Spaceflight results in a number of adaptations to skeletal muscle including atrophy and shifts towards faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations microarray expression analysis was performed on gastrocnemius from mice flown on the STS-108 shuttle flight (11 days 19 hours) versus mice maintained on earth for the same period. Additionally to identify changes that were due to unloading and reloading microarray analyses were conducted on calf muscle from ground-based mice subjected to hindlimb suspension (12 days) and mice subjected to hindlimb suspension plus a brief period of reloading (3.5 hours) to simulate the time between landing and sacrifice of the spaceflight mice.

In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a ground-based Rotating Wall Vessel (RWV) bioreactor. Our results show that 42 miRNAs were differentially expressed in MMG-incubated PBLs compared with 1g-incubated ones. Among these miR-9-5p miR-9-3p miR-155-5p miR-150-3p and miR-378-3p were the most dysregulated. To improve the detection of functional miRNA-mRNA pairs we performed gene expression profiles on the same samples assayed for miRNA profiling and we integrated miRNA and mRNA expression data. The functional classification of miRNA-correlated genes evidenced significant enrichments in the biological processes of immune/inflammatory response signal transduction regulation of response to stress regulation of programmed cell death and regulation of cell proliferation. We identified the correlation between miR-9-3p miR-155-5p miR-150-3p and miR-378-3p expression with that of genes involved in immune/inflammatory response (eg. IFNG and IL17F) apoptosis (eg. PDCD4 and PTEN) and cell proliferation (eg. NKX3-1 and GADD45A). Experimental assays of cell viability and apoptosis induction validated the results obtained by bioinformatics analyses demonstrating that in human PBLs the exposure to reduced gravitational force increases the frequency of apoptosis and decreases cell proliferation. microRNA expression profiling were carried out on total RNA extracted from PBLs of twelve healthy donors at the end of 24h-incubation time in MMG and in 1g conditions. Analyses were performed by using the Human miRNA Microarray kit (V2) (Agilent Technologies) that allows the detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs. By comparing the expression profile of MMG-incubated vs. 1g-incubated PBLs of the same donor we found 42 differentially expressed miRNAs 25 up-regulated and 17 down-regulated.

Space radiations and microgravity both could cause DNA damage in cells but the effects of microgravity on DNA damage response to space radiations are still controversial. A mRNA microarray and microRNA microarray in dauer larvae of Caenorhabditis elegans (C. elegans) that endured space xef xac x82ight environment and space radiations environment during 16.5-day Shenzhou-8 space mission were performed. In our study wild type and ced-1 mutant strains of C.elegans endured three conditions during shenzhou-8 spaceflight mission including spaceflight static condition(ss) spaceflight 1-g centrifugal condition(sc) and ground control condition(gc). Limited to the quantity of worm samples we performed technical-repeat test but not sample-repeat test.Accordingly xef xbc x8csix miRNA microarrays were performed.

Spaceflight is known to affect immune cell populations. In particular splenic B-cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after spaceflight an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires. We can now identify immunoglobulin gene- segment usage junctional regions and modifications that contribute to specificity and diversity. Due to limitations on the International Space Station alternate sample collection and storage methods must be employed. Our group compared Illumina MiSeq xc2 xae sequencing data from multiple sample preparation methods in normal C57Bl/6J mice to validate that sample preparation and storage would not bias the outcome of antibody repertoire characterization. In this report we also compared sequencing techniques and a bioinformatic workflow on the data output when we assessed the IgH and Ig xce xba variable gene usage. Our bioinformatic workflow has been optimized for Illumina HiSeq xc2 xae and MiSeq xc2 xae datasets and is designed specifically to reduce bias capture the most information from Ig sequences and produce a data set that provides other data mining options.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing and spatially resolved transcriptional profiling data from cerebellums from 5 WT flight animals, and 5 WT ground control animals. Cerebellums from the right hemisphere were embedded and cryosectioned. Cryosections were either processed for bulk RNA sequencing or placed on gene expression arrays, stained and imaged. Imaging was followed by tissue permeabilization to release mRNA molecules from cells for capture onto the array surface. Subsequently, spatial transcriptomics libraries were prepared and sequenced.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

Transforming growth factor beta-1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR) both a carcinogen and a therapeutic agent induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/ml) or double-treated. All double-treated (IR+TGFbeta) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin beta-catenin and ZO-1 remodeling of the actin cytoskeleton and increased expression of mesenchymal markers N-cadherin fibronectin and vimentin. Furthermore double-treatment increased cell motility promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT even though IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this Erk phosphorylation were transiently induced by irradiation persisted in irradiated cells treated with TGFbeta and treatment with U0126 a Mek inhibitor blocked the EMT phenotype. Together these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression. Experiment Overall Design: Nonmalignant human mammary epithelial MCF10A cells (passages 106 and 108) were seeded at cloning density in 35mm dishes (10^5 cells/dish). Cell culture medium consisted of 3ml/dish of MGEM serum free medium (Cambrex Inc.) supplemented or not with 400pg/ml recombinant Transforming Growth Factor-beta. Cells were irradiated or not 5h post plating using 160 KV X-ray with a total dose of 2Gy. Sham IR-treated TGFbeta-treated and double-treated (IR+TGFbeta) MCF10A cells were harvested 8 days post-IR. Briefly cells were washed with PBS denatured in Trizol scraped off the dish and subjected to chloroform extraction. After centrifugation the upper phase was precipitated with an equal volume of isopropanol. RNA precipitates were resuspended in RNase free water and further purified on RNeasy columns (Qiagen Germany). RNA quality was assessed on an Agilent Bio-Analyzer. The dataset analyzed by microarray included biological duplicates for each treatment in two independent experiments and three sham treated samples. Microarray data were generated at the Lawrence Berkeley National Laboratory Molecular Profiling Laboratory (http://hta.lbl.gov) using a high-throughput automated GeneChip system (Affymetrix). Briefly target preparation HT_HG-U133A array plate hybridization setup washing and staining were performed on an Affymetrix robotic system (GCAS) using version 2.1 protocols. Scanning (protocol version 2.2.09) was performed on a CCD-based high throughput scanner (Affymetrix). Samples were analyzed and clustered with the (UNO) One Color GenetrafficTM software version 3.2-12 (Iobion Informatics LLC Stratagene La Jolla CA). Genes whose expression was specifically altered by treatment were defined as those in which dye ratio was more than 1.75-fold ( mean log2ratio >0.8) from baseline in at least three out of the four treated samples compared to the three sham samples. Significance analysis tests (p<0.05) were performed using Excel between sham samples and either IR TGFbeta or TGFbeta+IR samples.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing data from left cerebral hemispheres from 4 WT flight animals and 5 WT ground control animals, and single nuclei transcriptomics and epigenomics data from left cerebral hemispheres from 5 WT flight animals, and 5 WT ground control animals.

The objective of the Rodent Research-10 mission (RR-10) was to investigate how spaceflight affects the cellular and molecular mechanisms of normal bone tissue regeneration in space. To this end, ten (10) 14-15 weeks-old female B6129SF2/J Wild Type (WT), and ten (10) 14-15 weeks-old female B6;129S2-Cdkn1atm1Tyj/J (p21-null) mice received a pre-flight subcutaneous injection of the bone marker (Alizarin Red), and were then delivered to the ISS aboard SpaceX-21. At 7 days before euthanasia, all 20 mice received an intraperitoneal (IP) injection with a bone formation marker (Calcein). At 48 +/- 2 hours before euthanasia, all 20 mice received an IP injection with a second dose of Calcein as well as a cell proliferation marker (BrdU). Then, following 28-29 days in microgravity, the Flight mice were euthanized. Following removal of hindlimbs, carcasses were wrapped in aluminum foil, preserved in the CryoChiller, and stored at -80 C or colder until return to Earth. In addition to the Flight group, three ground control groups were also part of the study: Basal (representing the pre-launch state), Vivarium (standard vivarium housing for the same duration of time as flight), and Ground (flight habitat in the International Space Station Environment Simulator, ISSES). Twenty mice (10 of each strain) were included in each of these control groups (except Vivarium which included 12 of each strain). These were treated, euthanized and processed on the same schedule and in the same manner as the flight samples. This study includes bulk RNA sequencing ribodepleted gene expression data from 10 Basal animals (5 WT and 5 p21-null), 9 Flight animals (4 WT and 5 p21-null), 10 Ground animals (5 WT and 5 p21-null), and 10 Vivarium animals (5 WT and 5 p21-null).

The purpose of the present work was to examine gene expression patterns in a rat keratinocyte line exposed to a 56Fe ion beam Experiment Overall Design: The cells were exposed to 1.01 geV/nucleon 56Fe ions generated by the NASA Space Radiation Laboratory facility. Data from Affymetrix rat microarrays (RAT 230_2) were processed by BRB ArrayTools 3.3.0 software and the Gene Ontogeny (GO) database was utilized to categorize significantly responding genes.