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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Phylogenetic analyses and reclassification of the oleaginous marine species Nannochloris sp. “desiccata" (Trebouxiophyceae, Chlorophyta), formerly Chlorella desiccata , supported by a high-quality genome assembly

Microalgae are diverse, with many gaps remaining in phylogenetic and physiological understanding. Thus, studying new microalgae species increases our broader comprehension of biological diversity, and evaluation of new candidates as algal production platforms can lead to improved productivity under a variety of cultivation conditions. Chlorella is a genus of fast-growing species often isolated from freshwater habitats and cultivated as a source of nutritional supplements. However, the use of freshwater increases competition with other freshwater needs. We identified Chlorella desiccata to be worthy of further investigation as a potential algae production strain, due to its isolation from a marine environment and its promising growth and biochemical composition properties. Long-read genomic sequencing was conducted for C. desiccata UTEX 2526, resulting in a high-quality, near chromosome level, diploid genome with an assembly length of 21.55 Mbp in only 18 contigs. We also report complete circular mitochondrial and chloroplast genomes. Phylogenomic and phylogenetic analyses using nuclear, chloroplast, 18S rRNA, and actin sequences revealed that this species clades within strains currently identified as Nannochloris (Trebouxiophyceae, Chlorophyta), leading to its reclassification as Nannochloris sp. “desiccata” UTEX 2526. The mode of cell division for this species is autosporulation, differing from the type species N. bacillaris. As has occurred across multiple microalgae genera, there are repeated examples of Nannochloris species reclassification in the literature. This high-quality genome assembly and phylogenetic analysis of the potential algal production strain Nannochloris sp. “desiccata” UTEX 2526 provides an important reference and useful tool for further studying this region of the phylogenetic tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification of key steps in the evolution of anaerobic methanotrophy in Candidatus Methanovorans (ANME-3) archaea

Despite their large environmental impact and multiple independent emergences, the processes leading to the evolution of anaerobic methanotrophic archaea (ANME) remain unclear. This work uses comparative metagenomics of a recently evolved but understudied ANME group, “Candidatus Methanovorans” (ANME-3), to identify evolutionary processes and innovations at work in ANME, which may be obscured in earlier evolved lineages. We identified horizontal transfer of hdrA homologs and convergent evolution in carbon and energy metabolic genes as potential early steps in Methanovorans evolution. We also identified the erosion of genes required for methylotrophic methanogenesis along with horizontal acquisition of multiheme cytochromes and other loci uniquely associated with ANME. The assembly and comparative analysis of multiple Methanovorans genomes offers important functional context for understanding the niche-defining metabolic differences between methane-oxidizing ANME and their methanogen relatives. Furthermore, this work illustrates the multiple evolutionary modes at play in the transition to a globally important metabolic niche.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na + ). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as N ε -acetyl-β-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genetic Predictive Factors for Nonsusceptible Phenotypes and Multidrug Resistance in Expanded-Spectrum Cephalosporin-Resistant Uropathogenic Escherichia coli from a Multicenter Cohort: Insights into the Phenotypic and Genetic Basis of Coresistance

Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum β-lactamase (ESBL) phenotype, with bla CTX-M-15 , bla CTX-M-27 , bla CTX-M-55 , and bla CTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained bla CMY-2 , which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum β-lactamases, bla TEM-1B and bla OXA-1 , were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of bla CTX-M-15 , bla OXA-1 , and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of bla CTX-M-15 , bla OXA-1 , and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of bla CTX-M-55 , bla TEM-1B , and the ST131 genotype as predictors of MDR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic model predictions enable targeted microbiome manipulation through precision prebiotics

While numerous health-beneficial interactions between host and microbiota have been identified, there is still a lack of targeted approaches for modulating these interactions. Thus, we here identify precision prebiotics that specifically modulate the abundance of a microbiome member species of interest. In the first step, we show that defining precision prebiotics by compounds that are only taken up by the target species but no other species in a community is usually not possible due to overlapping metabolic niches. Subsequently, we use metabolic modeling to identify precision prebiotics for a two-member Caenorhabditis elegans microbiome community comprising the immune-protective target species Pseudomonas lurida MYb11 and the persistent colonizer Ochrobactrum vermis MYb71. We experimentally confirm four of the predicted precision prebiotics, L-serine, L-threonine, D-mannitol, and γ-aminobutyric acid, to specifically increase the abundance of MYb11. L-serine was further assessed in vivo, leading to an increase in MYb11 abundance also in the worm host. Overall, our findings demonstrate that metabolic modeling is an effective tool for the design of precision prebiotics as an important cornerstone for future microbiome-targeted therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

maranasgroup/scRBA

Saccharomyces cerevisiae is an important model organism and a workhorse in bioproduction. Here, we reconstructed a compact and tractable genome-scale resource balance analysis (RBA) model (i.e., named scRBA) to analyze metabolic fluxes and proteome allocation in a computationally efficient manner. Resource capacity models such as scRBA provide the quantitative means to identify bottlenecks in biosynthetic pathways due to enzyme, compartment size, and/or ribosome availability limitations. ATP maintenance rate and in vivo apparent turnover numbers (kapp) were regressed from metabolic flux and protein concentration data to capture observed physiological growth yield and proteome efficiency and allocation, respectively. Estimated parameter values were found to vary with oxygen and nutrient availability. Overall, this work (i) provides condition-specific model parameters to recapitulate phenotypes corresponding to different extracellular environments, (ii) alludes to the enhancing effect of substrate channeling and post-translational activation on in vivo enzyme efficiency in glycolysis and electron transport chain, and (iii) reveals that the Crabtree effect is underpinned by specific limitations in mitochondrial proteome capacity and secondarily ribosome availability rather than overall proteome capacity.

Dinh, Hoang↗
DOE OSTI2021

Zymomonas diversity and potential for biofuel production

Abstract Background Zymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation. Results Using clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction–modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency. Conclusions Our findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates.

09 BIOMASS FUELS↗
DOE OSTI2020

Kraken2 Metagenomic Virus Database

The Database: Kraken2 [1] database built from a classification tree containing over 700k metagenomic viruses from JGI IMG/VR [2]. (1) Wood, D. E., Lu, J., and Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biol., 20(1), 1–13. doi: 10.1186/s13059-019-1891-0 (2) Paez-Espino D, Chen I-MA, Palaniappan K, Ratner A, Chu K, Szeto E, et al. IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses. Nucleic Acids Res. 2017;45:D457-65. For Paper: Title: A k-mer based approach for virus classification in metatranscriptomic and metagenomic samples identifies viral associations in the Populus phytobiome and autism brains Abstract Background Viruses are an underrepresented taxa in the study and identification of microbiome constituents; however, they play an important role in health, microbiome regulation, and transfer of genetic material. Only a few thousand viruses have been isolated, sequenced, and assigned a taxonomy, which further limits the ability to identify and quantify viruses in the microbiome. Additionally, the vast diversity of viruses represents a challenge for classification, not only in constructing a viral taxonomy, but also in identifying similarities between a virus' genotype and its phenotype. However, the diversity of viral sequences can be leveraged to classify their sequences in metagenomic and metatranscriptomic samples. Methods To identify viruses in transcriptomic and genomic samples, we developed a dynamic programming algorithm for creating a classification tree out of 715,672 metagenome viruses. To create the classification tree, we clustered proportional similarity scores generated from the k-mer profiles of each of the metagenome viruses. We then integrated the viral classification tree with the NCBI taxonomy for use with ParaKraken, a metagenomic/transcriptomic classifier. Results To illustrate the breadth of our utility for classifying viruses with ParaKraken, we analyzed data from a plant metagenome study identifying the differences between two Populus genotypes in three different compartments and on a human metatranscriptome study identifying the differences between Autism Spectrum Disorder patients and controls in post mortem brain biopsies. In the Populus study, we identified genotype and compartment specific viral signatures, while in the Autism study we identified a significant increased abundance of eight viral sequences in Autism brain biopsies. Conclusion Viruses represent an important aspect of the microbiome. The ability to classify viruses represents the first step in being able to better understand their role in the microbiome. The viral classification method presented here allows for more complete identification of viral sequences for use in identifying associations between viruses and the host and viruses and other microbiome members. Acknowledgements and Funding This research used resources of the Oak Ridge Leadership Computing Facility at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725. This research was also supported by the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program, the Office of Biological and Environmental Research (BER) in the U.S. Department of Energy Office of Science, and by the Department of Energy, Laboratory Directed Research and Development funding (ProjectID 8321), at the Oak Ridge National Laboratory. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US DOE under contract DE-AC05-00OR22725. This research used resources of the Compute and Data Environment for Science (CADES).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Dissemination of blaNDM-5 and mcr-8.1 in carbapenem-resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae in an animal breeding area in Eastern China

Animal farms have become one of the most important reservoirs of carbapenem-resistant Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. “One Health”-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of “One-Health,” 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene bla NDM-5 , which was flanked by the same core genetic structure (IS 5 - bla NDM-5 - ble MBL - trpF - dsbD -IS 26 -IS Kox3 ) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS 903 - orf - mcr-8.1-copR-baeS-dgkA - orf -IS 903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying- bla NDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.

Yang, Chengxia↗
DOE OSTI2023

Synergistic interactions between anammox and dissimilatory nitrate reducing bacteria sustains reactor performance across variable nitrogen loading ratios

Anaerobic ammonium oxidizing (anammox) bacteria are utilized for high efficiency nitrogen removal from nitrogen-laden sidestreams in wastewater treatment plants. The anammox bacteria form a variety of competitive and mutualistic interactions with heterotrophic bacteria that often employ denitrification or dissimilatory nitrate reduction to ammonium (DNRA) for energy generation. These interactions can be heavily influenced by the influent ratio of ammonium to nitrite, NH 4 + :NO 2 - , where deviations from the widely acknowledged stoichiometric ratio (1:1.32) have been demonstrated to have deleterious effects on anammox efficiency. Thus, it is important to understand how variable NH 4 + :NO 2 - ratios impact the microbial ecology of anammox reactors. We observed the response of the microbial community in a lab scale anammox membrane bioreactor (MBR) to changes in the influent NH 4 + :NO 2 - ratio using both 16S rRNA gene and shotgun metagenomic sequencing. Ammonium removal efficiency decreased from 99.77 ± 0.04% when the ratio was 1:1.32 (prior to day 89) to 90.85 ± 0.29% when the ratio was decreased to 1:1.1 (day 89–202) and 90.14 ± 0.09% when the ratio was changed to 1:1.13 (day 169–200). Over this same timespan, the overall nitrogen removal efficiency (NRE) remained relatively unchanged (85.26 ± 0.01% from day 0–89, compared to 85.49 ± 0.01% from day 89–169, and 83.04 ± 0.01% from day 169–200). When the ratio was slightly increased to 1:1.17–1:1.2 (day 202–253), the ammonium removal efficiency increased to 97.28 ± 0.45% and the NRE increased to 88.21 ± 0.01%. Analysis of 16S rRNA gene sequences demonstrated increased relative abundance of taxa belonging to Bacteroidetes, Chloroflexi, and Ignavibacteriae over the course of the experiment. The relative abundance of Planctomycetes, the phylum to which anammox bacteria belong, decreased from 77.19% at the beginning of the experiment to 12.24% by the end of the experiment. Analysis of metagenome assembled genomes (MAGs) indicated increased abundance of bacteria with nrfAH genes used for DNRA after the introduction of lower influent NH 4 + :NO 2 - ratios. The high relative abundance of DNRA bacteria coinciding with sustained bioreactor performance indicates a mutualistic relationship between the anammox and DNRA bacteria. Understanding these interactions could support more robust bioreactor operation at variable nitrogen loading ratios.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

CyanoCyc cyanobacterial web portal

CyanoCyc is a web portal that integrates an exceptionally rich database collection of information about cyanobacterial genomes with an extensive suite of bioinformatics tools. It was developed to address the needs of the cyanobacterial research and biotechnology communities. The 277 annotated cyanobacterial genomes currently in CyanoCyc are supplemented with computational inferences including predicted metabolic pathways, operons, protein complexes, and orthologs; and with data imported from external databases, such as protein features and Gene Ontology (GO) terms imported from UniProt. Five of the genome databases have undergone manual curation with input from more than a dozen cyanobacteria experts to correct errors and integrate information from more than 1,765 published articles. CyanoCyc has bioinformatics tools that encompass genome, metabolic pathway and regulatory informatics; omics data analysis; and comparative analyses, including visualizations of multiple genomes aligned at orthologous genes, and comparisons of metabolic networks for multiple organisms. CyanoCyc is a high-quality, reliable knowledgebase that accelerates scientists’ work by enabling users to quickly find accurate information using its powerful set of search tools, to understand gene function through expert mini-reviews with citations, to acquire information quickly using its interactive visualization tools, and to inform better decision-making for fundamental and applied research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

Three dimensional (3D) ultra-structural imaging is an important tool for unraveling the organizational structure of individual chromosomes at various stages of the cell cycle. Performing hitherto uninvestigated ultra-structural analysis of the human genome at prophase, we used serial block-face scanning electron microscopy (SBFSEM) to understand chromosomal architectural organization within 3D nuclear space. Acquired images allowed us to segment, reconstruct, and extract quantitative 3D structural information about the prophase nucleus and the preserved, intact individual chromosomes within it. Our data demonstrate that each chromosome can be identified with its homolog and classified into respective cytogenetic groups. Thereby, we present the first 3D karyotype built from the compact axial structure seen on the core of all prophase chromosomes. The chromosomes display parallel-aligned sister chromatids with familiar chromosome morphologies with no crossovers. Furthermore, the spatial positions of all 46 chromosomes revealed a pattern showing a gene density-based correlation and a neighborhood map of individual chromosomes based on their relative spatial positioning. A comprehensive picture of 3D chromosomal organization at the nanometer level in a single human lymphocyte cell is presented.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
NASA NTRS2003

Search-based optimization

The problem of determining the minimum cost hypothetical ancestral sequences for a given cladogram is known to be NP-complete (Wang and Jiang, 1994). Traditionally, point estimations of hypothetical ancestral sequences have been used to gain heuristic, upper bounds on cladogram cost. These include procedures with such diverse approaches as non-additive optimization of multiple sequence alignment, direct optimization (Wheeler, 1996), and fixed-state character optimization (Wheeler, 1999). A method is proposed here which, by extending fixed-state character optimization, replaces the estimation process with a search. This form of optimization examines a diversity of potential state solutions for cost-efficient hypothetical ancestral sequences and can result in greatly more parsimonious cladograms. Additionally, such an approach can be applied to other NP-complete phylogenetic optimization problems such as genomic break-point analysis. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

Non-NASA Center↗
NASA NTRS1995

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

NASA Discipline Radiation Health↗
NASA NTRS2019

Predicting Cancer Risk from Ionizing Radiation

The ability to predict cancer risk associated with exposure to low doses of high-LET ionizing radiation (IR) remains a challenge. Epidemiological methods lack the sensitivity and power to provide detailed risk estimates for cancer and ignore individual variance in IR sensitivity. We have hypothesized that DNA repair capacity can be used as a marker to evaluate and differentiate individual radiation sensitivity. More specifically, this work is based on the concept that the combined time-dose dependence of radiation-induced foci (RIF) of p53-binding protein 1 (53BP1) following low-LET exposure contains sufficient information to infer sensitivity to any other LET. Our hypothesis was tested in 15 different mouse strains as well as in primary human immune cells. We first approached individual ionizing radiation sensitivity in a mouse model by culturing primary skin fibroblasts extracted from 76 mice of 15 different genetic backgrounds and exposing them to HZE particles and X-rays. This work is one of the most extensive studies on the kinetics and possible genetic underpinnings of radiation-induced DNA damage and repair. Our results is in agreement with a DNA repair model we previously postulated, where nearby DNA double strand breaks (DSB) in the nucleus are brought together for more efficient repair, leading to RIF clustering. Such mechanism was evidenced by a specific dose and LET dependence of RIF numbers. Briefly, RIF quantification after low-LET X-ray exposure showed an asymptotic saturation for doses between 1 Gy and 4 Gy 4 hours post-irradiation across all 15 strains. The clustering of DSB across all strains also led to more RIF/Gy for lower LET (X-ray and 350 MeV/n Ar) than for higher LET (600 MeV/n Fe) 4 hours post-exposure. Considering the fact that the number of DSB/Gy should be independent of LET, our data suggest there are more DSB in individual RIF as the LET increases. RIF numbers for 24 and 48 hours post-exposure led to the inverse trend, with more remaining RIF/Gy for higher LET (by 600 MeV/n Fe). This result suggests cells have more difficulty resolving RIF from higher LET as they the number DSB/RIF increases. Note that for most conditions, the variance of RIF/Gy was small within individual animals of the same strain and large between strains, suggesting a strong genetics component. Furthermore, we present our preliminary data from an ongoing study on human genetic associations with IR sensitivity. To address the human variability in responses to HZE particle irradiation in a maximally comprehensive manner, we are in the process of collecting and isolating primary blood mononuclear cells from 768 healthy subjects of European descent, 18-75 years of age, 50/50 male/female distribution. We have analyzed 53BP1+ RIF formation as well as oxidative stress and cell death in primary cells from 192 subjects in response to the same HZE particles as used in mice: 600 MeV/n Fe, 350 MeV/n Ar and 350 MeV/n Si, 1.1 and 3 particles/100m2, 4 and 24 hours after irradiation. We will next complete the quantification of HZE particle-induced DNA and cellular damage in the remaining subjects and compare it to their responses to low-LET irradiation. Finally, we will perform GWAS analysis to identify human genomic associations with IR sensitivity and potential targets for biomarker development.

High-LET radiation↗
NASA NTRS2002

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages. (c) 2002 Elsevier Science (USA).

Non-NASA Center↗
DOE OSTI2022

BayFlux v1.0

This is an open source software library for performing Bayesian genome scale 13C Metabolic Flux Analysis (Bayesian 13C MFA). This is a new type of metabolic modeling that quantifies flux in a probabilistic manner at the genome scale.

Garcia Martin, Hector↗
DOE OSTI2022

A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations

The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines ( Triticum aestivum , CDC Stanley x CDC Landmark), wheat-barley ( T . aestivum x Hordeum vulgare ) and wheat-wheatgrass ( Triticum durum x Thinopyrum intermedium ) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

60 APPLIED LIFE SCIENCES↗