SEARCH · Search NASA

Results for “sequence analysis”

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

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 865 records · Page 48

All sources NASA NTRS DOE OSTI

DOE OSTI2019

Construction and comparison of three reference-quality genome assemblies for soybean

Here, we report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Association of Diet and Antimicrobial Resistance in Healthy U.S. Adults

Antimicrobial resistance (AMR) represents a significant source of morbidity and mortality worldwide, with expectations that AMR-associated consequences will continue to worsen throughout the coming decades. Since resistance to antibiotics is encoded in the microbiome, interventions aimed at altering the taxonomic composition of the gut might allow us to prophylactically engineer microbiomes that harbor fewer antibiotic resistant genes (ARGs). Diet is one method of intervention, and yet little is known about the association between diet and antimicrobial resistance. To address this knowledge gap, we examined diet using the food frequency questionnaire (FFQ; habitual diet) and 24-h dietary recalls (Automated Self-Administered 24-h [ASA24 ® ] tool) coupled with an analysis of the microbiome using shotgun metagenome sequencing in 290 healthy adult participants of the United States Department of Agriculture (USDA) Nutritional Phenotyping Study. We found that aminoglycosides were the most abundant and prevalent mechanism of AMR in these healthy adults and that aminoglycoside-O-phosphotransferases (aph3-dprime) correlated negatively with total calories and soluble fiber intake. Individuals in the lowest quartile of ARGs (low-ARG) consumed significantly more fiber in their diets than medium- and high-ARG individuals, which was concomitant with increased abundances of obligate anaerobes, especially from the family Clostridiaceae, in their gut microbiota. Finally, we applied machine learning to examine 387 dietary, physiological, and lifestyle features for associations with antimicrobial resistance, finding that increased phylogenetic diversity of diet was associated with low-ARG individuals. These data suggest diet may be a potential method for reducing the burden of AMR.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Reevaluating the Salty Divide: Phylogenetic Specificity of Transitions between Marine and Freshwater Systems

Marine and freshwater microbial communities are phylogenetically distinct, and transitions between habitat types are thought to be infrequent. We compared the phylogenetic diversity of marine and freshwater microorganisms and identified specific lineages exhibiting notably high or low similarity between marine and freshwater ecosystems using a meta-analysis of 16S rRNA gene tag-sequencing data sets. As expected, marine and freshwater microbial communities differed in the relative abundance of major phyla and contained habitat-specific lineages. At the same time, and contrary to expectations, many shared taxa were observed in both habitats. Based on several metrics, we found that Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Betaproteobacteria contained the highest number of closely related marine and freshwater sequences, suggesting comparatively recent habitat transitions in these groups. Using the abundant alphaproteobacterial group SAR11 as an example, we found evidence that new lineages, beyond the recognized LD12 clade, are detected in freshwater at low but reproducible abundances; this evidence extends beyond the 16S rRNA locus to core genes throughout the genome. Our results suggest that shared taxa are numerous, but tend to occur sporadically and at low relative abundance in one habitat type, leading to an underestimation of transition frequency between marine and freshwater habitats. Rare taxa with abundances near or below detection, including lineages that appear to have crossed the salty divide relatively recently, may possess adaptations enabling them to exploit opportunities for niche expansion when environments are disturbed or conditions change.

16S rRNA↗

DOE OSTI2015

Edge Bioinformatics

Edge Bioinformatics is a developmental bioinformatics and data management platform which seeks to supply laboratories with bioinformatics pipelines for analyzing data associated with common samples case goals. Edge Bioinformatics enables sequencing as a solution and forward-deployed situations where human-resources, space, bandwidth, and time are limited. The Edge bioinformatics pipeline was designed based on following USE CASES and specific to illumina sequencing reads. 1. Assay performance adjudication (PCR): Analysis of an existing PCR assay in a genomic context, and automated design of a new assay to resolve conflicting results; 2. Clinical presentation with extreme symptoms: Characterization of a known pathogen or co-infection with a. Novel emerging disease outbreak or b. Environmental surveillance

Lo, Chien-Chi↗

DOE OSTI2024

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations inTP53,PLXNB3, Zinc finger genes, and fewer mutations inSDC2,CDH1,PIK3CA,SVIL, andPTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, includingKIAA1522,DCK,FOXO3,MYO9B,ARID1A,EPRS,ZC3HAV1, andRBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Oncology↗

DOE OSTI2021

Mongooses ( Urva auropunctata ) as reservoir hosts of Leptospira species in the United States Virgin Islands, 2019–2020

During 2019–2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory methods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppkgene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens examined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Strategy for Safety-Related Digital I&C Qualification

Digital I&C qualification continues to be a major impediment to addressing the reliability and obsolescence issues of the legacy analog I&C systems of the operating LWR fleet. In particular, the issue of digital common cause failure (CCF) has been difficult to address and has been the reason that some nuclear plant licensees have deferred upgrades of these critical plant systems and have rather opted to maintain them with considerable engineering and maintenance efforts. This is getting more and more difficult in the face of declining analog technology suppliers and dwindling supplies of spare parts and support for many critical components. To remain a viable energy source for the nation, the nuclear fleet must reduce its operating costs to a competitive level. Digital technology is the foremost means of implementing performance improvements and cost reductions for this purpose. This has been demonstrated over and over again in other industry sectors, as well as in conventional generation facilities. However, due to the special nature of nuclear energy, these digital I&C qualification barriers remain and must be resolved in order to modernize these plants and transform the business model to one that is competitive. The purpose of this report is to present an assessment of digital I&C qualification issues and address gaps in qualification methods and processes that would potentially benefit from Department of Energy (DOE)-sponsored research and development. Two new qualification methods are described that are recommended for further Department of Energy-sponsored research and development. They are 1) Testability – the exhaustive (100%) testing of certain digital devices addressing all combinations of inputs and internal states, and 2) Elimination of CCF triggers – ensuring that any latent digital defects are not concurrently triggered in redundant and back-up safety systems. In addition this report presents a strategy for full nuclear plant modernization based on four interrelated elements that together address the remaining barriers to prudent implementation. These four elements are 1) End-State Architecture, 2) Cost-Benefit Analysis, 3) Regulatory Approval, and 4) Implementation Sequence Plan.

42 ENGINEERING↗

DOE OSTI2026

Microreactor Core Transportation Cask Model Description for Criticality Safety Validation Basis Assessment (Rev. 1)

Criticality safety analyses are completed on transportation casks used for microreactor whole core shipment to provide examples of models and analyses to industry, regulators, and nuclear community at large to be used in verification and validation analyses of similar applications. The microreactors considered are based on a Gas-Cooled Microreactor (GCMR) and a Heat-Pipe Microreactor (HPMR), both utilize HALEU fuel in the form of TRISO particles and various other design options considered in industry microreactor designs. Variant design options of GCMR and HPMR were also investigated to provide a wider application range for each technology. Criticality safety analyses for the GCMR and HPMR packages were performed using the CSAS6 sequence of SCALE 6.3.2 with the ENDF/B-VII.1-based continuous energy neutron libraries. Different scenarios were investigated, including normal operation and water flooded conditions to represent nominal and hypothetical accident scenarios. Sensitivity and similarity analyses are also performed using the TSUNAMI sequence of SCALE 6.3.2, and the similarity analysis uses all the experiments from the ICSBEP Handbook with Intermediate and mixed Enriched Uranium (IEU) and Low Enriched Uranium (LEU) systems, together with additional experiments that are sponsored by the DNCSH program. Many experiments were found marginally similar to the GCMR and HPMR models, with similarity index (ck) values greater than 0.8 but less than 0.9. Among all the experiments analyzed, no case has a ck value greater than 0.9, indicating that additional critical experiments might be needed to further validate the criticality safety models for microreactors transport packages.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2026

Microreactor Core Transportation Cask Model Description for Criticality Safety Validation Basis Assessment (Rev. 2)

Criticality safety analyses are completed on transportation casks used for microreactor whole core shipment to provide examples of models and analyses to industry, regulators, and nuclear community at large to be used in verification and validation analyses of similar applications. The microreactors considered are based on a Gas-Cooled Microreactor (GCMR) and a Heat-Pipe Microreactor (HPMR), both utilize HALEU fuel in the form of TRISO particles and various other design options considered in industry microreactor designs. Variant design options of GCMR and HPMR were also investigated to provide a wider application range for each technology. Criticality safety analyses for the GCMR and HPMR packages were performed using the CSAS6 sequence of SCALE 6.3.2 with the ENDF/B-VII.1-based continuous energy neutron libraries. Different scenarios were investigated, including normal operation and water flooded conditions to represent nominal and hypothetical accident scenarios. Sensitivity and similarity analyses are also performed using the TSUNAMI sequence of SCALE 6.3.2, and the similarity analysis uses all the experiments from the ICSBEP Handbook with High Enriched Uranium (HEU), Intermediate and mixed Enriched Uranium (IEU) and Low Enriched Uranium (LEU) systems. Many ICSBEP experiments were found marginally similar, with similarity index (ck) values greater than 0.8, to the GCMR and HPMR cask models, especially in flooded conditions that may be more constraining due to reduced reactivity margins. Some experiments using TRISO fuel particles and graphite moderator, including the recent THETA and Deimos experiments from LANL, together with some IRPhEP experiments, were also used for the similarity analyses. These TRISO-fueled and graphite moderated experiments behaved similarly to the GCMR models, with maximum ck value greater than 0.9, but they are less similar to the HPMR models. This observation indicates that additional critical experiments might be needed to further validate the criticality safety models, especially for heat-pipe based microreactor transport packages and for non-flooded transportation configurations.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2022

Astronauts Plasma-Derived Exosomes Induced Aberrant EZH2-Mediated H3K27me3 Epigenetic Regulation of the Vitamin D Receptor

There are unique stressors in the spaceflight environment. Exposure to such stressors may be associated with adverse effects on astronauts' health, including increased cancer and cardiovascular disease risks. Small extracellular vesicles (sEVs, i.e., exosomes) play a vital role in intercellular communication and regulate various biological processes contributing to their role in disease pathogenesis. To assess whether spaceflight alters sEVs transcriptome profile, sEVs were isolated from the blood plasma of 3 astronauts at two different time points: 10 days before launch (L-10) and 3 days after return (R+3) from the Shuttle mission. AC16 cells (human cardiomyocyte cell line) were treated with L-10 and R+3 astronauts-derived exosomes for 24 h. Total RNA was isolated and analyzed for gene expression profiling using Affymetrix microarrays. Enrichment analysis was performed using Enrichr. Furthermore, transcription factor (TF) enrichment analysis using the ENCODE/ChEA Consensus TF database identified gene sets related to the polycomb repressive complex 2 (PRC2) and Vitamin D receptor (VDR) in AC16 cells treated with R+3 compared to cells treated with L-10 astronauts-derived exosomes. Further analysis of the histone modifications using datasets from the Roadmap Epigenomics Project confirmed enrichment in gene sets related to the H3K27me3 repressive mark. Interestingly, analysis of previously published H3K27me3–chromatin immunoprecipitation sequencing (ChIP-Seq) ENCODE datasets showed enrichment of H3K27me3 in the VDR promoter. Collectively, our results suggest that astronaut-derived sEVs may epigenetically repress the expression of the VDR in human adult cardiomyocytes by promoting the activation of the PRC2 complex and H3K27me3 levels.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Airborne Bacteria in Earth's Lower Stratosphere Resemble Taxa Detected in the Troposphere: Results From a New NASA Aircraft Bioaerosol Collector (ABC)

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights—four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Some taxa were more abundant in the flight samples than the ground samples, including OTUs from families Lachnospiraceae , Ruminococcaceae and Erysipelotrichaceae as well as the following genera: Clostridium, Mogibacterium, Corynebacterium, Bacteroides, Prevotella, Pseudomonas, and Parabacteroides . Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Furthermore considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Adaptive Evolution of Sphingobium hydrophobicum C1 T in Electronic Waste Contaminated River Sediment

Electronic waste (e-waste) has caused a severe worldwide pollution problem. Despite increasing isolation of degradative microorganisms from e-waste contaminated environments, the mechanisms underlying their adaptive evolution in such habitats remain unclear. Sphingomonads generally have xenobiotic-degrading ability and may play important roles in bioremediation. Sphingobium hydrophobicum C1 T , characterized with superior cell surface hydrophobicity, was recently isolated from e-waste contaminated river sediment. To dissect the mechanisms driving its adaptive evolution, we evaluated its stress resistance, sequenced its genome and performed comparative genomic analysis with 19 other Sphingobium strains. Strain C1 T can feed on several kinds of e-waste-derived xenobiotics, exhibits a great resistance to heavy metals and possesses a high colonization ability. It harbors abundant genes involved in environmental adaptation, some of which are intrinsic prior to experiencing e-waste contamination. The extensive genomic variations between strain C1 T and other Sphingobium strains, numerous C1 T -unique genes, massive mobile elements and frequent genome rearrangements reflect a high genome plasticity. Positive selection, gene duplication, and especially horizontal gene transfer drive the adaptive evolution of strain C1 T . Moreover, presence of type IV secretion systems may allow strain C1 T to be a source of beneficial genes for surrounding microorganisms. This study provides new insights into the adaptive evolution of sphingomonads, and potentially guides bioremediation strategies.

comparative genomics↗

DOE OSTI2017

VLT/Magellan spectroscopy of 29 strong lensing selected galaxy clusters

We present an extensive spectroscopic follow-up campaign of 29 strong lensing (SL) selected galaxy clusters discovered primarily in the Second Red-Sequence Cluster Survey (RCS-2). Our spectroscopic analysis yields redshifts for 52 gravitational arcs present in the core of our galaxy clusters, which correspond to 35 distinct background sources that are clearly distorted by the gravitational potential of these clusters. These lensed galaxies span a wide redshift range of 0.8 ≤ z ≤ 2.9, with a median redshift of z{sub s} = 1.8 ± 0.1. We also measure reliable redshifts for 1004 cluster members, allowing us to obtain robust velocity dispersion measurements for 23 of these clusters, which we then use to determine their dynamical masses by using a simulation-based σ{sub DM} − M{sub 200} scaling relation. The redshift and mass ranges covered by our SL sample are 0.22 ≤ z ≤ 1.01 and 5×10{sup 13}⩽M{sub 200}/h{sub 70}{sup −1} M{sub ⊙}⩽1.9×10{sup 15}, respectively. We analyze and quantify some possible effects that might bias our mass estimates, such as the presence of substructure, the region where cluster members are selected for spectroscopic follow-up, the final number of confirmed members, and line-of-sight effects. We find that 10 clusters of our sample with N{sub mem} ≳ 20 show signs of dynamical substructure. However, the velocity data of only one system is inconsistent with a uni-modal distribution. We therefore assume that the substructures are only marginal and not of comparable size to the clusters themselves. Consequently, our velocity dispersion and mass estimates can be used as priors for SL mass reconstruction studies and also represent an important step toward a better understanding of the properties of the SL galaxy cluster population.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2015

Edge Bioinformatics

Lo, Chien-Chi↗

DOE OSTI2025

Exploring the Potential of ASHRAE Guideline 36 in District Cooling Systems: Preprint

ASHRAE Guideline 36-2021 (G36) provides high-performance control sequences for all-air and hydronic HVAC systems, and it has proven effective at reducing energy use for single building all-air equipment. The most recent version of G36 included control sequences for water-cooled chiller plants, which can be used in district cooling systems (DCSs). Despite not being a traditional application of G36 controls, DCSs could benefit from energy savings that G36 control sequences have demonstrated in single building systems. We therefore investigate the possibility to extend G36 chiller plant control from a single building to district cooling using a real-world district cooling system in Boulder, Colorado as a case study. Four timeframes representing low to peak loads are simulated and analyzed to compare energy use of the district cooling system for three control sequences. Results show that although G36 chiller plant sequences can be applied to DCSs, further analysis and potentially control optimization is needed to ensure energy savings compared to default controls.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

DOE OSTI2018

Distorted antibody repertoire developed in the absence of pre-B cell receptor formation

Highlights: • Leaky B cells in SLC-deficient mice circumvent the pre-BCR checkpoint. • Antibody repertoires differing in VDJ-usage exist in leaky B cells in λ5{sup −}/{sup −} mice. • Leaky B cells do not originate from B1 or particular B2 cells. • The CDR-H3 middle loop in λ5{sup −}/{sup −} bone marrow B cells is enriched in arginines. The pre-B cell receptor (pre-BCR), consisting of the μ heavy chain (μHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5{sup −/−}) mice using next-generation sequencing. In λ5{sup −/−} mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of μ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5{sup −/−} mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5{sup −/−} mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5{sup −/−} mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Single-Genome Sequencing of Hepatitis C Virus in Donor-Recipient Pairs Distinguishes Modes and Models of Virus Transmission and Early Diversification

Despite the recent development of highly effective anti-hepatitis C virus (HCV) drugs, the global burden of this pathogen remains immense. Control or eradication of HCV will likely require the broad application of antiviral drugs and development of an effective vaccine. A precise molecular identification of transmitted/founder (T/F) HCV genomes that lead to productive clinical infection could play a critical role in vaccine research, as it has for HIV-1. However, the replication schema of these two RNA viruses differ substantially, as do viral responses to innate and adaptive host defenses. These differences raise questions as to the certainty of T/F HCV genome inferences, particularly in cases where multiple closely related sequence lineages have been observed. To clarify these issues and distinguish between competing models of early HCV diversification, we examined seven cases of acute HCV infection in humans and chimpanzees, including three examples of virus transmission between linked donors and recipients. Using single-genome sequencing (SGS) of plasma vRNA, we found that inferred T/F sequences in recipients were identical to viral sequences in their respective donors. Early in infection, HCV genomes generally evolved according to a simple model of random evolution where the coalescent corresponded to the T/F sequence. Closely related sequence lineages could be explained by high multiplicity infection from a donor whose viral sequences had undergone a pretransmission bottleneck due to treatment, immune selection, or recent infection. These findings validate SGS, together with mathematical modeling and phylogenetic analysis, as a novel strategy to infer T/F HCV genome sequences.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Towards resolving protein structures at the atomic scale using atom probe tomography

In the field of Structural Biology, Atom Probe Tomography (APT) is in the nascent stages of development wherein coarse-grained visuals of proteins have been captured. The characterization of organic samples or biomolecules through the technique is currently limited to the detection of a few dominant signatures. The problem of indecipherable characterization can inherently be traced back to multiple forms of technique-specific responses to organic samples and consequent triggers leading to organic-sample and sample-medium interactions. While it is possible for captured manifestations of protein reconstructions to seemingly appear intact from a basic visual purview, the parameter-protein associative responses throughout the structure as a direct consequence of the inherent workings of the technique (until harmonized with organic sample complexity and behavior) and field evaporation-based factors make non-aberrative atomic associations infeasible. The work focused on identifying and theorizing the (above stated and other) fundamental mechanisms that stronghold the study of intricate atomic to higher order associations in proteins through APT. Attempts at structure elucidation of the cryogenic sample under study, through indirect associations (and methods) based on other imaging techniques, further revealed the distinct and highly distortive nature at the atomic scale deterring structural tunability and thus characterization of APT based cryogenic samples under analysis. As a direct counter to the atomic scale characterization problem, by taking the experiment-specific uncertainties, and probable APT-centric organic sample-based variabilities into account, a basic result is extracted and presented. Through mass-spectrometric and computational analysis, specific individual amino acids (Sulfur-containing protein-bound amino acids) in proteins and aspects of protein structure (probable backbone fragments, partial sequence - partial backbone portions) have been identified and characterized. Under analysis considerations, a few of the simplest known and easily inferable segments that favor structural deteriorations in the reconstructions are stated. Additionally, to overcome technique-specific deterrents to the characterization of biomolecules in cryogenic sample medium, the development of a protein-labeling strategy tailored to APT is suggested.

59 BASIC BIOLOGICAL SCIENCES↗