study area and provided a robust tool for population assignment. Population assignment tests were conducted using the program RUBIAS. Result Population assignment tests were successful for all pure WCT of unknown origin despite potential influence from hatchery lineages across the study area. Of 83 migratory WCT captured below AFD, approximately 80% were assigned to tributaries upstream of AFD with a posterior assignment probability of at least 90%. Only one fish was assigned to a tributary downstream of AFD. Conclusion Our results indicate that AFD disrupts the natural metapopulation dynamics of WCT populations in the basin. Passage for WCT at this barrier would reestablish metapopulation connectivity within the basin by allowing migratory individuals to make genetic contributions to populations upstream of the dam.

Wells, Craig D. (ORCID:0000000258513250)↗