The Cosmic Ray Exposure Sequencing Science (CRESS) payload system is a proof of concept experiment to assess the genomic impact of space radiation on seeds. CRESS was designed as a secondary payload for the December 2016 high-altitude high-latitude and long-duration balloon flight carrying the Boron And Carbon Cosmic Rays in the Upper Stratosphere (BACCUS) experimental hardware. Investigation of the biological effects of Galactic Cosmic Radiation (GCR) particularly those of ions with High-Z and Energy (HZE) is of interest due to the genomic damage this type of radiation inflicts. The biological effects of upper-stratospheric mixed radiation above Antarctica (ANT) were sampled using Arabidopsis thaliana seeds and were compared to those resulting from a controlled simulation of GCR at Brookhaven National Laboratory (BNL) and to laboratory control seed. The payload developed for Antarctica exposure was broadly designed to 1U CubeSat specifications (10cmx10cmx10cm <1.33kg) maintained 1 atm internal pressure and carried an internal cargo of four seed trays (about 580,000 seeds) and twelve CR-39 Solid-State Nuclear Track Detectors (SSNTDs). The irradiated seeds were recovered sterilized and grown on Petri plates for phenotypic screening. BNL and ANT M0 seeds showed significantly reduced germination rates and elevated somatic mutation rates when compared to non-irradiated controls with the BNL mutation rate also being significantly higher than that of ANT. Genomic DNA from mutants of interest was evaluated with whole-genome sequencing using PacBio SMRT technology. Sequence data revealed the presence of an array of genome structural variants in the genomes of M0 and M1 mutant plants.

Understanding the molecular mechanisms by which plants sense and adapt to changes in the space environment is essential for generating plants that are better adapted to withstand space flight microgravity and other adverse conditions encountered in space. The objective of our spaceflight experiment x93Plant Signaling in Microgravity x94 (carried out on the International Space Station ISS) was to compare transcript profiles of wild type and transgenic InsP 5-ptase plants with compromised InsP3 signaling. The transgenic Arabidopsis plants constitutively express the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) an enzyme that specifically hydrolyzes the lipid-derived second messenger inositol 1,4,5-trisphosphate (InsP3). These transgenic plants exhibit normal growth and morphology; however their responses to environmental stimuli including gravity and drought are altered. Seedlings were grown for 5 days under continuous light in experimental containers placed in the European Modular Cultivation system (EMCS) onboard the ISS. The EMCS consists of two rotors within a controlled chamber allowing for a x931g x94 control in space. After sample retrieval from the ISS RNA was isolated from shoot and root tissue and subjected to RNA sequencing. Two-way comparisons of micro g versus x931 x94g have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings.

Mice were flown onboard STS-135 and returned to Earth for analysis. Cerebellums were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Samples were shipped to UCI Genomics High Throughput Facility for analysis.

This study describes the transcriptional response of P. aeruginosa PAO1 to low-Earth orbit environmental conditions. Our aim was to assess whether the microgravity environment of spaceflight could induce virulence traits in P. aeruginosa. To this end P. aeruginosa cultures were grown in space and the expression profile was compared with ground control samples (both in biological triplicate). Characterization of bacterial behavior in the microgravity environment of spaceflight is of importance towards risk assessment and prevention of infectious disease during long-term missions. Further this research field unveils new insights into connections between low fluid-shear regions encountered by pathogens during their natural infection process in vivo and bacterial virulence. This study is the first to characterize the global transcriptomic and proteomic response of an opportunistic pathogen that is actually found in the space habitat Pseudomonas aeruginosa. Overall P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins with Hfq identified as a global transcriptional regulator in the response to this environment. Since Hfq was also induced in spaceflight-grown Salmonella typhimurium Hfq represents the first spaceflight-induced regulator across the bacterial species border. The major P. aeruginosa virulence-related genes induced in spaceflight conditions were the lecA and lecB lectins and the rhamnosyltransferase (rhlA) involved in the production of rhamnolipids. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data of this organism grown in microgravity-analogue conditions using the rotating wall vessel (RWV) bioreactor technology. Interesting similarities were observed among others with regard to Hfq regulation and oxygen utilization. While LSMMG-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism P. aeruginosa cultured in spaceflight adopted an anaerobic mode of growth in which denitrification was presumably most prominent. Differences in hardware between spaceflight and LSMMG experiments in combination with more pronounced low fluid shear and mixing in spaceflight when compared to LSMMG conditions were hypothesized to be at the origin of these observations. Collectively our data suggest that spaceflight conditions could induce the transition of P. aeruginosa from an opportunistic organism to potential pathogen results that are of importance for infectious disease risk assessment and prevention both during spaceflight missions and in the clinic.

Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms underpinning the effects of microgravity on the immune system. The goal was to validate the Drosophila model for understanding alterations of innate immune responses in humans due to spaceflight. Five containers of flies with ten female and five male fruit flies in each container were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours with a new generation reared in microgravity. RNA was extracted on the day of shuttle landing from whole body animals (3rd instar larvae and adults) hybridized to Drosophila 2.0 Affymetrix genome arrays and the expression level of all genes was normalized against the gene expression level from the corresponding developmental stage animals raised on ground. Spaceflight altered the expression of larval genes involved in the maturation of plasmatocytes (macrophages) and their phagocytic response as well as the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria and of lysozymes antimicrobial peptide pathway and immune stress genes hallmarks of humoral immunity. Larval microarrays (FL 6 samples) are based on RNA extracted from 6 independent sets of 50 mid 3rd instar larvae reared in microgravity and collected on the day of landing after 12 days and 18.5 hours on the space shuttle and the same number of control larvae raised on ground (GL 6 samples). Adults microarrays (F1 3 samples) are based on RNA from 3 sets of 20 adult females each that emerged during spaceflight and within 4 hours of landing and the same number of adult females from the corresponding ground control containers (G1 3 samples).

These investigations studied the fundamentals of how plants perceive gravity and develop in microgravity. It informs how gene regulation is altered by spaceflight conditions.

Gene expression profile of two-week-old etiolated Arabidopsis seedlings under microgravity on board space flight BRIC16 were compared with ground grown control in both wild-type and act2-3 mutant plants.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi and tongue (as a new potential in-flight negative control) adaptation to microgravity at global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice (False Discovery Rrate < 0,05 and fold change < -2 and > +2) while only a small number of genes were found differentially regulated in tongue muscle ( BF vs. BG = 27 genes). Overall Design: C57BL/N6 mice were randomly divided in 3 groups: Bion Flown (BF) mice flown aboard the Bion M1 biosatellite in microgravity environment for 30 days; Bion Ground (BG) mice housed in the same habitat of flown animals but exposed to earth gravity; and Flight Control (FC) mice housed in a standard animal facility.

We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b CD31 CD44 Ly6C Ly6G F4/80 Mac2 c-Fos as well as c-Fms. In addition RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b CD31 F4/80 Mac2 Ly6C and c-Fos. However there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.

Female C57BL/6CR mice were flown onboard STS-135 for 13 days and returned to Earth for analysis. Livers were collected within 3-4 hours of landing and snap frozen in liquid nitrogen. Liver tissue samples that were used for microarray analysis for GLDS-25 were provided to GeneLab. GeneLab extracted RNA added ERCC control spike-in to the samples and performed RNA-Seq analysis.