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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 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
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 OSTI2018

Hepatocyte-specific deletion of Brg1 alleviates methionine-and-choline-deficient diet (MCD) induced non-alcoholic steatohepatitis in mice

Highlights: • Brg1 expression is up-regulated by pro-NASH stimuli in vitro and in vivo. • Hepatocyte-specific Brg1 deletion alleviates non-alcoholic steatohepatitis in mice. • Brg1 is essential for the interaction between MRTF-A and NF-κB. • Brg1 promotes the activation of SREBP1c target genes in mice. Uncontrolled inflammatory response and augmented lipid accumulation represent two key pathophysiological events in the pathogenesis of non-alcoholic steatohepatitis (NASH). NF-κB and SREBP1c program transcriptional regulation of cellular inflammatory response and lipid metabolism, respectively. The epigenetic mechanism underlying NF-κB-dependent pro-inflammatory transcription and SREBP1c-dependent pro-lipogenic transcription remains incompletely understood. In the present study we investigated the involvement of Brg1, a chromatin remodeling protein, in NASH pathogenesis in a methionine-and-choline deficient diet (MCD) induced mouse model. Brg1 expression was up-regulated in the liver in mice fed on the MCD diet and in primary hepatocytes exposed to free fatty acids. Liver injury and hepatic inflammation attenuated in hepatocyte-specific Brg1 knockout (CKO) mice fed on the MCD diet compared to the wild type (WT) littermates. Likewise, synthesis of pro-inflammatory mediators was down-regulated in primary hepatocytes isolated from CKO mice compared to WT mice, which resulted in reduced macrophage chemotaxis. Brg1 contributed to the transcription of pro-inflammatory mediators possibly by regulating the interaction between NF-κB and its co-factor MRTF-A. On the other hand, accumulation of triglyceride and cholesterol was ameliorated in MCD-fed CKO mice with a concomitant reduction of SREBP1c target genes. Brg1 interacted with SREBP1c and modulated the transcription of SREB1c target genes in the liver in response to MCD feeding by influencing active histone modifications. In conclusion, targeting Brg1 may yield novel anti-NASH therapeutics by simultaneously normalizing hepatic inflammatory status and metabolic profile in NASH patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Mass spectrometry-based technologies for probing the 3D world of plant proteins

Abstract Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions—which are more difficult to study—dynamically regulate the proteome’s 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based “structural” proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function.

Plant Sciences↗
DOE OSTI2017

Cyanobacterial carbon metabolism: Fluxome plasticity and oxygen dependence: Cyanobacterial Carbon Metabolism

Synechocystis sp. strain PCC 6803 has been widely used as a photo-biorefinery chassis. Based on its genome annotation, this species contains a complete TCA cycle, an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose phosphate pathway (OPPP), and an Entner–Doudoroff pathway (EDP). To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic conditions, we performed 13 C metabolic flux analysis, metabolite pool size analysis, gene knockouts, and heterologous expressions. The results revealed a cyclic mode of flux through the OPPP. Small, but non-zero, fluxes were observed through the TCA cycle and the malic shunt. Independent knockouts of 6-phosphogluconate dehydrogenase (gnd) and malic enzyme (me) corroborated these results, as neither mutant could grow under dark heterotrophic conditions. Our data also indicate that Synechocystis 6803 metabolism relies upon oxidative phosphorylation to generate ATP from NADPH under dark or insufficient light conditions. The pool sizes of intermediates in the TCA cycle, particularly acetyl-CoA, were found to be several fold lower in Synechocystis 6803 (compared to E. coli metabolite pool sizes), while its sugar phosphate intermediates were several-fold higher. Moreover, negligible flux was detected through the native, or heterologous, EDP in the wild type or Δgnd strains under heterotrophic conditions. Comparing photoautotrophic, photomixotrophic, and heterotrophic conditions, the Calvin cycle, OPPP, and EMPP in Synechocystis 6803 possess the ability to regulate their fluxes under various growth conditions (plastic), whereas its TCA cycle always maintains at low levels (rigid). This work also demonstrates how genetic profiles do not always reflect actual metabolic flux through native or heterologous pathways. Biotechnol. Bioeng. 2017;114: 1593–1602. © 2017 Wiley Periodicals, Inc.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT-encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T-DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacety CoA precursor required for the cytosol-localized, mevalonate-derived isoprenoid biosynthetic pathway.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Fluorescence-labeled liposome accumulation in injured colon of a mouse model of T-cell transfer-mediated inflammatory bowel disease

Drug delivery systems maximize the efficacy of drugs by improving their pharmacokinetic profiles, pharmacodynamic effects, or both and reducing their adverse effects. One of the most advanced, clinically available formulations are liposome-encapsulated drugs. In this study, we aimed to determine if liposomes can selectively deliver compounds in gastrointestinal diseases. Initially, we evaluated the correlation between the diarrhea score and accumulation of fluorescence (FL)-labeled liposome using in vivo imaging systems in various disease states of an inflammatory bowel disease mouse model. The result showed that FL-labeled liposome accumulation and colon tissue weight, which reflect the disease state were highly and positively correlated. Then, to confirm the accumulation of liposomes at injured sites of the colon, we administered both FL-labeled liposomes and luminescence probes for detecting reactive oxygen species (ROS) to the mouse model. The imaging data showed that liposome accumulation tended to coincide with ROS detected sites and the correlation coefficient indicated a significantly positive correlation between liposome accumulation and ROS detection levels. Finally, we evaluated the involvement of macrophages in the uptake mechanism of the liposomes by analyzing the relationship between FL-labeled liposome accumulation and macrophage marker gene expression levels. The result showed that the expression of each macrophage marker gene and liposome accumulation showed a significant positive correlation. Therefore, the macrophages considerably contributed to the uptake mechanism of the liposomes. These data suggest that liposomes could be an attractive delivery tool for enhancing the accumulation of drug candidates through macrophages in injured colonic tissues. This approach is expected to provide new treatment options for patients with colitis.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1997

A low molecular weight artificial RNA of unique size with multiple probe target regions

Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E. coli. These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring. Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs. This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence. This aRNA is 160 nucleotides in length and again accumulates to high levels in the E. coli cytoplasm without incorporating into ribosomes. The 3xPen aRNA illustrates two improvements in detection. First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs. Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization. These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences. It thus may be possible to detect them directly. An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Non-NASA Center↗
NASA NTRS2016

Sex-specific Effects of Unpredictable Variable Prenatal Stress: Implications for Mammalian Developmental Programming During Spaceflight

During initial exposure and adaptation to the microgravity environment, adult mammals exhibit elevated stress, mediated by the Hypothalamic-Pituitary-Adrenal (HPA) axis. In our previous studies of pregnant rats exposed to 2-g hypergravity via continuous centrifugation, we reported changes in neuroendocrine profiles including decreased corticosterone and a concomitant increase in body mass and leptin in adult male offspring. Prenatally stressed adult offspring have been shown to exhibit an elevated stress response in adulthood, therefore we hypothesized that these changes resulted from stress exposure during fetal development. Future studies examining reproduction, gestation, and development on-orbit need to consider the unique stressors of vehicle launch, the space environment, and landing on the development of the HPA axis in animals born and raised in microgravity. In this study, we utilize Unpredictable Variable Prenatal Stress (UVPS) to simulate the stressors of spaceflight by exposing dams to three different stressors: (1) White Noise, (2) Strobe Light, and (3) Tube Restraint. Stressors were applied from Gestational Day 0 (G0), following an unpredictable schedule (morning [0600-1200hrs]; afternoon [1200-1800hrs]; evening [1800-2400hrs] in 15, 30, or 60 minute durations alongside non-stressed (NS) control dams. Following parturition, pups were fostered to non-manipulated, newly parturient dams to control for differential maternal care. On postnatal day 90 (P90), we harvested the hypothalamus, pituitary, and adrenal glands, and analyzed mRNA expression of the following genes via RT-qPCR: 1) melanocortin-2 receptor (MC2R), POMC, corticotropin-releasing hormone (CRH) in the pituitary; 2) glucocorticoid receptor (NR3C1), pro-opiomelanocortin (POMC), corticotropin-releasing hormone (CRH), brain-derived neurotropic factor (BDNF), in the hypothalamus; and 3) MC2R, tyrosine hydroxylase (TH), steroidogenic acute regulatory protein (STAR), cytochrome P450scc enzyme (CYP) in the adrenal. The identification of sex-specific fetal programming effects on adult stress response is a key step in determining potential animal behavior on-orbit, and will guide future multi-generational studies in microgravity.

prenatal stress↗
NASA NTRS2004

Biodosimetry as a New Paradigm for Determination of Radiation Risks and Risk-Mitigation in Astronauts Exposed to Space Radiation

Predicting risk of cancer in astronauts exposed to space radiation is challenging partly because uncertainties of absorption of dose and the processing of dose-related damage at the cellular level degrade the confidence of predicting the expression of cancer. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the macromolecular profiles related to malignant transformation in cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported, This is the demonstration of two dose-responsive field-effects of enhanced protein-expression. In one case, expression of keratin 18 (K18) in cultures of human mammary epithelial cells (HMEC) irradiated with cesium-137 gamma-rays is enhanced following exposure of log phase cells to relatively low doses of 30 to 90 cGy. K18 has been reported by a marker for tumor staging and for apoptosis. In the second case, expression of connexin 43 (Cx43) is increased in irradiated stationary phase cultures of HMEC, indicating enhanced formation of gap junctions. Gap junctions have been reported to be involved in bystander effects following irradiation. It is a biodosimeter for assessing radiogenic damage. It is suggested further that such biomolecular dosimetry may introduce a new paradigm for assessing cancer risk and risk-mitigation in individuals, a requirement for managing radiation health in astronauts during extended missions in space. This new paradigm is built upon the statistical power provided by the use of functional genomics and proteomics represented in combined gene- and protein-expression assays.

Richmond, Robert↗
DOE OSTI2025

Transcript profiling of plastid ferrochelatase two mutants reveals that chloroplast singlet oxygen signals lead to global changes in RNA profiles and are mediated by Plant U-Box 4

Abstract Background In response to environmental stresses, chloroplasts generate reactive oxygen species, including singlet oxygen ( 1 O 2 ), an excited state of oxygen that regulates chloroplast-to-nucleus (retrograde) signaling, chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. TheArabidopsis thaliana plastid ferrochelatase two(fc2) mutant conditionally accumulates 1 O 2 , and Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, is involved in propagating 1 O 2 signals for chloroplast turnover and cellular degradation. Thus, thefc2andfc2 pub4mutants are useful genetic tools to elucidate these signaling pathways. Previous studies have focused on the role of 1 O 2 in promoting cellular degradation infc2mutants, but its impact on retrograde signaling from mature chloroplasts (the major site of 1 O 2 production) is poorly understood. Results To gain mechanistic insights into 1 O 2 signaling pathways, we compared transcriptomes of adult wt,fc2, andfc2 pub4plants. The accumulation of 1 O 2 infc2plants broadly repressed genes involved in chloroplast function and photosynthesis, while inducing genes and transcription factors involved in abiotic and biotic stress, the biosynthesis of jasmonic acid (JA) and salicylic acid (SA), microautophagy, and senescence. Elevated JA and SA levels were observed in 1 O 2 -stressedfc2plants.pub4reversed most of this 1 O 2 -induced gene expression and reduced the JA content infc2plants. Thepub4mutation also blocked JA-induced senescence pathways in the dark. However, fc2 pub4 plantsmaintained constitutively elevated levels of SA even in the absence of bulk 1 O 2 accumulation. Conclusions Together, this work demonstrates that infc2plants, 1 O 2 leads to a robust retrograde signal that may protect cells by downregulating photosynthesis and ROS production while simultaneously mounting a stress response involving SA and JA. The induction of microautophagy and senescence pathways indicate that 1 O 2 -induced cellular degradation is a genetic response to this stress, and the bulk of this transcriptional response is modulated by the PUB4 protein. However, the effect ofpub4on hormone synthesis and signaling is complex and indicates that an intricate interplay of SA and JA are involved in promoting stress responses and programmed cell death during photo-oxidative damage.

Plant Sciences↗
DOE OSTI2018

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium distachyon transcriptional profiling data, we identified a B. distachyon trihelix family transcription factor (BdTHX1) that is highly coexpressed with the B. distachyon CSLF6 gene (BdCSLF6), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature B. distachyon seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of BdCSLF6 as one of the most enriched sequences. The binding of BdTHX1 to the BdCSLF6 intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase (BdXTH8) is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. B. distachyon shoots regenerated from transformed calli overexpressing BdTHX1 showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. Finally, these results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2011

Evidence-Based Annotation of Gene Function in Shewanella oneidensis MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions

Most genes in bacteria are experimentally uncharacterized and cannot be annotated with a specific function. Given the great diversity of bacteria and the ease of genome sequencing, high-throughput approaches to identify gene function experimentally are needed. Here, we use pools of tagged transposon mutants in the metal-reducing bacterium Shewanella oneidensis MR-1 to probe the mutant fitness of 3,355 genes in 121 diverse conditions including different growth substrates, alternative electron acceptors, stresses, and motility. We find that 2,350 genes have a pattern of fitness that is significantly different from random and 1,230 of these genes (37% of our total assayed genes) have enough signal to show strong biological correlations. We find that genes in all functional categories have phenotypes, including hundreds of hypotheticals, and that potentially redundant genes (over 50% amino acid identity to another gene in the genome) are also likely to have distinct phenotypes. Using fitness patterns, we were able to propose specific molecular functions for 40 genes or operons that lacked specific annotations or had incomplete annotations. In one example, we demonstrate that the previously hypothetical gene SO_3749 encodes a functional acetylornithine deacetylase, thus filling a missing step in S. oneidensis metabolism. Additionally, we demonstrate that the orphan histidine kinase SO_2742 and orphan response regulator SO_2648 form a signal transduction pathway that activates expression of acetyl-CoA synthase and is required for S. oneidensis to grow on acetate as a carbon source. Lastly, we demonstrate that gene expression and mutant fitness are poorly correlated and that mutant fitness generates more confident predictions of gene function than does gene expression. The approach described here can be applied generally to create large-scale gene-phenotype maps for evidencebased annotation of gene function in prokaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Molecular basis of carcinogenicity of tungsten alloy particles

The tungsten alloy of 91% tungsten, 6% nickel and 3% cobalt (WNC 91–6–3) induces rhabdomyosarcoma when implanted into a rat thigh muscle. To investigate whether this effect is species-specific human HSkMc primary muscle cells were exposed to WNC 91–6–3 particles and responses were compared with those from a rat skeletal muscle cell line (L6-C11). Toxicity was assessed by the adenylate kinase assay and microscopy, DNA damage by the Comet assay. Caspase 3 enzyme activity was measured and oligonucleotide microarrays were used for transcriptional profiling. WNC 91–6–3 particles caused toxicity in cells adjacent to the particles and also increased DNA strand breaks. Inhibition of caspase 3 by WNC 91–6–3 occurred in rat but not in human cells. In both rat and human cells, the transcriptional response to WNC 91–6–3 showed repression of transcripts encoding muscle-specific proteins with induction of glycolysis, hypoxia, stress responses and transcripts associated with DNA damage and cell death. In human cells, genes encoding metallothioneins were also induced, together with genes related to angiogenesis, dysregulation of apoptosis and proliferation consistent with pre-neoplastic changes. An alloy containing iron, WNF 97–2–1, which is non-carcinogenic in vivo in rats, did not show these transcriptional changes in vitro in either species while the corresponding cobalt-containing alloy, WNC 97–2–1 elicited similar responses to WNC 91–6–3. Tungsten alloys containing both nickel and cobalt therefore have the potential to be carcinogenic in man and in vitro assays coupled with transcriptomics can be used to identify alloys, which may lead to tumour formation, by dysregulation of biochemical processes. - Highlights: • Use of transcriptomics to identify likely carcinogenic tungsten alloys in vitro • Cobalt containing alloys cause oxidative stress, DNA-damage and perturb apoptosis. • Presence of cobalt causes changes in gene expression consistent with carcinogenesis. • Development of an in vitro screening platform for tungsten alloy toxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Divergence in Gene Regulation Contributes to Sympatric Speciation of Shewanella baltica Strains

Niche partitioning and sequence evolution drive genomic and phenotypic divergence, which ultimately leads to bacterial diversification. This study investigated the genomic composition of two Shewanella baltica clades previously identified through multilocus sequencing typing and recovered from the redox transition zone in the central Baltic Sea. Comparative genomic analysis revealed significantly higher interclade than intraclade genomic dissimilarity and that a subset of genes present in clade A were associated with potential adaptation to respiration of sulfur compounds present in the redox transition zone. The transcriptomic divergence between two representative strains of clades A and D, OS185 and OS195, was also characterized and revealed marked regulatory differences. We found that both the transcriptional divergence of shared genes and expression of strain-specific genes led to differences in regulatory patterns between strains that correlate with environmental redox niches. For example, under anoxic conditions of respiratory nitrate ammonification, OS185—the strain isolated from a nitrate-rich environment—upregulated nearly twice the number of shared genes upregulated by OS195—the strain isolated from an H 2 S-containing anoxic environment. On the other hand, OS195 showed stronger induction of strain-specific genes, especially those associated with sulfur compound respiration, under thiosulfate-reducing conditions. A positive association between the level of transcriptional divergence and the level of sequence divergence for shared genes was also noted. Our results provide further support for the hypothesis that genomic changes impacting transcriptional regulation play an important role in the diversification of ecologically distinct populations. IMPORTANCE: This study examined potential mechanisms through which co-occurring Shewanella baltica strains diversified to form ecologically distinct populations. At the time of isolation, the strains studied composed the major fraction of culturable nitrate-reducing communities in the Baltica Sea. Analysis of genomic content of 13 S. baltica strains from two clades representing different ecotypes demonstrated that one clade specifically possesses a number of genes that could favor successful adaptation to respire sulfur compounds in the portion of the water column from which these strains were isolated. In addition, transcriptional profiling of fully sequenced strains representative of these two clades, OS185 and OS195, under oxygen-, nitrate-, and thiosulfate-respiring conditions demonstrated that the strains exhibit relatively similar transcriptional responses during aerobic growth but more-distinct transcriptional responses under nitrate- and thiosulfate-respiring conditions. Results from this study provide insights into how genomic and gene regulatory diversification together impacted the redox specialization of the S. baltica strains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Preparation for and Performance of a Pseudomonas aeruginosa Biofilm Experiment On Board the International Space Station

Biofilms are a problem on Earth given their ability to degrade the materials upon which they grow and due to their relevance to infections. Remarkably, 65% and 80% of infections and chronic diseases on Earth are associated with biofilms, respectively. In space, these problems' impact is higher because the crew's lives and mission success depend on nominal operation of mechanical systems. Furthermore, the isolated confined environment nature of spaceflight may increase the rates of disease transmission. In the case of the International Space Station (ISS), biofilms are an identified problem on the Environmental Control and Life Support System (ECLSS), namely on the water processor assembly (WPA). In late 2019, the Space Biofilms experiment launched towards ISS to (i) characterize the mass, thickness, morphology, and gene expression of biofilms formed in space with respect to matched Earth controls, (ii) interrogate the expression of antimicrobial resistance genes, and (iii) test novel materials as potential biofilm control strategies for future ECLSS components. For this, 288 bacterial samples were prepared prior to the launch of the Northrop Grumman CRS-12 mission from NASA's Wallops Flight Facility. The samples were integrated into the spaceflight hardware, BioServe's Fluid Processing Apparatus (FPA) packed in sets of eight in Group Activation Packs (GAP). Half of these samples were activated and terminated on orbit by NASA astronauts Jessica Meir and Christina Koch, while the remaining half were processed equivalently on Earth. The spaceflight bacterial samples of Space Biofilms returned on board SpaceX' CRS-19 Dragon spacecraft, in early 2020. We here describe the test campaign implemented to verify the experiment design and confirm it would enable us to achieve the project's scientific goals. This campaign ended with the Experiment Verification Test (EVT), from which we here present example morphology and transcriptomic results. We describe in detail the sample preparation prior to flight, including cleaning and sterilization of the coupons of six materials (SS316, passivated-SS316, lubricant impregnated surface, catheter-grade silicone with and without a nanotopography, and cellulose membrane), loading and integration of growth media, bacterial inoculum, fixative and preservative to enable experiment termination on orbit. Additionally, we describe the performance of the experiment on board the ISS, including crew activities, use of assets, temperature profile, and experiment timeline; all leading to a successful spaceflight experiment.

Pamela Flores↗
DOE OSTI2024

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Genetic mutations in adipose triglyceride lipase and myocardial up-regulation of peroxisome proliferated activated receptor-γ in patients with triglyceride deposit cardiomyovasculopathy

Highlights: •Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare severe heart disease. •PPARγ is up-regulated in myocardium in patients with TGCV. •Possible vicious cycle for fatty acid may be involved in pathophysiology of TGCV. -- Abstract: Adipose triglyceride lipase (ATGL, also known as PNPLA2) is an essential molecule for hydrolysis of intracellular triglyceride (TG). Genetic ATGL deficiency is a rare multi-systemic neutral lipid storage disease. Information regarding its clinical profile and pathophysiology, particularly for cardiac involvement, is still very limited. A previous middle-aged ATGL-deficient patient in our institute (Case 1) with severe heart failure required cardiac transplantation (CTx) and exhibited a novel phenotype, “Triglyceride deposit cardiomyovasculopathy (TGCV)”. Here, we tried to elucidate molecular mechanism underlying TGCV. The subjects were two cases with TGCV, including our second case who was a 33-year-old male patient (Case 2) with congestive heart failure requiring CTx. Case 2 was homozygous for a point mutation in the 5′ splice donor site of intron 5 in the ATGL, which results in at least two types of mRNAs due to splicing defects. The myocardium of both patients (Cases 1 and 2) showed up-regulation of peroxisome proliferated activated receptors (PPARs), key transcription factors for metabolism of long chain fatty acids (LCFAs), which was in contrast to these molecules’ lower expression in ATGL-targeted mice. We investigated the intracellular metabolism of LCFAs under human ATGL-deficient conditions using patients’ passaged skin fibroblasts as a model. ATGL-deficient cells showed higher uptake and abnormal intracellular transport of LCFA, resulting in massive TG accumulation. We used these findings from cardiac specimens and cell-biological experiments to construct a hypothetical model to clarify the pathophysiology of the human disorder. In patients with TGCV, even when hydrolysis of intracellular TG is defective, the marked up-regulation of PPARγ and related genes may lead to increased uptake of LCFAs, the substrates for TG synthesis. This potentially vicious cycle of LCFAs could explain the massive accumulation of TG and severe clinical course for this rare disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Metaproteomics Identifies the Protein Machinery Involved in Metal and Radionuclide Reduction in Subsurface Microbiomes and Elucidates Mechanisms and U(VI) Reduction Immobilization

The overall goal for this funded project was to develop and exploit environmental metaproteomics tools to identify biomarkers for monitoring microbial activity affecting U speciation at U-contaminated sites, correlate metaproteomics profiles with geochemical parameters and U(VI) reduction activity (or lack thereof), elucidate mechanisms contributing to U(VI) reduction, and provide remediation project managers with additional information to make science-based site management decisions for achieving cleanup goals more efficiently. Although significant progress has been made in elucidating the microbiology contribution to metal and radionuclide reduction, the cellular components, pathway(s), and mechanisms involved in U trans-formation remain poorly understood. Recent advances in (meta)proteomics technology enable detailed studies of complex samples, including environmental samples, which differ between sites and even show considerable variability within the same site (e.g., the Oak Ridge IFRC site). Additionally, site-specific geochemical conditions affect microbial activity and function, suggesting generalized assessment and interpretations may not suffice. This research effort integrated current understanding of the microbiology and biochemistry of U(VI) reduction and capitalize on advances in proteomics technology made over the past few years. Field-related analyses used Oak Ridge IFRC field ground water samples from locations where slow-release substrate biostimulation has been implemented to accelerate in situ U(VI) reduction rates. Our overarching hypothesis was that the metabolic signature in environmental samples, as deciphered by the metaproteome measurements, would show a relationship with U(VI) reduction activity. Since metaproteomic and metagenomic characterizations were computationally challenging and time-consuming, we used a tiered approach that combines database mining, controlled laboratory studies, U(VI) reduction activity measurements, phylogenetic analyses, and gene expression studies to support the metaproteomics characterizations. Growth experiments of target microorganisms (Anaeromyxobacter, Shewanella, Geobacter) revealed tremendous respiratory versatility, as evidenced by the ability to utilize a range of electron donors (e.g. acetate, hydrogen, pyruvate, lactate, succinate, formate) and electron acceptors (e.g. nitrate, fumarate, halogenated phenols, ferric iron, nitrous oxide, etc.). In particular, the dissimilatory metabolic reduction of metals, including radionuclides, by target microorganisms spurred interest for in situ bioremediation of contaminated soils and sediments. Distinct c-type cytochrome expression patterns were observed in target microorganisms grown with the different electron acceptors. For each target microorganism, the core proteome covered almost all metabolic pathways represented by their corresponding pan-proteomes. Unique proteins were detected for each target microorganism, and their expression and possible functionalities were linked to specific growth conditions through proteomics measurements. Optimization of the proteomic tools included in-depth comprehensive metagenomic and metaproteomic analyses on a limited number of samples. The optimized metaproteomic analyses were then applied to Oak Ridge IFRC field samples from the slow-release substrate biostimulation. Metaproteomic analysis and pathway mapping results demonstrated the distinct effects of metal and non-metal growth conditions on the proteome expression. With these metaproteomic tools, we identified which previously hypothetical metabolic pathways were active during the analyzed time points of the slow release substrate biostimulation. Thus, we demonstrated the utility of these tools for site assessment, efficient implementation of bioremediation and long-term monitoring. This research of detailed protein analysis linked with metal reduction activity did (1) show that c-type cytochrome isoforms, previously associated with radionuclide reduction activity, are suitable biomarkers, (2) identify new biomarker targets for site assessment and bioremediation monitoring, and (3) provide new information about specific proteins and mechanisms involved in U(VI) reduction and immobilization. This expanded metagenomic and metaproteomic toolbox contributed to implementing science-driven site management with broad benefits to the DOE mission.

54 ENVIRONMENTAL SCIENCES↗