The wild type Col-0 plants and the Sku-6 mutant plants of the Col-0 background were germinated on ISS or on the ground and the gene expression profiles in roots at 4 days or 8 days were established. The Sku6 gene (At2g03680) codes for a protein that localizes to microtubule within the cortical array and putatively acting as an intramolecular linker. The development of Col-0 and Sku-6 plants on orbit differs from that on the ground as demonstrated by the comparison of the gene expression profiles between 4 days old to 8 days old plant in the two environments. However the development of Sku-6 mutant plants also differs from Col-0 at either age and in either environment suggesting the role of the genetic background in these developmental decisions. The 4 days old Sku-6 roots in orbit engaged more genes than the 4 days old Col-0 roots in orbit but also more than the 4 days old Sku-6 roots on the ground. Overall the 4 days old roots differentially expressed more genes in spaceflight relative to ground than the 8 days old roots of either genotype.

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR) and in particular the high linear energy transfer (LET) heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation and primarily affected sites in open chromatin regions including enhancers promoters and edges ( shores ) of CpG islands. The 28Si ion-exposure had mixed effects inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments whereas X rays induced mostly hypomethylation primarily at sites in gene bodies and intergenic regions. Significantly the methylation status of 56Fe ion irradiation sensitive sites but not those affected by X ray or 28Si ions could discriminate tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus high LET radiation exposure leaves a lasting imprint on the epigenome and affects sites relevant to human lung cancer. The 56Fe ion signature may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space. Genome wide DNA methylation profiling of normal human bronchial epithelial cells irradiated with varying doses of 28Si-ion radiation ( 300 MeV/u at 0 0.3 1.0 Gy) 56Fe-ion radiation (600 MeV/u at 0 0.1 0.3 1.0 Gy) or X rays (320 kV at 0 1.0 Gy). Triplicate control and irradiated samples were incubated and sampled at 4 timepoints between 2 and 62 days. The Illumina Infinium 450k Human DNA methylation Beadchip was used to obtain DNA methylation profiles across >485,000 CpGs from collected samples. Samples include: 56Fe ions 4 doses x 4 time points x 3 replicates (4 removed in QC) = 44 samples; 28Si ions = 3 doses x 4 time points x 3 replicates = 36 samples; X ray 2 doses x 4 time points x 3 replicates (2 removed in QC)= 22 samples. Overall design: Bisulphite converted DNA from the 102 samples were hybridized to the Illumina Infinium 450k Human Methylation Beadchip.

We investigated differentially regulated genes in human myelomonocytic U937 cells in 20s and 5min microgravity and in hypergravity and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. Overall design: Human myelomonocytic U937 cells were exposed to altered gravity during a parabolic flight.

We investigated differentially regulated genes in human myelomonocytic U937 cells in 20s and 5min microgravity and in hypergravity and compared expression profiles to identify potential gravity-regulated genes and adaptation processes. Overall design: Human myelomonocytic U937 cells were exposed to altered gravity during a sounding rocket mission.

Controlled hypobaria presents biology with an environment that is never encountered in terrestrial ecology yet the apparent components of hypobaria are stresses typical of terrestrial ecosystems. High altitude for example presents terrestrial hypobaria always with hypoxia as a component stress since the relative partial pressure of O2 is constant in the atmosphere. Laboratory-controlled hypobaria however allows the dissection of pressure effects away from the effects typically associated with altitude in particular hypoxia as the partial pressure of O2 can be varied. In this study whole transcriptomes of plants grown in ambient (97 kPa/pO2 = 21 kPa) atmospheric conditions were compared to those of plants transferred to five different atmospheres of varying pressure and oxygen composition for 24 h: 50 kPa/pO2 = 10 kPa 25 kPa/pO2 = 5 kPa 50 kPa/pO2 = 21 kPa 25 kPa/pO2 = 21 kPa or 97 kPa/pO2 = 5 kPa. The plants exposed to these environments were 10 day old Arabidopsis seedlings grown vertically on hydrated nutrient plates. In addition 5 day old plants were also exposed for 24 h to the 50 kPa and ambient environments to evaluate age-dependent responses. The gene expression profiles from roots and shoots showed that the hypobaric response contained more complex gene regulation than simple hypoxia and that adding back oxygen to normoxic conditions did not completely alleviate gene expression changes in hypobaric responses.

Here in this study we systematically examined the patterns of DNA methylation and hydroxy-methylation with its functional implications in gene regulation for the cultured TK6 lymphoblastoid cells upon exposure to micro-gravity conditions. The results reported here indicate that simulated microgravity alters methylation patterns in a limited way and subsequently the expression of genes involved in stress response like ATF3 FBXO17 MAP3K13 and VCL in TK6 cells. Overall design: Examination of 2 different DNA modifications with 2 replicates each for 1 cell type.

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed and even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation. CARA Seed Lines and Planting: Three seed lines Wild-Type Wassilewskija (Ws) Columbia-0 (Col-0) and Col-0 PhyD (phyD) Mutants were tested for viability sterility and ability to maintain dormancy before the launch. Tested batches of seeds were planted on phytagel plates as one genotype per plate for gene expression analysis in replicates of three. One set was planted for the flight and one for ground control. The plates were wrapped such that every surface of the plate was covered by two layers of Duvetyn Black-Out cloth (Seattle Fabrics) (Sng et al 2014). The plates were stored 4 xb0 C until launch and was then launched in a cold-stow bag to maintain the plates at 4 xb0 C until integration and activation on the ISS. On Orbit Operations and harvest: The dormant plates were activated on station by removing the Black-Out cloth wrapping 12 days after launch. The plates were then placed on a fabric that was mounted in the US Laboratory module on the wall adjoining the MELFI freezer and secured using Velcro. The plants were allowed to grow on orbit for 11 days; some in the ambient light of ISS and some in the dark. The dark-grown plates were first activated by exposing the seeds to light for 4 hours and then re-wrapped in Black-Out cloth for the duration of the growth period. A corresponding set of seedlings were grown as ground control in KSC. At 11 days seedlings were photographed harvested into KFT containing RNAlater solutions and returned for post-flight analysis. All plates were harvested into KFTs with their counterpart (e.g. Light 1 was harvested with Dark 1). Once the plants were placed in the KFTs the KFT was actuated with RNAlater to preserve the sample. At 24 hours post-harvest KFTs were then transferred to MELFI the -32 xb0C freezer. Following SpaceX-3 splashdown in the Pacific Ocean the KFTs transferred to the Cold Stowage charter plane at

Controlled hypobaria presents biology with an environment that is never encountered in terrestrial ecology yet the apparent components of hypobaria are stresses typical of terrestrial ecosystems. High altitude for example presents terrestrial hypobaria always with hypoxia as a component stress since the relative partial pressure of O2 is constant in the atmosphere. Laboratory-controlled hypobaria however allows the dissection of pressure effects away from the effects typically associated with altitude in particular hypoxia as the partial pressure of O2 can be varied. In this study whole transcriptomes of plants grown in ambient (97 kPa/pO2 = 21 kPa) atmospheric conditions were compared to those of plants transferred to five different atmospheres of varying pressure and oxygen composition for 24 h: 50 kPa/pO2 = 10 kPa 25 kPa/pO2 = 5 kPa 50 kPa/pO2 = 21 kPa 25 kPa/pO2 = 21 kPa or 97 kPa/pO2 = 5 kPa. The plants exposed to these environments were 10 day old Arabidopsis seedlings grown vertically on hydrated nutrient plates. In addition 5 day old plants were also exposed for 24 h to the 50 kPa and ambient environments to evaluate age-dependent responses. The gene expression profiles from roots and shoots showed that the hypobaric response contained more complex gene regulation than simple hypoxia and that adding back oxygen to normoxic conditions did not completely alleviate gene expression changes in hypobaric responses.