SEARCH · Engineering Papers

Results for “Gene”

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

SourceResearch connectionFrom yearThrough year

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

At least 343 records · Page 19

All sources NASA NTRS DOE OSTI

NASA NTRSDate not supplied

Oxygen Deficiency in Spaceflight & its Impact on Plants’ Adaptive Changes

The goal of this study was to investigate the effects of hypoxic conditions in spaceflight. The distribution of genes involved with hypoxia in Arabidopsis thaliana and Brassica rapa were analyzed with the results from past spaceflight experiments to evaluate genes for future studies. Transcriptomes data of two different spaceflight studies of Arabidopsis thaliana from the NASA GeneLab database, GLDS-7 and GLDS-17, were compared. DNA microarrays were utilized for transcription profiling to conduct these studies. For GLDS-7, the response in spaceflight was studied with approaches that collected gene expression data. Leaves, hypocotyls, and root tissues were compared to the whole plant. For GLDS-17, seedlings and undifferentiated cultured cells were placed in the Biological Research in Canisters (BRIC), specifically BRIC-16. The genes related to hypoxia in Arabidopsis thaliana from these two studies were compared to genes in Brassica rapa with the TOAST database to evaluate similarities. When transcriptomes were analyzed for GLDS-7 and 17, genes that were considered significant had p-values ≤ 0.05 and log fold change values ≤ -1 or ≥1. Sixteen genes fulfilled the criteria. The genes related to hypoxia were alcohol dehydrogenase, elongation factor, ethylene-responsive factor, GUS, heat-shock proteins, NAP, RAP2.12, and RD20. The genes most impacted by spaceflight were heat-shock proteins. These genes were compared with Brassica rapa through Arabidopsis Ensemble Orthology from the TOAST Database. Similarities were seen in alcohol dehydrogenase, elongation factor, ethylene-responsive factor, heat-shock proteins, NAP, and RAP2.12. Overall, transcription profiling indicates that plants’ survival in spaceflight is dependent on adaptive changes with gene expression. This study also indicates that there are similarities in gene expression between Arabidopsis thaliana and Brassica rapa with comparable gene expression. Future studies could include analyzing additional species to understand which genes could be modified to ensure better yield of space crops amid hypoxic conditions.

hypoxia↗

NASA NTRSDate not supplied

Biological Insights at the Interface of Multiple Arabidopsis Legacy Datasets

The NASA GeneLab database includes an open-access collection of datasets yielded by space biology experiments. Six Gene Lab Data Sets (GLDS’s) performed in Arabidopsis were selected for analysis (7/17/44/121/205/213), all of which included transcriptome data from spaceflight and ground control environments. Hardware, ecotype, environmental conditions, and other experimental conditions varied, allowing the observations of overarching gene expression impacts of microgravity on plant life without focusing on effects of specific experimental conditions. Using GeneLab pre-processed datasets as the basis for the study, RNA microarray data were analyzed to identify genes that showed altered expression in microgravity when compared to control samples for each individual GLDS. All differentially expressed genes were compared to locate differentially expressed genes common between spaceflight experiments. The most noteworthy result is that not one gene shared differential expression among the six GLDS’s. However, gene expression was not influenced randomly by the microgravity environment, as there were several gene ontology terms that were significantly enriched across all experiments. These included 20 significantly enriched biological processes, and although the genes which enriched each term varied, there were many cases of specific genes common to clusters of multiple GLDS’s. Gene expression such as NAC92 and ERF011 or membrane structural element FFP6 provide insight and direction toward understanding the plant response to spaceflight. Characterizing these common processes and the shared differentially expressed genes has demonstrated potential targets for further study to understand and modulate the biological response of plants in microgravity. Life on Earth has never been subjected to the absence of gravity as a selective pressure, so observing how life forms react to a microgravity environment could provide insight to our shared fundamental biological processes. It is also feasible that the genetic modification of specific genes linked to the microgravity response could improve health and yield of space crops.

Joseph Emhof↗

NASA NTRS2006

Immune Response in Microgravity: Genetic Basis and Countermeasure Development Implications

Impairment of the immunity in astronauts and cosmonauts even in shortterm flights is a recognized risk. Longterm orbital space missions and anticipated interplanetary flights increase the concern for more pronounced effects on the immune system with potential clinical consequences. Studies in true and modeled microgravity (MG) have demonstrated that MG directly affects numerous lymphocyte functions. The purpose of this study was to screen for genes involved in lymphocytes response to modeled microgravity (MMG) that could explain the functional and structural changes observed earlier. The microgravity-induced changes in gene expression were analyzed by microarray DNA chip technology. CD3and IL2activated Tcells were cultured in 1g (static) and modeled microgravity (NASA Rotating Wall Vessel bioreactor) conditions for 24 hours. Total RNA was extracted using the RNeasy isolation kit (Qiagen, Valencia, CA). Microarray experiments were performed utilizing Affymetrix Gene Chips (U133A), allowing testing for 18,400 human genes. To decrease the biological variation and aid in detecting microgravity-associated changes, experiments were performed in triplicate using cells obtained from three different donors. Exposure to modeled microgravity resulted in alteration of 89 genes, 10 of which were upregulated and 79 down-regulated. Altered genes were categorized by their function, structural role and by association with metabolic and regulatory pathways. A large proportion was found to be involved in fundamental cellular processes: signal transduction, DNA repair, apoptosis, and multiple metabolic pathways. There was a group of genes directly related to immune and inflammatory responses (IL7R, granulysin, proteasome activator subunit 2, peroxiredoxin 4, HLADRA, lymphocyte antigen 75, IL18R and DOCK2 genes). Among these genes only one (IL7R) was upregulated, the rest were downregulated. The upregulation of the IL7 receptor gene was confirmed by RT PCR. Three genes with altered expression were identified in the apoptosis related group (Granzyme B, APO2 ligand and Beta3endonexin). All of them were downregulated. Gene expression changes in MG might appear pivotal in identifying potential molecular targets for countermeasure development. (Supported by NRA OLMSA02 and NSCORT NAG54072 grants).

Risin, Diana↗

NASA NTRS2014

Radiation Quality Effects on Transcriptome Profiles in 3-d Cultures After Particle Irradiation

In this work, we evaluate the differential effects of low- and high-LET radiation on 3-D organotypic cultures in order to investigate radiation quality impacts on gene expression and cellular responses. Reducing uncertainties in current risk models requires new knowledge on the fundamental differences in biological responses (the so-called radiation quality effects) triggered by heavy ion particle radiation versus low-LET radiation associated with Earth-based exposures. We are utilizing novel 3-D organotypic human tissue models that provide a format for study of human cells within a realistic tissue framework, thereby bridging the gap between 2-D monolayer culture and animal models for risk extrapolation to humans. To identify biological pathway signatures unique to heavy ion particle exposure, functional gene set enrichment analysis (GSEA) was used with whole transcriptome profiling. GSEA has been used extensively as a method to garner biological information in a variety of model systems but has not been commonly used to analyze radiation effects. It is a powerful approach for assessing the functional significance of radiation quality-dependent changes from datasets where the changes are subtle but broad, and where single gene based analysis using rankings of fold-change may not reveal important biological information. We identified 45 statistically significant gene sets at 0.05 q-value cutoff, including 14 gene sets common to gamma and titanium irradiation, 19 gene sets specific to gamma irradiation, and 12 titanium-specific gene sets. Common gene sets largely align with DNA damage, cell cycle, early immune response, and inflammatory cytokine pathway activation. The top gene set enriched for the gamma- and titanium-irradiated samples involved KRAS pathway activation and genes activated in TNF-treated cells, respectively. Another difference noted for the high-LET samples was an apparent enrichment in gene sets involved in cycle cycle/mitotic control. It is plausible that the enrichment in these particular pathways results from the complex DNA damage resulting from high-LET exposure where repair processes are not completed during the same time scale as the less complex damage resulting from low-LET radiation.

Patel, Z. S.↗

NASA NTRS2005

Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance

This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.

NASA Discipline Cell Biology↗

NASA NTRS2003

Gravity Plays an Important Role in Muscle Development and the Differentiation of Contractile Protein Phenotype

Several muscles in the body exist mainly to work against gravity. Whether gravity is important in the development of these muscles is not known. By examining the basic proteins that compose muscle, questions about the role of gravity in muscle development can be answered. Myosin heavy chains (MHCs) are a family of proteins critically important for muscle contraction. Several types of MHCs exist (e.g., neonatal, slow, fast), and each type is produced by a particular gene. Neonatal MHCs are produced early in life. Slow MHCs are important in antigravity muscles, and fast MHCs are found in fast-twitch power muscles. The gene that is turned on or expressed will determine which MHC is produced. Early in development, antigravity skeletal muscles (muscles that work against gravity) normally produce a combination of the neonatal/embryonic MHCs. The expression of these primitive MHCs is repressed early in development; and the adult slow and fast MHC genes become fully expressed. We tested the hypothesis that weightbearing activity is critical for inducing the normal expression of the slow MHC gene typically expressed in adult antigravity muscles. Also, we hypothesized that thyroid hormone, but not opposition to gravity, is necessary for expressing the adult fast IIb MHC gene essential for high-intensity muscle performance. Groups of normal thyroid and thyroid-deficient neonatal rats were studied after their return from the 16-day Neurolab mission and compared to matched controls. The results suggest: (1) Weightlessness impaired body and limb skeletal muscle growth in both normal and thyroid-deficient animals. Antigravity muscles were impaired more than those used primarily for locomotion andor nonweightbearing activity. (2) Systemic and muscle expression of insulin-like growth factor-I (IGF-I), an important body and tissue growth factor, was depressed in flight animals. (3) Normal slow, type I MHC gene expression was markedly repressed in the normal thyroid flight group. (4) Fast IIb MHC gene expression was enhanced in fast-twitch muscles of normal thyroid animals exposed to spaceflight; however, thyroid deficiency markedly repressed expression of this gene independently of spaceflight. In summary, the absence of gravity, when imposed at critical stages of development, impaired body and skeletal muscle growth, as well as expression of the MHC gene family of motor proteins. This suggests that normal weightbearing activity is essential for establishing body and muscle growth in neonatal animals, and for expressing the motor gene essential for supporting antigravity functions.

Adams, Gregory A.↗

NASA NTRS2005

Efficient Credit Assignment through Evaluation Function Decomposition

Evolutionary methods are powerful tools in discovering solutions for difficult continuous tasks. When such a solution is encoded over multiple genes, a genetic algorithm faces the difficult credit assignment problem of evaluating how a single gene in a chromosome contributes to the full solution. Typically a single evaluation function is used for the entire chromosome, implicitly giving each gene in the chromosome the same evaluation. This method is inefficient because a gene will get credit for the contribution of all the other genes as well. Accurately measuring the fitness of individual genes in such a large search space requires many trials. This paper instead proposes turning this single complex search problem into a multi-agent search problem, where each agent has the simpler task of discovering a suitable gene. Gene-specific evaluation functions can then be created that have better theoretical properties than a single evaluation function over all genes. This method is tested in the difficult double-pole balancing problem, showing that agents using gene-specific evaluation functions can create a successful control policy in 20 percent fewer trials than the best existing genetic algorithms. The method is extended to more distributed problems, achieving 95 percent performance gains over tradition methods in the multi-rover domain.

Agogino, Adrian↗

NASA NTRSDate not supplied

Effects of Simulated Microgravity and LINC-Complex on Chromosome 8 of Human Mammary Cells

Previously, human breast epithelial cells were studied under simulated microgravity to determine the role of the LINC-complex in maintaining nuclear morphology and gene expression. Data from that study was used in this project to understand how the LINC-complex influences gene expression at chromosome 8 under simulated microgravity. Differentially expressed genes found in clusters typically indicate a molecular relationship amongst them. Visual and data analyses (GO, pathways) identified 51 genes within clusters on chromosome 8 across various simulated microgravity conditions that did not share any molecular relationship. Additional results also determined that cells with a disrupted LINC-complex experienced more differential gene expression than the control, and that extended periods of simulated microgravity exacerbated differential gene expression in chromosome 8 of LINC-disrupted cells. These results suggest that the stressful simulated microgravity environment coupled with disrupted biological processes, such as in the case of the LINC-complex, can increase differential gene expression in chromosomes. Future studies where additional analyses are performed to further analyze gene expression of clusters under simulated microgravity could potentially elucidate the molecular relationship amongst affected genes.

Simulated microgravity↗

NASA NTRS1994

The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.

NASA Discipline Developmental Biology↗

NASA NTRSDate not supplied

MULTI-OMICS ANALYSIS OF THE IMPACT OF CHRONIC LOW-DOSE RADIATION AND HINDLIMB SUSPENSION ON MURINE BRAIN AND RETINA

The space environment includes hazards like radiation and microgravity which can adversely affect biological systems. We assessed multi-omics multi-tissue NASA GeneLab datasets where 6-month-old female mice were gamma irradiated (IR) and/or hindlimb unloaded (HLU) for 21 days. Whole transcriptome shotgun sequencing (RNA-Seq) and reduced representation bisulfite sequencing (RRBS) of brain and retina samples collected at 4 months post-exposure was performed to better characterize the retinal and neurological responses to spaceflight. We compared epigenomic and transcriptomic profiles within each exposure group for both tissue types to identify correlation (Pearson’s correlation test; p-value < 0.05) between gene expression and DNA methylation levels that may be related to transcriptional regulation. We then obtained genes with methylation-expression correlation that also showed differences in mean expression or dispersion between exposed and control groups (adjusted p-value < 0.25; relaxed to denote ‘hypothesis’) in the brain (37 genes in HLU, 4 in IR, and 156 in HLU+IR) or retina (92 genes in HLU, 1 in IR, and 55 in HLU+IR). Enriched Gene Ontology (GO) terms for these genes are listed in Table 1 for HLU and HLU+IR for both tissue types. No enriched terms and only a few genes were detected with IR-only exposure in the brain (Chmp1a, Limd1, Rab40b, Ubc) and retina (retinoblastoma binding protein Rbbp7). Cellular components related to synapse were enriched in both tissue types. Previous analysis of differentially expressed genes in the retina after 1 month of HLU+IR showed enrichment in the somatodendritic compartment of the neuron, which was also observed in the brain 4 months post-exposure. Interestingly, genes related to ubiquitination showed correlation between methylation and expression and were differentially expressed or dispersed in different exposure groups indicating that this pathway may play an important role in multi-stressor response (Figure 1). The current multi-omics and multi-tissue analysis interrogates the epigenomic and transcriptomic impacts of radiation and hindlimb unloading, in isolation and in combination, on the retina and the brain. The results provide insight into the adaptive response to individual spaceflight hazard analogs and their interplay, as well as hypotheses to be further tested for understanding spaceflight-induced neurological and vision effects.

Prachi Kothiyal↗

NASA NTRSDate not supplied

Transcriptomics-based Machine Learning Analysis Predicts Space-Exposed Murine Livers

Limited sample sizes, high data dimensionality, and sensitivity to technical and biological variability of next generation sequencing (NGS), typically limits machine learning (ML) approaches in spaceflight studies that include radiation effects. However, pooling smaller studies while addressing intra- and inter-study variabilities allows for ML predictive modeling. Here, integration methods were applied to whole transcriptome shotgun sequencing (RNA-seq) data from six mouse liver GeneLab datasets (GLDS) (n ranging from 6 to 39 samples) from with a total of 81 spaceflight and ground-control samples to determine top features (i.e. genes) relevant to spaceflight including the effect of radiation exposure. RNASeq counts were normalized for each study, then merged and scaled across all datasets. Data dimensionality was reduced using a minimum redundancy maximum relevance (MRMR) methodology. Redundancy and relevance were computed using the Pearson correlation and F-statistic, respectively. The top 100 MRMR features were used to predict spaceflight vs. ground-control samples using Random Forest (RF), Support Vector Machine (SVM), and Linear Discriminant Analysis (LDA) classifiers with 5-fold cross validation (CV). Principal component analysis (PCA) on the complete feature set versus the MRMR features shows separation between spaceflight samples and ground controls (Figure 1A). The ML-based gene sets were compared against differential gene expression results obtained with DESeq2 from individual GLDS. Using all features or randomly sampled subsets at matching set sizes with MRMR, a maximum classifier accuracy of 69% was shown on the test set over 5 folds. For all classifiers, CV training using at least the top 30 MRMR genes show minimum 89% accuracy and 0.95 AUC value on the test set over 5 folds (Figure 1B). Baseline set analysis on differentially expressed genes (DEGs) identified using padj ≤ 0.05 show 295 DEGs that overlap at least two studies and 13 DEGs that overlap three studies (Figure 1C). Set analysis between the top 100 MRMR features and the DEGs showed 47 genes that overlap at least one study and 24 genes that overlap two studies. Over-representation analysis showed overlapping biological processes related to fatty acid and lipid metabolism which may indicate these processes in the response to spaceflight stressors. MRMR feature selection for the selected ML methods improve performance relative to a classifier built on all features or randomly sampled subsets. Permutation feature importance within the decorrelated MRMR features showed concordance in feature ranking between ML methods. A challenge of applying ML methods across heterogeneous NGS data is accounting for signal:noise. Here, signal validation across studies was shown by intersecting sets between top MRMR genes and DEGs from DESeq2 analysis. Non-intersecting sets introduce opportunity to explore genes relevant to differentiating space flight exposed groups and implementing ML methods across existing NGS datasets may overcome sample size limitations.

Machine Learning↗

NASA NTRSDate not supplied

Transcriptomics-based Machine Learning (ML) Analysis Predicts Space-Exposed Murine Livers

Limited sample sizes, high data dimensionality, and sensitivity to technical and biological variability of next generation sequencing (NGS), typically limits machine learning (ML) approaches in spaceflight studies that include radiation effects. However, pooling smaller studies while addressing intra- and inter-study variabilities allows for ML predictive modeling. Here, integration methods were applied to whole transcriptome shotgun sequencing (RNA-seq) data from six mouse liver GeneLab datasets (GLDS) (n ranging from 6 to 39 samples) from with a total of 81 spaceflight and ground-control samples to determine top features (i.e. genes) relevant to spaceflight including the effect of radiation exposure. RNASeq counts were normalized for each study, then merged and scaled across all datasets. Data dimensionality was reduced using a minimum redundancy maximum relevance (MRMR) methodology. Redundancy and relevance were computed using the Pearson correlation and F-statistic, respectively. The top 100 MRMR features were used to predict spaceflight vs. ground-control samples using Random Forest (RF), Support Vector Machine (SVM), and Linear Discriminant Analysis (LDA) classifiers with 5-fold cross validation (CV). Principal component analysis (PCA) on the complete feature set versus the MRMR features shows separation between spaceflight samples and ground controls (Figure 1A). The ML-based gene sets were compared against differential gene expression results obtained with DESeq2 from individual GLDS. Using all features or randomly sampled subsets at matching set sizes with MRMR, a maximum classifier accuracy of 69% on the test set over 5 folds. For all classifiers, CV training using at least the top 30 MRMR genes show minimum 89% accuracy and 0.95 AUC value on the test set over 5 folds (Figure 1B). Baseline set analysis on differentially expressed genes (DEGs) identified using padj ≤ 0.05 show 295 DEGs that overlap at least two studies and 13 DEGs that overlap three studies (Figure 1C). Set analysis between the top 100 MRMR features and the DEGs showed 47 genes that overlap at least one study and 24 genes that overlap two studies. Over-representation analysis showed overlapping biological processes related to fatty acid and lipid metabolism which may indicate these processes in the response to spaceflight stressors. MRMR feature selection for the selected ML methods improve performance relative to a classifier built on all features or randomly sampled subsets. Permutation feature importance within the decorrelated MRMR features showed concordance in feature ranking between ML methods. A challenge of applying ML methods across heterogeneous NGS data is accounting for signal:noise. Here, signal validation across studies was shown by intersecting sets between top MRMR genes and DEGs from DESeq2 analysis. Non-intersecting sets introduce opportunity to explore genes relevant to differentiating space flight exposed groups and implementing ML methods across existing NGS datasets may overcome sample size limitations.

Machine Learning↗

NASA NTRSDate not supplied

Transcriptomics-based Machine Learning Analysis Predicts Space-Exposed Murine Livers

Machine Learning↗

NASA NTRS1995

Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

Non-NASA Center↗

NASA NTRS2005

Early immune response and regulation of IL-2 receptor subunits

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Non-NASA Center↗

NASA NTRS1999

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Rosenthal, Nadia A.↗

NASA NTRS1998

Assessment of changes in microbial community structure during operation of an ammonia biofilter with molecular tools

Biofiltration has been used for two decades to remove odors and various volatile organic and inorganic compounds in contaminated off-gas streams. Although biofiltration is widely practiced, there have been few studies of the bacteria responsible for the removal of air contaminants in biofilters. In this study, molecular techniques were used to identify bacteria in a laboratory-scale ammonia biofilter. Both 16S rRNA and ammonia monooxygenase (amoA) genes were used to characterize the heterotrophic and ammonia-oxidizing bacteria collected from the biofilter during a 102-day experiment. The overall diversity of the heterotrophic microbial population appeared to decrease by 38% at the end of the experiment. The community structure of the heterotrophic population also shifted from predominantly members of two subdivisions of the Proteobacteria (the beta and gamma subdivisions) to members of one subdivision (the gamma subdivision). An overall decrease in the diversity of ammonia monooxygenase genes was not observed. However, a shift from groups dominated by organisms containing Nitrosomonas-like and Nitrosospira-like amoA genes to groups dominated by organisms containing only Nitrosospira-like amoA genes was observed. In addition, a new amoA gene was discovered. This new gene is the first freshwater amoA gene that is closely affiliated with Nitrosococcus oceanus and the particulate methane monooxygenase gene from the methane oxidizers belonging to the gamma subdivision of the Proteobacteria.

NASA Discipline Life Support Systems↗

NASA NTRS2011

System Re-set: High LET Radiation or Transient Musculoskeletal Disuse Cause Lasting Changes in Oxidative Defense Pathways Within Bone

Six months post-IR, there were no notable changes in skeletal expression of 84 principal genes in the p53 signaling pathway due to low dose IR (0.5Gy), HU, or both. In contrast, numerous genes relevant to oxidative stress were regulated by the treatments, typically in a direction indicative of increased oxidative stress and impaired defense. IR and HU independently reduced (between 0.46 to 0.88 fold) expression levels of Noxa1, Gpx3, Prdx2, Prdx3, and Zmynd17. Surprisingly, transient HU alone (sham-irradiated) decreased expression of several redox-related genes (Gpx1,Gstk1, Prdx1, Txnrd2), which were not affected significantly by IR alone. Irradiation increased (1.13 fold) expression of a gene responsible for production of superoxides by neutrophils (NCF2). Of interest, only combined treatment with HU and IR led to increased expression levels of Ercc2, (1.19 fold), a DNA excision repair enzyme. Differences in gene expression levels may reflect a change in gene expression on a per cell basis, a shift in the repertoire of specific cell types within the tissue, or both. Serum nitrite/nitrate levels were elevated to comparable levels (1.6-fold) due to IR, HU or both, indicative of elevated systemic nitrosyl stress. CONCLUSIONS The magnitude of changes in skeletal expression of oxidative stress-related genes six months after irradiation and/or transient unloading tended to be relatively modest (0.46-1.15 fold), whereas the p53 pathway was not affected. The finding that many different oxidative stress-related genes differed from controls at this late time point implicates a generalized impairment of oxidative defense within skeletal tissue, which coincides with both profound radiation damage to osteoprogenitors/stem cells in bone marrow and impaired remodeling of mineralized tissue.

Kumar, Akhilesh↗