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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 973 records · Page 54
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria

A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Distinct Luminal-Type Mammary Carcinomas Arise from Orthotopic Trp53-Null Mammary Transplantation of Juvenile versus Adult Mice

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. Lastly, these data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Diel Transcriptional Oscillations of a Plastid Antiporter Reflect Increased Resilience of Thalassiosira pseudonana in Elevated CO 2

Acidification of the ocean due to high atmospheric CO 2 levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatom Thalassiosira pseudonana under elevated CO 2 . Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na + (K + )/H + antiporter that is significantly upregulated at >800 ppm CO 2 , with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO 2 conditions. In future acidified oceanic conditions, dramatic oscillation with >10-fold change between nighttime (high) and daytime (low) transcript abundances of the antiporter was associated with increased resilience of T. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO 2 conditions in marine environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Deletion of ApoM gene induces apoptosis in mouse kidney via mitochondrial and endoplasmic reticulum stress pathways

Highlights: • Deletion of ApoM gene caused abnormal changes in mitochondria in mouse kidney. • Deletion of ApoM gene caused endoplasmic reticulum stress in mouse kidney. • Effect of ApoM gene deletion on apoptosis was determined in vivo and in vitro. • Deletion of ApoM gene caused upregulation of renal apoptosis in mouse. Apolipoprotein M (ApoM) is involved in lipid metabolism, and especially is involved in reverse cholesterol transport. However, the relationship between ApoM and apoptosis has been rarely reported. This study aimed to investigate the effect of ApoM on apoptosis using an ApoM gene-deficient mice (ApoM{sup −/−}) model and a mouse mesangial cell model with suppressed ApoM gene expression. First, we observed by transmission electron microscopy that mitochondrial damage and endoplasmic reticulum stress were abnormally altered in the kidneys of ApoM{sup −/−} mice compared with wild-type mice, showing mitochondrial swelling, vacuolization, myeloid changes, and expansion of the rough endoplasmic reticulum. At the molecular level, the expression of pro-apoptotic related proteins such as AIF, Bax, chop, clever-caspase 3, clever-caspase 7, clever-caspase 9, and clever-caspase 12 increased, and the expression of anti-apoptotic protein Bcl-2 decreased. Secondly, by interfering with the expression of the ApoM gene in mouse mesangial cells, we found that, compared with the control group (NC-si), the cells of the experimental group (siApoM) showed decreased cell viability, nuclear chromatin condensation, nuclear lysis, and an increased proportion of early apoptotic cells. The results in cells at the molecular level were consistent with those at the tissue level. These data indicated that the deletion of the ApoM gene led to upregulation of apoptosis in mouse kidney tissues and mesangial cells through the mitochondrial and endoplasmic reticulum pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of CisPt-triggered DDR leads to cytoprotection. • Lovastatin attenuates CisPt-induced kidney DNA damage and apoptosis in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Transcript, protein and metabolite temporal dynamics in the CAM plant Agave

Already a proven mechanism for drought resilience, crassulacean acid metabolism (CAM) is a specialized type of photosynthesis that maximizes water-use efficiency (WUE) via an inverse (compared to C 3 and C 4 photosynthesis-performing species) day/night pattern of stomatal closure/opening to shift CO 2 uptake to the night, when evapotranspiration rates are low. A systems-level understanding of temporal molecular and metabolic controls is needed to define the cellular behavior that underpins CAM. Here, we report high-resolution temporal behaviors of transcript, protein and metabolite abundances across a CAM diel cycle and, where applicable, compare those observations to the well-established C 3 model plant, Arabidopsis thaliana. A mechanistic finding that emerged is that CAM operates with a diel redox poise that is shifted relative to that in Arabidopsis thaliana. Moreover, we identified widespread rescheduled expression of genes associated with signal transduction mechanisms that regulate stomatal opening/closing. Controlled production and degradation of transcripts and proteins represents a timing mechanism by which to regulate cellular function, yet how this molecular timekeeping regulates CAM physiology remains unclear. In this paper, we provide new insights into complex post-transcriptional and -translational hierarchies that govern CAM in Agave. These data sets together provide a resource to inform efforts to engineer more water-use efficient CAM pathway traits into economically valuable C 3 crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

The flagella of ‘ Candidatus Liberibacter asiaticus’ and its movement in planta

Summary Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘ Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin‐encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin‐encoding genes. Only flgJ Atu but not flgJ Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull‐down assays demonstrated that FlgJ Las interacts with FlgB but not with FliE. Ectopic expression of flaA Las in A. tumefaciens mutants restored the swimming motility of ∆ flaA mutant and ∆ flaAD mutant, but not that of the null mutant ∆ flaABCD . No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta . In addition, western blotting using flagellin‐specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta . The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

Andrade, Maxuel O.↗
DOE OSTI2016

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

The SlZRT1 Gene Encodes a Plasma Membrane-Located ZIP (Zrt-, Irt-Like Protein) Transporter in the Ectomycorrhizal Fungus Suillus luteus

Zinc (Zn) is an essential micronutrient but may become toxic when present in excess. In Zn-contaminated environments, trees can be protected from Zn toxicity by their root-associated micro-organisms, in particular ectomycorrhizal fungi. The mechanisms of cellular Zn homeostasis in ectomycorrhizal fungi and their contribution to the host tree’s Zn status are however not yet fully understood. The aim of this study was to identify and characterize transporters involved in Zn uptake in the ectomycorrhizal fungus Suillus luteus, a cosmopolitan pine mycobiont. Zn uptake in fungi is known to be predominantly governed by members of the ZIP (Zrt/IrtT-like protein) family of Zn transporters. Four ZIP transporter encoding genes were identified in the S. luteus genome. By in silico and phylogenetic analysis, one of these proteins, SlZRT1, was predicted to be a plasma membrane located Zn importer. Heterologous expression in yeast confirmed the predicted function and localization of the protein. A gene expression analysis via RT-qPCR was performed in S. luteus to establish whether SlZRT1 expression is affected by external Zn concentrations. SlZRT1 transcripts accumulated almost immediately, though transiently upon growth in the absence of Zn. Exposure to elevated concentrations of Zn resulted in a significant reduction of SlZRT1 transcripts within the first hour after initiation of the exposure. Altogether, the data support a role as cellular Zn importer for SlZRT1 and indicate a key role in cellular Zn uptake of S. luteus. Further research is needed to understand the eventual contribution of SlZRT1 to the Zn status of the host plant.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

A spontaneously arising mutation in connexin32 with repeated passage of FRTL-5 cells coincides with increased growth rate and reduced thyroxine release

In this study we examine changes in the cellular properties of FRTL-5 cells as a function of passage number, with particular emphasis on gap junction expression, karyotype, morphology, growth rate and thyroxine (T(4)) release. Early passage FRTL-5 follicular cells transfer dye through gap junctions from injected cell(s) to third-order neighboring cells and beyond within their respective follicles and have immuno-detectable connexin32 (Cx32) type gap junctional plaques in their lateral contacting plasma membranes. By contrast, FRTL-5 cells established as monolayers, or as follicles from cultures passed more than 15 times, did not transfer microinjected Lucifer Yellow dye to contiguous neighboring cells and did not express any immuno-detectable rat thyroid specific connexins (Cx43, Cx32 or Cx26). Western blots confirmed that total, membrane and cytosolic Cx32 protein was present only in early pass follicular cultures. To better understand the passage-dependent loss of Cx32 expression, RT-PCR primers were made to the most unique sequences of the rat Cx32 molecule, the cytoplasmic and carboxyl-terminal regions. These primers were used to screen FRTL-5 RNA from cultures of various passage numbers. The results revealed that later passage cultures had a single base deletion in the middle of the Cx32 cytoplasmic loop region at nucleotide position 378. This base deletion was in the middle position of the codon for amino acid 116, which is normally a CAC (histidine) but read with the frame shift was a CCC (proline). The four amino acids that followed this deletion were also altered with the fourth one becoming UAA, the ochre translation stop codon. This premature stopping of translation resulted in a truncation of 60% of the protein, which included the remaining cytoplasmic loop, third and fourth transmembrane regions and the carboxyl-terminus. The later passage cultures did not produce a carboxyl-terminal RT-PCR product, indicating that the mRNA was also truncated. These regions of the Cx32 molecule contain the sequences and epitopes to which probes and antibodies are directed, and as such alterations of these regions with repeated passage explains reports by others that FRTL-5 cells do not express Cx32, and implies that cultures used for these assessments were passed more than 15 times. To determine if genetic or epigenetic abnormalities existed in FRTL-5 cells we performed chromosome spreads from various passage cultures. FRTL-5 cells have been reported to be diploid and more recently non-diploid; however, we found them to be fully tetraploid. This tetraploidy appears to be unstable in that later passes are tetraploid plus two or three extra chromosomes. There were no obvious translocations, breaks or large-scale interstitial deletions of any chromosomes in the FRTL-5 cultures tested. As FRTL-5 cells were repeatedly passed their morphology changed. Monolayer areas spread from beneath the follicles, and the follicles became flattened in appearance. These physical changes were coincident with dramatically increased growth rates. Early cultures (passed 3-12 times) divided on average every 49+/-1 h, whereas later passes (passes 20-25) divided every 28+/-3 h. To correlate these changes with a measure of thyroid function we assayed T(4) output. Early passage follicular cultures incubated for 6 h with sodium iodide, released on average 5.27+/- 0.33 ng/ml of T(4)/100 follicles. Later passes, or early passes treated with heptanol to down-regulate Cx32, released an average of 3.84+/-0.50 ng/ml of T(4)/100 follicles. There was a 27% difference in T(4) release between early follicular cultures, that were coupled by Cx32, and late or down-regulated early follicular cultures, that were uncoupled (P<0.0001). Collectively, the physical changes documented in this study were coincident with the loss of functional Cx32. This implies a relationship between the loss of intercellular communication and changes in morphogenic appearance, growth rate and reduced thyroid function and supports the previously postulated, tumor-suppressor role for Cx32. FRTL-5 cultures from low passage numbers are an excellent model of primary thyroid cells. However, many reports in the literature ascribe features to FRTL-5 cells that are mutually inconsistent. These differences may be resolved in the future by addressing the passage number and the conditional differences of the cultures being studied.

NASA Discipline Radiation Health↗
NASA NTRS1992

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

NASA Discipline Developmental Biology↗
NASA NTRS2013

3D Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent VZV Infection

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

Goodwin, Thomas J.↗
DOE OSTI2016

Crystal structure of the kinase domain of human protein tyrosine kinase 6 (PTK6) at 2.33 Å resolution

Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50%.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation. - Highlights: • We report the first crystal structure of the kinase domain of human PTK6. • >95% pure recombinant protein was purified in two-steps protocol: affinity chromatography followed by gel filtration. • Kinase activity of the crystal construct is comparable to the full length enzyme. • This apo PTK6-KD is in DFG-in and αC-helix-out conformation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

BMP4 signaling in NPCs upregulates Bcl-xL to promote their survival in the presence of FGF-2

Highlights: • BMP4 promoted survival of neural stem/progenitor cells (NPCs) in the presence of FGF-2. • BMP4 activated Smad1/5/8 signaling and promoted Id1 and Bcl-xL expression. • BMP4 inhibited cytochrome c release from mitochondria via Bcl-xL expression. We previously reported that BMP4 does not promote proliferation or differentiation of CD44-positive astrocyte precursor cells (APCs) but greatly promotes their survival in the presence of fibroblast growth factor-2 (FGF-2). In this study, we examined if BMP4 acts as a survival factor also for neural stem/progenitor cells (NPCs) isolated from ganglionic eminence of neonatal mouse brain. We found BMP4 promotes survival but not proliferation or differentiation of these cells, just as in the case for CD44-positive APCs. Microarray analysis revealed some candidate molecules in the signaling pathway downstream of BMP4. Among them, we focused on Id1 (inhibitor of DNA-binding 1) and Bcl-xL in this study. Expression of both genes was promoted in the presence of BMP4, and this promotion was reduced by dorsomorphin, an inhibitor of BMP4 signaling. Furthermore, cytochrome c release from mitochondria was significantly reduced in the presence of BMP4, suggesting up-regulation of Bcl-xL activity by BMP4. Id1 siRNA reduced the expression of Bcl-xL, and negated survival promoting effect of BMP4. These data suggest that BMP4 promotes survival of NPCs by enhancing the anti-apoptotic function of Bcl-xL via BMP4-Smad1/5/8-Id1 signaling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Predictive Pathogen Biology: Genome-Based Prediction of Pathogenic Potential and Countermeasures Targets

Bacterial pathogens have numerous processes by which their genomic DNA is acquired or rearranged as part of their normal physiology (e.g., exchange of plasmids through conjugation) or by bacteriophage that parasitize bacteria and often insert into the bacterial genome as prophages. These processes occur with relatively high probability/frequency, and may lead to sudden changes in virulence, as new genetic material is added to the chromosome, or structural changes in the chromosome affect gene expression. We set out to devise methods to measure the rates of these processes in bacteria using next generation DNA sequencing. Using very deep sequencing on genomes we had assembled, using library preparation methods and bioinformatics tools designed to help find mobile elements and signs of their insertion, we were able to find numerous examples of attempted novel genome arrangements, revealing data that can be used to calculate rates of different mechanisms of genome change.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Somatic Mutation in Mice on the International Space Station (ISS): Guanine Substitution Suggests Link to Cancer Risk

We conducted comprehensive analysis of single nucleotide somatic mutations in mice exposed to microgravity and other factors aboard the International Space Station (ISS), using data archived in GeneLab. Animals in the experimental cohort consisted of mice that spent 37 days on the ISS within the Rodent Habitat. Ground control animals consisted of mice of identical age, sex, strain, in a terrestrial Rodent Habitat controlled for temperature, humidity and carbon dioxide levels, to match ISS conditions as closely as possible. RNA extracted from eye, liver, skeletal muscle, and kidney tissue specimens was subjected to next-generation sequencing to acquire primary data. Our analysis employed cutting-edge software developed at NASA Ames Research Center, executed on the NASA Ames Supercomputer and on another high-performance computer, for accurate variant calling of single point mutations. ISS-flown mice exhibited a notably heightened level of somatic mutation compared to control mice. The degree of somatic mutation correlated with the degree of gene expression across the four tissue types, i.e., the greatest rate of mutation accumulation was seen in highly expressed genes. We discovered that guanine substitutions were the most common type of somatic mutation. This observation is consistent with the hypothesis that DNA mutation events stem from reactive oxygen/nitrogen/chlorine species-mediated guanine oxidation induced by the spaceflight environment. Since guanine oxidation is a prominent feature of the DNA mutation landscape that accompanies malignant transformation, our findings suggest a possible link between the spaceflight environment and cancer risk that is independent of radiation carcinogenesis.

ISS↗
DOE OSTI2021

Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines

Highlights: • Amino acid metabolism is impacted in myoblasts treated with cancer cell conditioned media. • Inflammatory cytokines induce nitric oxide synthase 2 in myoblasts. • Elevated nitric oxide synthase 2 activity impairs myoblast proliferation and differentiation. Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Ca X ML: Chemistry‐informed machine learning explains mutual changes between protein conformations and calcium ions in calcium‐binding proteins using structural and topological features

Proteins' flexibility is a feature in communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. When binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit. Accurately determining the ionic charges of those ions is essential for understanding their role in such processes. However, it is unclear whether the limited experimental data available can be effectively used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. Our design created a computational tool of Ca X ML, which provided a framework of explainable machine learning model to annotate ionic charges of calcium ions in calcium-binding proteins in response to the chemical changes in an environment. Our framework will provide new insights into protein design for engineering functionality based on the limited size of scientific data in a genome space.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗