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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Transcriptional profiling of rat white adipose tissue response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), is highly lipophilic, leading to bioaccumulation. White adipose tissue (WAT) is a major site for energy storage, and is one of the organs in which TCDD accumulates. In laboratory animals, exposure to TCDD causes numerous metabolic abnormalities, including a wasting syndrome. We therefore investigated the molecular effects of TCDD exposure on WAT by profiling the transcriptomic response of WAT to 100 μg/kg of TCDD at 1 or 4 days in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. A comparative analysis was conducted simultaneously in identically treated TCDD-resistant Han/Wistar (Kuopio; H/W) rats one day after exposure to the same dose. We sought to identify transcriptomic changes coinciding with the onset of toxicity, while gaining additional insight into later responses. More transcriptional responses to TCDD were observed at 4 days than at 1 day post-exposure, suggesting WAT shows mostly secondary responses. Two classic AHR-regulated genes, Cyp1a1 and Nqo1, were significantly induced by TCDD in both strains, while several genes involved in the immune response, including Ms4a7 and F13a1 were altered in L-E rats alone. We compared genes affected by TCDD in rat WAT and human adipose cells, and observed little overlap. Interestingly, very few genes involved in lipid metabolism exhibited altered expression levels despite the pronounced lipid mobilization from peripheral fat pads by TCDD in L-E rats. Of these genes, the lipolysis-associated Lpin1 was induced slightly over 2-fold in L-E rat WAT on day 4. - Highlights: • Exposure to TCDD causes wasting syndrome in L-E rats but not in H/W rats. • We examined the transcriptome of TCDD-treated L-E and H/W rat white adipose tissue. • L-E WAT demonstrated altered abundance of several genes involved in immune response. • Few genes had altered abundance in both L-E rat WAT and human adipocytes (hMADS). • Pmepa1 was induced in both L-E WAT and differentiated hMADS cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Combined Imaging and RNA-Seq on a Microfluidic Platform for Viral Infection Studies

The goal of this work was to pioneer a novel, low-overhead protocol for simultaneously assaying cell-surface markers and intracellular gene expression in a single mammalian cell. The purpose of developing such a method is to be able to understand the mechanisms by which pathogens engage with individual mammalian cells, depending on their cell surface proteins, and how both host and pathogen gene expression changes are reflective of these mechanisms. The knowledge gained from such analyses of single cells will ultimately lead to more robust pathogen detection and countermeasures. Our method was aimed at streamlining both the upstream cell sample preparation using microfluidic methods, as well as the actual library making protocol. Specifically, we wanted to implement a random hexamer-based reverse transcription of all RNA within a single cell (as opposed to oligo dT-based which would only capture polyadenylated transcripts), and then use a CRISPR-based method called scDash to deplete ribosomal DNAs (since ribosomal RNAs make up the majority of the RNA in a mammalian cell). After significant troubleshooting, we demonstrate that we are able to prepare cDNA from RNA using the random hexamer primer, and perform the rDNA depletion. We also show that we can visualize individually stained cells, setting up the pipeline for connecting surface markers to RNA-sequencing profiles. Finally, we test a number of devices for various parts of the pipeline, including bead generation, optical barcoding and cell dispensing, and demonstrate that while some of these have potential, more work is needed to optimize this part of the pipeline.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament

Highlights: • Human periodontal ligament cells express karyopherin α (KPNA) of three subclasses. • BCL6 corepressor (BCOR) is translocated to the nucleus by KPNA2, 4, and 6. • BCOR interacts with KPNA through nuclear localization signal 1. • BCOR mutation causing OFCD syndrome downregulates the SMAD4-SHH-NFIC pathways. Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Cell wall response of field grown Populus to Septoria infection

Due to its ability to spread quickly and result in tree mortality, Sphaerulina musiva (Septoria) is one of the most severe diseases impacting Populus . Previous studies have identified that Septoria infection induces differential expression of phenylpropanoid biosynthesis genes. However, more extensive characterization of changes to lignin in response to Septoria infection is lacking. To study the changes of lignin due to Septoria infection, four field grown, naturally variant Populus trichocarpa exhibiting visible signs of Septoria infection were sampled at health, infected, and reaction zone regions for cell wall characterization. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and acid hydrolysis were applied to identify changes to the cell wall, and especially lignin. FTIR and subsequent principal component analysis revealed that infected and reaction zone regions were similar and could be distinguished from the non-infected (healthy) region. NMR results indicated the general trend that infected region had a higher syringyl:guaiacyl ratio and lower p -hydroxybenzoate content than the healthy regions from the same genotype. Finally, Klason lignin content in the infected and/or reaction zone regions was shown to be higher than healthy region, which is consistent with previous observations of periderm development and metabolite profiling. These results provide insights on the response of Populus wood characteristics to Septoria infection, especially between healthy and infected region within the same genotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Three differential expression profiles of miRNAs as potential biomarkers for lung adenocarcinoma

Highlights: • To identify the differential miRNAs expression between lung cancer and normal tissues. • Data analysis was based on a computational approach to detect survival-associated signatures. • Three-miRNA signature could be used as a prognostic marker in LACA. Owing to the poor prognosis, novel biomarkers for lung adenocarcinoma (LACA) are needed nowadays. The aim of the study was to identify the differential miRNAs expression between lung cancer and normal tissues and evaluate the prognostic values of the miRNAs. Multidimensional data of 528 samples were retrieved from The Cancer Genome Atlas archive. Data analysis was based on a computational approach to detect survival-associated molecular signatures. A total of 191 differentially expressed miRNAs were identified between LACA tissues and normal tissues, including 88 up-regulated and 103 down-regulated miRNAs. The Kaplan-Meier survival method revealed the prognostic function of the three miRNAs (miR-1293, miR-873 and miR-1914). Cox regression analysis showed that the three-miRNA signature was an independent prognostic factor in LACA. The functional enrichment analysis suggested that the target genes of three miRNAs may be involved in various pathways related to the cancer. This study demonstrated that the three-miRNA signature (miR-1293, miR-873 and miR-1914) could be used as a prognostic marker in LACA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Single-Cell RNA-Seq Reveals Transcriptomic Heterogeneity and Post-Traumatic Osteoarthritis-Associated Early Molecular Changes in Mouse Articular Chondrocytes

Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T . atroviride exudates. In contrast, P . simiae and P . putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δ tmk 3) gene of T . atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T . atroviride , bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δ tmk3 mutant. Unexpectedly, exudates from wild-type T . atroviride and the Δ tmk 3 mutant rescued purine auxotrophic mutants of H . seropedicae , K . michiganensis and P . simiae . Metabolomic analysis on exudates from wild-type T . atroviride and the Δ tmk 3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma -metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

59 BASIC BIOLOGICAL SCIENCES↗
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↗