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

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At least 991 records · Page 55
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

WA-TmG.2.0

Soil samples were collected in triplicate in the Fall of 2017 across three grassland locations having differences in historical annual precipitation. For each of the three field site soil collections, one deeply sequenced composite metagenome (> 1 terabase) was obtained and individually sequenced in replicate to provide three additional terabase metagenomes for replicate statistical comparison in addition to screening for viral soil contigs for assessing the impact of historical annual precipitation. This data package provides new knowledge of viral types, abundances, diversity, and auxiliary metabolic genes (AMGs) in grassland soils for predicting viral response to changes in their environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

SPRUCE Deep Peat Microbial Diversity, CO2 and CH4 Production in Response to Nutrient, Temperature, and pH Treatments during Incubation Studies

This dataset contains the results of a microcosm incubation study on deep peat collected from the Spruce and Peatland Responses Under Changing Environments (SPRUCE) experimental site within the Marcell Experimental Forest in northern Minnesota, USA in September 2014. Microcosms were monitored for carbon dioxide (CO2) and methane (CH4) production, and microbial community dynamics were assessed using quantitative polymerase chain reaction (qPCR) and amplicon sequencing. The experiment was designed with a full factorial design with elevated temperature, nitrogen (N), (P), and pH treatments was used with samples from each transect serving replicates. In all, 96 microcosms were constructed to account for the 16 treatment combinations (Nitrogen x Phosphorous x pH x temperature), 2 time points, and 3 replicates. Temperature treatments were 6 degrees C, to mimic the SPRUCE ambient plot temperatures, and 15 degrees C to mimic the SPRUCE +9 degrees C treatment. These data show that one year of deep peat warming, microbial community structure and abundance of bacterial, archaeal, fungal, and methanogenic populations showed strong vertical stratification across the peat depth profile yet no clear response to the temperature treatments (Kluber et al, 2020). In an effort to identify factors that may be limiting decomposition and microbial community change in deep peat, we conducted an anerobic microcosm incubation of deep peat (150-200 cm depth). This dataset contains one data file in comma-separate values (*.csv) format. Additional metadata are provided: a data dictionary and a file-level metadata file in comma-separate values (.csv) format and a user guide in PDF (*.pdf) format.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.

09 BIOMASS FUELS↗
DOE OSTI2015

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Francisella novicida Two-Component System Response Regulator BfpR Modulates iglC Gene Expression, Antimicrobial Peptide Resistance, and Biofilm Production

Response regulators are a critical part of the two-component system of gene expression regulation in bacteria, transferring a signal from a sensor kinase into DNA binding activity resulting in alteration of gene expression. In this study, we investigated a previously uncharacterized response regulator in Francisella novicida, FTN_1452 that we have named BfpR (Biofilm-regulating Francisella protein Regulator, FTN_1452). In contrast to another Francisella response regulator, QseB/PmrA, BfpR appears to be a negative regulator of biofilm production, and also a positive regulator of antimicrobial peptide resistance in this bacterium. The protein was crystallized and X-ray crystallography studies produced a 1.8 Å structure of the BfpR N-terminal receiver domain revealing interesting insight into its potential interaction with the sensor kinase. Structural analysis of BfpR places it in the OmpR/PhoP family of bacterial response regulators along with WalR and ResD. Proteomic and transcriptomic analyses suggest that BfpR overexpression affects expression of the critical Francisella virulence factor iglC, as well as other proteins in the bacterium. We demonstrate that mutation of bfpR is associated with an antimicrobial peptide resistance phenotype, a phenotype also associated with other response regulators, for the human cathelicidin peptide LL-37 and a sheep antimicrobial peptide SMAP-29. F. novicida with mutated bfpR replicated better than WT in intracellular infection assays in human-derived macrophages suggesting that the down-regulation of iglC expression in bfpR mutant may enable this intracellular replication to occur. Response regulators have been shown to play important roles in the regulation of bacterial biofilm production. We demonstrate that F. novicida biofilm formation was highly increased in the bfpR mutant, corresponding to altered glycogen synthesis. Waxworm infection experiments suggest a role of BfpR as a negative modulator of iglC expression with de-repression by Mg 2+ . In this study, we find that the response regulator BfpR may be a negative regulator of biofilm formation, and a positive regulator of antimicrobial peptide resistance in F. novicida.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Meta gene set enrichment analyses link miR-137-regulated pathways with schizophrenia risk

Background: A single nucleotide polymorphism (SNP) within MIR137, the host gene for miR-137, has been identified repeatedly as a risk factor for schizophrenia. Previous genetic pathway analyses suggest that potential targets of this microRNA (miRNA) are also highly enriched in schizophrenia-relevant biological pathways, including those involved in nervous system development and function. Methods: In this study, we evaluated the schizophrenia risk of miR-137 target genes within these pathways. Gene set enrichment analysis of pathway-specific miR-137 targets was performed using the stage 1 (21,856 subjects) schizophrenia genome wide association study data from the Psychiatric Genomics Consortium and a small independent replication cohort (244 subjects) from the Mind Clinical Imaging Consortium and Northwestern University. Results: Gene sets of potential miR-137 targets were enriched with variants associated with schizophrenia risk, including target sets involved in axonal guidance signaling, Ephrin receptor signaling, long-term potentiation, PKA signaling, and Sertoli cell junction signaling. The schizophrenia-risk association of SNPs in PKA signaling targets was replicated in the second independent cohort. Conclusions: These results suggest that these biological pathways may be involved in the mechanisms by which this MIR137 variant enhances schizophrenia risk. SNPs in targets and the miRNA host gene may collectively lead to dysregulation of target expression and aberrant functioning of such implicated pathways. Pathway-guided gene set enrichment analyses should be useful in evaluating the impact of other miRNAs and target genes in different diseases.

Genetics & Heredity↗
DOE OSTI2021

Deep Sequencing of MHC-Adapted Viral Lines Reveals Complex Recombinational Exchanges With Endogenous Retroviruses Leading to High-Frequency Variants

Experimental evolution (serial passage) of Friend virus complex (FVC) in mice demonstrates phenotypic adaptation to specific host major histocompatibility complex (MHC) genotypes. These evolved viral lines show increased fitness and virulence in their host-genotype-of-passage, but display fitness and virulence tradeoffs when infecting unfamiliar host MHC genotypes. Here, we deep sequence these viral lines in an attempt to discover the genetic basis of FVC adaptation. The principal prediction for genotype-specific adaptation is that unique mutations would rise to high frequency in viral lines adapted to each host MHC genotype. This prediction was not supported by our sequencing data as most observed high-frequency variants were present in each of our independently evolved viral lines. However, using a multi-variate approach to measure divergence between viral populations, we show that populations of replicate evolved viral lines from the same MHC congenic mouse strain were more similar to one another than to lines derived from different MHC congenic mouse strains, suggesting that MHC genotype does predictably act on viral evolution in our model. Sequence analysis also revealed rampant recombination with endogenous murine leukemia virus sequences (EnMuLVs) that are encoded within the BALB/c mouse genome. The highest frequency variants in all six lines contained a 12 bp insertion from a recombinant EnMuLV source, suggesting such recombinants were either being favored by selection or were contained in a recombinational hotspot. Interestingly, they did not reach fixation, as if they are low fitness. The amount of background mutations linked to FVC/EnMuLV variable sites indicated that FVC/EnMuLV recombinants had not reached mutation selection equilibrium and thus, that EnMuLV sequences are likely continuously introgressing into the replicating viral population. These discoveries raise the question: is the expression of EnMuLV sequences in mouse splenocytes that permit recombination with exogenous FVC a pathogen or host adaptation?

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Unveiling a Drift Resistant Cryptotope within Marburgvirus Nucleoprotein Recognized by Llama Single-Domain Antibodies

Marburg virus (MARV) is a highly lethal hemorrhagic fever virus that is increasingly re-emerging in Africa, has been imported to both Europe and the US, and is also a Tier 1 bioterror threat. As a negative sense RNA virus, MARV has error prone replication which can yield progeny capable of evading countermeasures. To evaluate this vulnerability, we sought to determine the epitopes of 4 llama single-domain antibodies (sdAbs or VHH) specific for nucleoprotein (NP), each capable of forming MARV monoclonal affinity reagent sandwich assays. Here, we show that all sdAb bound the C-terminal region of NP, which was produced recombinantly to derive X-ray crystal structures of the three best performing antibody-antigen complexes. The common epitope is a trio of alpha helices that form a novel asymmetric basin-like depression that accommodates each sdAb paratope via substantial complementarity-determining region (CDR) restructuring. Shared core contacts were complemented by unique accessory contacts on the sides and overlooks of the basin yielding very different approach routes for each sdAb to bind the antigen. The C-terminal region of MARV NP was unable to be crystallized alone and required engagement with sdAb to form crystals suggesting the antibodies acted as crystallization chaperones. While gross structural homology is apparent between the two most conserved helices of MARV and Ebolavirus, the positions and morphologies of the resulting basins were markedly different. Naturally occurring amino acid variations occurring in bat and human Marburgvirus strains all mapped to surfaces distant from the predicted sdAb contacts suggesting a vital role for the NP interface in virus replication. As an essential internal structural component potentially interfacing with a partner protein it is likely the C-terminal epitope remains hidden or “cryptic” until virion disruption occurs. Conservation of this epitope over 50 years of Marburgvirus evolution should make these sdAb useful foundations for diagnostics and therapeutics resistant to drift.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus

Dendritic cells (DCs) are professional antigen-presenting cells, which are key components of the immune system and involved in early immune responses. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with avian influenza virus (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death. For this study, chicken bone marrow-derived DC (ckBM-DC)s were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphologies, and evaluate virus replication. A strong proinflammatory response was observed at 8 hours post infection, via upregulation of chicken interleukin-1β and stimulation of the interferon response pathway. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The increase in activation of infected DCs may be indicative of a dysregulated immune response typically seen with HPAI infections.

Immunology↗
DOE OSTI2016

Presence of Viral RNA and Proteins in Exosomes from Cellular Clones Resistant to Rift Valley Fever Virus Infection

Rift Valley Fever Virus (RVFV) is a RNA virus that belongs to the genus Phlebovirus, family Bunyaviridae. It infects humans and livestock and causes Rift Valley fever. RVFV is considered an agricultural pathogen by the USDA, as it can cause up to 100% abortion in cattle and extensive death of newborns. In addition, it is designated as Category A pathogen by the CDC and the NIAID. In some human cases of RVFV infection, the virus causes fever, ocular damage, liver damage, hemorrhagic fever, and death. There are currently limited options for vaccine candidates, which include the MP-12 and clone 13 versions of RVFV. Viral infections often deregulate multiple cellular pathways that contribute to replication and host pathology. We have previously shown that latent human immunodeficiency virus-1 (HIV-1) and human T-cell lymphotropic virus-1 (HTLV-1) infected cells secrete exosomes that contain short viral RNAs, limited number of genomic RNAs, and viral proteins. These exosomes largely target neighboring cells and activate the NF-κB pathway, leading to cell proliferation, and overall better viral replication. In this manuscript, we studied the effects of exosome formation from RVFV infected cells and their function on recipient cells. We initially infected cells, isolated resistant clones, and further purified using dilution cloning. We then characterized these cells as resistant to new RVFV infection, but sensitive to other viral infections, including Venezuelan Equine Encephalitis Virus (VEEV). These clones contained normal markers (i.e., CD63) for exosomes and were able to activate the TLR pathway in recipient reporter cells. Interestingly, the exosome rich preparations, much like their host cell, contained viral RNA (L, M, and S genome). The RNAs were detected using qRT-PCR in both parental and exosomal preparations as well as in CD63 immunoprecipitates. Viral proteins such as N and a modified form of NSs were present in some of these exosomes. Finally, treatment of recipient cells (T-cells and monocytic cells) showed drastic rate of apoptosis through PARP cleavage and caspase 3 activation from some but not all exosome enriched preparations. Collectively, these data suggest that exosomes from RVFV infected cells alter the dynamics of the immune cells and may contribute to pathology of the viral infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides , Ruminococcus , and Streptococcus . All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/ Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Physiology of Highly Radioresistant Escherichia coli After Experimental Evolution for 100 Cycles of Selection

Ionizing radiation (IR) is lethal to most organisms at high doses, damaging every cellular macromolecule via induction of reactive oxygen species (ROS). Utilizing experimental evolution and continuing previous work, we have generated the most IR-resistant Escherichia coli populations developed to date. After 100 cycles of selection, the dose required to kill 99% the four replicate populations (IR9-100, IR10-100, IR11-100, and IR12-100) has increased from 750 Gy to approximately 3,000 Gy. Fitness trade-offs, specialization, and clonal interference are evident. Long-lived competing sub-populations are present in three of the four lineages. In IR9, one lineage accumulates the heme precursor, porphyrin, leading to generation of yellow-brown colonies. Major genomic alterations are present. IR9 and IR10 exhibit major deletions and/or duplications proximal to the chromosome replication terminus. Contributions to IR resistance have expanded beyond the alterations in DNA repair systems documented previously. Variants of proteins involved in ATP synthesis (AtpA), iron-sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA) each contribute to IR resistance in IR9-100. Major genomic and physiological changes are emerging. An isolate from IR10 exhibits protein protection from ROS similar to the extremely radiation resistant bacterium Deinococcus radiodurans, without evident changes in cellular metal homeostasis. Selection is continuing with no limit to IR resistance in evidence as our E. coli populations approach levels of IR resistance typical of D. radiodurans.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2022

Cryptic Methane-Cycling by Methanogens During Multi-Year Incubation of Estuarine Sediment

As marine sediments are buried, microbial communities transition from sulfate-reduction to methane-production after sulfate is depleted. When this biogenic methane diffuses into the overlying sulfate-rich sediments, it forms a sulfate-methane transition zone (SMTZ) because sulfate reducers deplete hydrogen concentrations and make hydrogenotrophic methanogenesis exergonic in the reverse direction, a process called the anaerobic oxidation of methane (AOM). Microbial participation in these processes is often inferred from geochemistry, genes, and gene expression changes with sediment depth, using sedimentation rates to convert depth to time. Less is known about how natural sediments transition through these geochemical states transition in real-time. We examined 16S rRNA gene amplicon libraries and metatranscriptomes in microcosms of anoxic sediment from the White Oak River estuary, NC, with three destructively sampled replicates with methane added (586-day incubations) and three re-sampled un-amended replicates (895-day incubations). Sulfate dropped to a low value (∼0.3 mM) on similar days for both experiments (312 and 320 days, respectively), followed by a peak in hydrogen, intermittent increases in methane-cycling archaea starting on days 375 and 362 (mostly Methanolinea spp. and Methanosaeta spp., and Methanococcoides sp. ANME-3), and a methane peak 1 month later. However, methane δ 13 C values only show net methanogenesis 6 months after methane-cycling archaea increase and 4 months after the methane peak, when sulfate is consistently below 0.1 mM and hydrogen increases to a stable 0.61 ± 0.13 nM (days 553–586, n = 9). Sulfate-reducing bacteria (mostly Desulfatiglans spp. and Desulfosarcina sp. SEEP-SRB1) increase in relative abundance only during this period of net methane production, suggesting syntrophy with methanogens in the absence of sulfate. The transition from sulfate reduction to methane production in marine sediments occurs through a prolonged period of methane-cycling by methanogens at low sulfate concentrations, and steady growth of sulfate reducers along with methanogens after sulfate is depleted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Inhibition of tick-borne encephalitis virus in cell cultures by ribavirin

Tick-borne encephalitis virus (TBEV) belonging to arboviruses is a major member of zoonotic pathogens. TBEV infection causes severe human encephalitis without specific antiviral drugs. Due to its use of antiviral drug against a wide range of viruses, we investigated antiviral effect of ribavirin against TBEV in susceptible human cell lines A549 and SH-SY5Y. Ribavirin displayed minor cytotoxicity on multiple cell lines. Ribavirin obviously impaired TBEV replication and protected the infected cells from cytopathic effect. Importantly, ribavirin markedly inhibited TBEV propagation, as evidenced by impairment of TBEV production and viral RNA replication. Treatment with ribavirin (co-treatment and post-treatment) led to a dose-dependent reduction in TBEV titers as well as the viral RNA levels. Antiviral protein myxovirus resistance A mRNA expression was significantly up-regulated and signal transducer and activator of transcription 3 was activated in TBEV-infected A549 cells upon the ribavirin treatment. Induction of inflammatory cytokine tumor necrosis factor alpha by TBEV was decreased in A549 cells with the treatment of ribavirin, whereas interleukin 1 beta release appeared to be unaffected. These results suggest that ribavirin might represent a promising safe and effective antiviral drug against TBEV.

Tang, Wan-Da↗
DOE OSTI2024

Evaluation of Apparent Metabolizable Energy and Apparent Ileal Amino Acid Digestibility of Spirulina (Arthrospira platensis) in Broiler Chickens and Laying Hens

Our study objective was to determine the apparent metabolizable energy (AME) and apparent ileal amino acid digestibility (AIAAD) of spirulina fed to broiler chickens and laying hens using the difference method. In both experiments, birds were either fed corn-soybean meal basal diets, containing no spirulina and formulated to provide the nutrient requirements of either broilers or layers, or fed test diets consisting of 25% spirulina and 75% of the appropriate basal diet. Titanium dioxide was added to all diets as an indigestible marker. The diets were fed to 10 replicate cages/treatment of broilers and 15 replicate cages/treatment of laying hens. The AME of spirulina for broilers was significantly lower (p < 0.05) (2368 ± 104 kcal/kg, as fed) than for laying hens (3144 ± 173 kcal/kg, as fed), suggesting bird type and age may influence energy utilization. The AIAAD of spirulina fed to broiler chickens did not differ from that of laying hens (p > 0.05), except for valine, alanine, and glycine, which were all significantly higher in laying hens (p < 0.05). Overall, the AIAAD for nonessential amino acids averaged 81.1%, with no significant difference between essential and nonessential amino acids. Differences in spirulina nutrient content cited in the literature support further research to determine the optimal inclusion of this alternative ingredient in broiler and layer diets.

Agriculture↗
DOE OSTI2022

On the Rapid Calculation of Binding Affinities for Antigen and Antibody Design and Affinity Maturation Simulations

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Improving Experimental Design through Uncertainty Analysis

In this paper, the development of a fission-gas collecting and physical-analysis-enabling instrument was proposed for small-volume determination. Analysis specifications require a design capable of accurately and repeatably determining volumes in the range of 0.07–2.5 mL. This system relies on a series of gas expansions originating from a cylinder with known internal volume. The combined gas law is used to derive the unknown volumes from these expansions. Initial system designs included one of two known volumes, 11.85 ± 0.34 mL and 5.807 ± 0.078 mL, with a manifold volume of 32 mL. Results obtained from modeling this system’s operation showed that 0.07 mL can be determined with a relative expanded uncertainty greater than 300% (k = 2) for a single replicate, which was unacceptable for the proposed experimental design. Initial modeling showed that the volume connecting the known volume and rodlet, i.e., the manifold volume, and the sensitivity of the pressure sensor were key contributors to the expanded uncertainty of the measured rodlet volume. The system’s design limited the available options for pressure sensors, so emphasis was placed on the design of the manifold volume. The final system design reduced the manifold volume to 17 mL. These changes in design, combined with replicate analysis, were able to reduce the relative expanded uncertainty by ±12% (k = 2) for the 0.07 mL volume.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗