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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
NASA NTRS2015

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

ISS tools↗
NASA NTRS2014

WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015.

quantitative PCR↗
NASA NTRS2013

Conducting On-orbit Gene Expression Analysis on ISS: WetLab-2

WetLab-2 will enable expanded genomic research on orbit by developing tools that support in situ sample collection, processing, and analysis on ISS. This capability will reduce the time-to-results for investigators and define new pathways for discovery on the ISS National Lab. The primary objective is to develop a research platform on ISS that will facilitate real-time quantitative gene expression analysis of biological samples collected on orbit. WetLab-2 will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on orbit. WetLab-2 will significantly expand the analytical capabilities onboard ISS and enhance science return from ISS.

quantitative PCR↗
NASA NTRS2017

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

microgravity↗
NASA NTRS2004

Gene Expression Profile Analysis as a Prognostic Indicator of Normal Tissue Response to Simulated Space Radiations

This project was funded as a pilot project to determine the feasibility of using gene expression profiles to characterize the response of human cells to exposure to particulate radiations such as those encountered in the spaceflight environment. We proposed to use microarray technology to examine the gene expression patterns of a bank of well-characterized human fibroblast cell cultures. These fibroblast cultures were derived from breast or head and neck cancer patients who exhibited normal, minimal, or severe normal tissue reactions following low LET radiation exposure via radiotherapy. Furthermore, determination of SF2 values from fibroblasts cultured from these individuals were predictive of risk for severe late reactions. We hypothesized that by determining the expression of thousands of genes we could identify gene expression patterns that reflect how normal tissues respond to high Z and energy (HZE) particles, that is, that there are molecular signatures for HZE exposures. We also hypothesized that individuals who are intrinsically radiosensitive may elicit a unique response. Because this was funded as a pilot project we focused our initial studies on logistics and appropriate experimental design, and then to test our hypothesis that there is a unique molecular response to specific particles, in this case C and Fe, for primary human skin fibroblasts.

Story, Michael↗
NASA NTRS1998

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Non-NASA Center↗
NASA NTRS2001

Ecogenomics: Ensemble Analysis of Gene Expression in Microbial Communities

The hierarchical organization of microbial ecosystems determines process rates that shape Earth's environment, create the biomarker sedimentary and atmospheric signatures of life, and define the stage upon which major evolutionary events occurred. In order to understand how microorganisms have shaped the global environment of Earth and, potentially, other worlds, we must develop an experimental paradigm that links biogeochemical processes with ever-changing temporal and spatial distributions of microbial populations and their metabolic properties. Additional information is contained in the original extended abstract.

Sogin, Mitchell↗
NASA NTRS2016

Gene Expression and Structural Skeletal Responses to Long-Duration Simulated Microgravity in Rats

Spaceflight has deleterious effects on skeletal structure and function, specifically causingprofound loss in bone mass, density, and strength, as well as changes in expression levels of genes related to oxidative stress [Hyeon et al., Smith et al.]. It is known that bone resorption remains elevated after spaceflight and that bone density and strength fail to recover completely even years following spaceflight [Smith et al., Carpenter et al.]. However, our current understanding of the signaling pathways and molecular mechanisms that control bone loss and that link oxidative stress, bone resorption, and mechanical unloading of skeletal tissue is incomplete. Here, we aim to examine skeletal responses to simulated long-duration spaceflight on bone loss using the ground-based hindlimb unloading (HU) model in adult (9 months old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative-defense genes and pro-osteoclastogenic factors, showing progression and eventual plateau during long-term unloading, and that transient changes at early timepoints in these pathways precede skeletal adaptations to long-duration unloading. We will identify oxidativestress and bone resorption-related changes using global gene expression analysis (Affymetrix arrays) for both acute (within 14 days) and long-term timepoints (90 days). We will also use quantitative PCR to examine changes in expression of genes related to oxidative metabolism (e.g. Nrf2, SOD-1), bone turnover (resorption and formation markers, e.g. TRAP, osteocalcin respectively, SOST), and osteoclastogenesis (e.g. RANKL, OPG) at both early and late timepoints. We will then use detailed microarchitectural and structural analysis through microcomputed tomography to relate gene expression changes with structural changes in bone, expecting that plateaus in gene expression correlate with long-term changes in bone microarchitecture.

Rael, Victoria E.↗
NASA NTRSDate not supplied

NASA GeneLab Multi-study Visualization Portal

NASA GeneLab has helped advance the field of Space Biology by providing a public repository where researchers can store, share, analyze and visualize the results of space flight related omics experiments. The GeneLab data visualization portal allows any user, regardless of bioinformatics knowledge or access to computational resources, to interact with the experimental data, draw their own conclusions, and gain insights about the effects of space on living systems. These tools help democratize scientific research and foster the NASA Open Science initiative. The new multi-study feature of the GeneLab visualization platform allows users to mine study metadata from RNA sequencing (RNA-seq) experiments to identify samples of interest by filtering datasets based on organism, tissue, assay technology type, and/or factor. Once samples are selected from multiple datasets, users can combine and normalize the sample data, then utilize the visualization displays, including Principal Component Analysis (PCA) plots, to assess sample distributions. Finally, users can perform differential gene expression analysis on the combined data and visualize the results through PCA plots, Volcano plots, Pair plots, Heatmap, Ideogram and Gene Set Enrichment Analysis. All user-generated results and visualizations will be available for download. Here, we present a biological study using samples from multiple GeneLab RNA-seq datasets and analyzed using the multi-study visualization platform to demonstrate inter- and intra-study variability, as well as commonly differentially expressed genes between spaceflight and ground control conditions across datasets. This new feature opens a wide range of possibilities and opportunities for further development including combining other assay technology types and integration with batch effect correction techniques and machine learning applications. Overall, this tool allows users to increase the statistical power of individual experiments, validate hypothesis, identify patterns, and opens the door to new and exciting research.

space biology↗
NASA NTRSDate not supplied

Looking into Ocular Risks of Spaceflight through the Mouse Retina

Ocular alterations have been observed at anatomical levels in astronauts on long duration spaceflight missions, such as what would be required for missions to Mars. These alterations cause an array of signs which together constitute the Spaceflight-Associated Neuro-ocular Syndrome (SANS), one of the top risk priorities of the NASA Human Research Program. Not much is known about SANS at the cellular and molecular level, but studies in mice and rats have recently begun to yield observations on how the spaceflight environment might affect the eye’s biology. Preliminary data from shuttle mouse experiments, and more recently experiments on ISS, have shown changes in retinal physiology via histology and gene expression analysis. This study utilizes samples from the CASIS sponsored Rodent Research 8 Experiment (RRRM-1) tissue sharing opportunity, delivered to the ISS by SpaceX CRS-16 on 12/08/2018. Female BALB/cAnNTac mice were on the ISS for 45 days, while ground controls consisted ofa standard vivarium group and spaceflight habitat group. Here we investigate the molecular response of the mouse retina to identify genes and pathways affected by spaceflight conditions using histology and transcriptomic RNAseq data. This Differentially Expressed Gene (DEG) data was used for pathway analysis with Galaxy (Genelab) and Ingenuity Pathway Analysis (IPA). We identified pathways related to neuronal differentiation, cellular transport/movement, and wound healing. Some of the top DEGs have known relation to ophthalmic diseases. Though there were DEGs throughout the comparisons we tested, there was no clear effect of spaceflight. This could be due to sample processing, which required mice to be returned to Earth about a day before they were sacrificed, possibly allowing for readaptation affecting the retinal transcriptome. However, there was a clear effect of age, between the young (10-12 weeks) and old (32 weeks) groups, and between the baseline and end of experiment, about 46 days.

SANS↗
NASA NTRS2015

Wetlab-2 - Quantitative PCR Tools for Spaceflight Studies of Gene Expression Aboard the International Space Station

Wetlab-2 is a research platform for conducting real-time quantitative gene expression analysis aboard the International Space Station. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space. Currently, gene expression analyses of space flown biospecimens must be conducted post flight after living cultures or frozen or chemically fixed samples are returned to Earth from the space station. Post-flight analysis is limited for several reasons. First, changes in gene expression can be transient, changing over a timescale of minutes. The delay between sampling on Earth can range from days to months, and RNA may degrade during this period of time, even in fixed or frozen samples. Second, living organisms that return to Earth may quickly re-adapt to terrestrial conditions. Third, forces exerted on samples during reentry and return to Earth may affect results. Lastly, follow up experiments designed in response to post-flight results must wait for a new flight opportunity to be tested.

ISS research platform↗
NASA NTRSDate not supplied

Transcriptomic Analysis of Irradiated Mouse Retina Following Readaptation

Rodent models are used as analogs for studying the effects of spaceflight. NASA GeneLab provides access to relevant omics datasets generated from spaceflight and ground-based experiments allowing for additional retrospective analysis. In this study, we used GeneLab’s GLDS-203, a dataset generated by researchers at Loma Linda University to study the impact of prolonged unloading and/or low-dose radiation on mouse retina. We analyzed transcriptomics data from retina of mice irradiated with gamma-rays for 21 days followed by 7 days, 1 month, or 4 months of readaptation. We obtained raw gene counts from GeneLab and performed differential gene expression analysis after data normalization. For each of the three timepoints, we performed differential expression analysis to compare transcriptional profiles for retina from irradiated vs. non-irradiated (controls) mice, all exposed to gravity. We observed the highest number of differentially expressed genes at 7 days, followed by 1 month and 4 months. Enrichment analysis showed top pathways (adjusted p-value < 0.05) were related to transport along microtubule and photoreceptor cell development in the 7-day readaptation group. Fewer significantly enriched pathways were observed for the 1-month group and included mRNA metabolic processes and neuron differentiation. No significantly enriched pathways were found in the 4-month group. The Gene Ontology biological processes common between the 7 days and 1-month groups include visual perception, synapse organization, and perception of light stimulus. This analysis is part of a larger effort to characterize the molecular mechanisms involved in retinal readaptation following radiation exposure. Future analyses will include other related retina datasets in GeneLab repository to assess whether gene expression patterns are consistent across different study cohorts.

Prachi Kothiyal↗
NASA NTRSDate not supplied

Differential Gene Expression in A Cross-Feeding Two-Species Model Microbial Community Under Simulated Microgravity and Deep-Space Radiation

A long-term goal of space biology is to understand interspecies microbial interactions in space. Presently, little is known about the combined effect of microgravity and ionizing radiation on bacterial community response when species are interdependent through exchange of metabolites in fluid medium (cross-feeding). Microgravity is expected to slow interspecies mass transfer and growth in cross-feeding communities in the low-shear, diffusion-limited environment, while ionizing radiation may influence stress response to direct (DNA damage) and indirect damage (ROS). Using a well-understood, two-species (Escherichia coli and Salmonella enterica) microbial community engineered to be a model for studying cross-feeding, we simulated galactic cosmic rays (GCRsim) and microgravity to test the hypothesis: exposure to ionizing radiation causes cell damage or stress, altering transcriptomic community responses in metabolically interdependent cells, which is exacerbated by microgravity. We expect to see differential gene expression between cross-feeding and non-cross-feeding communities. We measured GCRsim effects on growth and gene expression in well-mixed versus simulated-microgravity conditions and in cross-feeding and non-cross-feeding medium. Microbial cultures were inoculated into liquid medium in rotating wall vessels (RWV) with different rotation rates: 5 RPM (simulated microgravity) and 50 RPM (well-mixed). The E. coli-S. enterica consortium, under simulated microgravity, were exposed to 500 mGy of Simplified 5-ion Galactic Cosmic Ray Simulation for 2 hours at Brookhaven National Lab. We harvested samples 40 minutes after irradiation for extraction and sequencing (NASA GeneLab). Here we present the differential gene expression analysis results, which reveal altered transcriptomic community responses, even where growth rate differences are not observed. Gene expression of these actively metabolizing microbial communities in GCRsim may illuminate molecular mechanisms of microbial interactions in space. Understanding how microbial community gene expression, metabolism, and other cellular processes are influenced by spaceflight stressors can inform the use of microbes in human life support for low Earth orbit missions and beyond.

microgravity↗
NASA NTRSDate not supplied

Histological and Transcriptomic Analysis of Spaceflight-Induced Ocular Changes in the Mouse Retina

Anatomical changes have been observed in astronauts’ eyes after long duration spaceflight missions. These alterations can lead to visual impairment which in part constitutes the spaceflight-associated neuroocular syndrome (SANS), one of the top risk priorities for deep space missions. The HRP Systems Biology (SysBio) Translation Project will apply systems biology approaches utilizing current human physiological spaceflight data, molecular results from rodents, and future research with a multi-level, multi-system, and multi-species perspective to augment the existing research plan to resolve the SANS risk. Not much is known about SANS at the cellular and molecular level, but studies in mice and rats have recently begun to determine how spaceflight might affect the biology of the eye. Preliminary studies of mice that flew on the Space Shuttle, and more recently the International Space Station (ISS), have shown changes in retinal physiology as assessed by histology and gene expression analysis. The study presented here obtained samples from the CASIS sponsored Rodent Research 8 Experiment delivered to the ISS by SpaceX CRS-16 on 12/08/2018. Female BALB/cAnNTac mice flew on the ISS for 45 days, while ground controls were housed in a standard vivarium or animal enclosure module. Sacrifice and sample acquisition occurred once mice returned to Earth, possibly allowing for readaptation affecting retinal homeostasis. We applied standard transcriptomic (RNAseq) and histological approaches to characterize genes and pathways in the mouse retina affected by spaceflight or age. The differentially expressed gene (DEG) data was analyzed using Galaxy (GeneLab) and Ingenuity Pathway Analysis. Significant DEGs between flight and ground samples were relatively few but biologically meaningful. Pathways identified related to neuronal differentiation, cellular transport/movement, and wound healing. Age effects were detected between the young (10–12 weeks) and old (32 weeks) groups and between the baseline and end of experiment (~46 days). The biological relevance of specific DEGs were confirmed through immunohistochemical evaluation using fixed histological sections of the eye from four flight group mice and four habitat control mice. Staining was performed specific for synaptophysin, glial fibrillary acidic protein (GFAP), and neurofilament in the retinal periphery, equator, and peripapillary regions. For synaptophysin staining, the innerplexiform and outerplexiform layers were scored; for GFAP staining, Mueller cells and perivascular astrocytes were scored. Results show flight samples typically had more staining of GFAP and neurofilament while, conversely, the habitat control group had more staining of synaptophysin.

C. Perez↗
NASA NTRS2013

Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.

The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is currently designing a module that will lyse the cells and extract RNA of sufficient quality for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. Current testing focuses on two promising commercial products and chemistries that allow for RNA extraction with minimal complexity and crew time.

quantitative PCR↗
NASA NTRSDate not supplied

Batch Effect Correction Methods for NASA GeneLab Transcriptomic Datasets

RNA sequencing (RNA-seq) data from space biology experiments promise to yield invaluable insights into the effects of spaceflight on terrestrial biology. However, sample numbers from each study are low due to limited crew availability, hardware, and space. To increase statistical power, spaceflight RNA-seq datasets from different missions are often aggregated together. However, this can introduce technical variation or "batch effects", often due to differences in sample handling, sample processing, and sequencing platforms. Several computational methods have been developed to correct for technical batch effects, thereby reducing their impact on true biological signals. In this study, we combined 7 mouse liver RNA-seq datasets from NASA GeneLab (part of the NASA Open Science Data Repository) to evaluate several common batch effect correction methods (ComBat and ComBat-seq from the sva R package, and Median Polish, Empirical Bayes, and ANOVA from the MBatch R package). We quantitatively evaluated the ability of these methods to correct for technical batch variables in space biology RNA-seq data using the following criteria: BatchQC, principal component analysis, dispersion separability criterion, log fold change correlation, and differential gene expression analysis. Each batch variable / correction method combination was then assessed using a custom scoring approach to identify the optimal correction method for the combined dataset, by geometrically probing the space of all allowable scoring functions to yield an aggregate volume-based scoring measure. Finally, we describe the way in which the GeneLab multi-study analysis and visualization portal will allow users to examine the presence or absence of batch effects using multiple metrics. If the user chooses to perform batch effect correction, the scoring approach described here can be implemented to identify the optimal correction method to use for their specific combined dataset prior to analysis.

Lauren M. Sanders↗
NASA NTRS2015

Oxidative Stress and Autophagy Responses of Osteocytes Exposed to Spaceflight-like Radiation.

Weightlessness and radiation, two of the unique elements of the space environment, causes a profound decrement in bone mass that mimics aging. This bone loss is thought to result from increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. Our current understanding of the signaling factors and mechanisms underlying bone loss is incomplete. However, it is known that oxidative stress, characterized by the excess production of free radicals, is elevated during radiation exposure. The goals of this study is to examine the response of osteocytes to spaceflight-like radiation and to identify signaling processes that may be targeted to mitigate bone loss in scenarios of space exploration, earth-based radiotherapy and accidental radiation exposure. We hypothesize that (1) oxidative stress, as induced by radiation, decreases osteocyte survival and increases pro-osteoclastogenic signals and that (2) autophagy is one of the key cellular defenses against oxidative stress. Autophagy is the process by which cellular components including organelles and proteins are broken down and recycled. To test our hypothesis, we exposed the osteocyte-like cell line, MLO-Y4, to 0.5, 1, and 2 Gy of simulated space radiation (Iron-56 radiation at 600 MeV/n) and assessed cell numbers, cell growth-associated molecules as well as markers of autophagy and oxidative stress at various time points post-irradiation. We observed a reduction in cell numbers in the groups exposed to 1 and 2 Gy of Iron-56 radiation. Collectively, flow cytometry and gene expression analysis revealed that radiation caused a shift in cell cycle distribution consistent with growth arrest. Compared to sham-treatment, 2 Gy of Iron-56 increased FoxO3, SOD1, and RANKL gene expression yet unexpectedly decreased LC3B-II protein levels at 4 and 24 hours post-IR. Taken together, these findings suggest that simulated space radiation invoke antioxidant, pro-osteoclastogenic, and growth arrest responses in osteocytes. The implications of reduced autophagy flux at the time points examined remain to be elucidated.

Biological Sciences↗