Arabidopsis were grown on horizontal or vertical clinostat for 4 8 or 12 days. Seedlings on horizontal clinostat were in simulated microgravity and seedlings on vertical clinostat are considered as a control. Comparison was made between plants grown on simulated microgravitry and vertical position. 6 dye-swap - treated vs untreated comparison

Arabidopsis were grown on horizontal or vertical clinostat for 4 8 or 12 days. Seedlings on horizontal clinostat were in simulated microgravity and seedlings on vertical clinostat are considered as a control. Comparison was made between plants grown on simulated microgravitry and vertical position. 6 dye-swap - treated vs untreated comparison

Understanding the possible impact of potential confounding factors is necessary for any approach to radiation biodosimetry. Potential confounding factors have not been fully addressed for gene expression-based biodosimetry approaches such as we are developing. To begin addressing this need we have used an ex vivo irradiated peripheral blood cell model to investigate the potential effect of smoking on the global radiation gene expression response and looked for genes that respond to radiation differently in smokers and non-smokers and also in males and females. The results indicate that only a small number of genes may be significantly confounded by either factor supporting the idea of developing peripheral blood gene expression strategies for radiation biodosimetry. Blood from each of 24 different donors was exposed to four doses of ionizing radiation (0 0.1 0.5 or 2 Gy) and analyzed using single-color microarray hybridization. The donors represented equal numbers of male and female smokers (1 or more packs a day) and non-smokers. There are 95 data sets in the study as the sample from one of the female smokers exposed to 2 Gy was lost.

Genome-wide transcriptional profiling shows that reducing gravity levels in the International Space Station (ISS) causes important alterations in Drosophila gene expression. However simulation experiments on ground without space constraints show weaker effects than space environment. A global and integrative analysis using the gene expression dynamics inspector (GEDI) self-organizing maps reveals a subtle response of the transcriptome using different populations and microgravity and hypergravity simulation devices. These results suggest that in addition to behavioural responses that can be detected also at the gene expression level the transcriptome is finely tuned to normal gravity. The alteration of this constant parameter on Earth can have effects on gene expression that depends both on the environmental conditions and the ground based facility used to compensate the gravity vector. Alternative and commons effects of mechanical facilities like the Random Positioning Machine and a centrifuge and strong magnetic field ones like a cryogenically cooled superconductive magnet are discussed. We compare the effects over the gene expression profile of different gender/age Drosophila imagoes in 3-4 days-long experiments under altered gravity conditions into three GBF (Ground Based Facilities for micro/hyper- gravity simulation) using whole genome microarray platforms. Descriptions of different GBFs (treatments): LDC means Large Diameter Centrifuge. Samples can be placed under three conditions: inside LDC (at certain g level) at the LDC rotational control and at external 1g control (outside the LDC). RPM means Random Positioning Machine. Samples can be placed under two conditions: inside RPM (at nearly 0g Microgravity level) and at external 1g control (outside the RPM). At the magnet means INSIDE the Magnetic levitator (another GBF). Samples can be placed under four conditions: inside Magnet 0g* (at microgravity with magnetic field) inside Magnet at 1g* (internal control with magnetic field) or inside the magnet 2g* (at hypergravity with magnetic field) and at external 1g control (outside the magnet)

Age plays a crucial role in the interplay between tumor and host; with further perturbations induced by irradiation. Proton irradiation on tumors induces biological modulations including inhibition of angiogenic and immune factors critical to hallmark processes impacting tumor development in addition to physical targeting advantages. These advantages have provided promising results for proton therapy in cancer. Additionally protons have implications for carcinogenesis risk of space travel (due to the high proportion of high energy protons in space radiation). Through a systems biology approach we investigated how host tissue (i.e. splenic tissue) of tumor-bearing mice is altered with age with or without whole-body proton exposure. Transcriptome analysis was performed on splenic tissue from adolescent (68 day) versus old (736 day) C57BL/6 male mice injected with Lewis lung carcinoma cells with or without three fractionations of 0.5Gy (1GeV) proton irradiation. Global transcriptome analysis indicated that proton irradiation of adolescent hosts caused significant signaling changes within splenic tissues that support carcinogenesis within the mice as compared to old subjects. Increases in cell cycling and immunosuppression in irradiated adolescent hosts with CDK2 MCM7 CD74 and RUVBL2 as the key players were involved in the regulatory changes in host environment response (i.e. spleen). These results suggest a significant biological component to proton irradiation operative through host age that would indicate a modulation of host s ability to support carcinogenesis in adolescence and the bestowal of resistance to immunosuppression carcinogenesis and genetic perturbation by old age.

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as collateral damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment HZE (1 GeV Fe26+ high mass high charge and high energy relativistic particles) and gamma photons on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs) but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response although they differ slightly in the timing degree and ATM-dependence of the response. The ATM-dependent DNA metabolism-related transcripts of the xd2DSB response xd3 were also induced by other DNA damaging agents but were not induced by conventional stresses. Both Gamma and HZE irradiation induced at 24 h post-irradiation ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response rather than DNA metabolism. In contrast only HZE-irradiated plants at 1.5 h after irradiation exhibited an additional and very extensive transcriptional response shared with plants experiencing extended night. This response was not apparent in gamma-irradiated plants. We treated 5-day-old WT and atm-1 seedlings of Arabidopsis thaliana with 100 Gy of Gamma radiation (over a span of 15 minutes) or 30 Gy of HZE (over a span of approximately 12 minutes). Gamma irradiations were completed at 8:40 am while HZE irradiations were conducted in two runs (due to space limitations) which were completed at 1:09 and 1:28pm respectively. Gamma treated seedlings were sampled at 10:10 am 11:40 am 2:55 pm 8:40 pm and 8:40 am. HZE treated seedlings were sampled at 2:39 pm 4:09 pm 7:24 pm 1:09 am and 1:09 pm. Un-irradiated WT and atm-1 control seedlings were sampled at 10:45 am on Day #1 and 9:15 am on Day #2. There are a total of 22 experimental or control conditions with two replicates per condition yielding 44 samples overall.

Analysis of human peripheral blood 48 hours after irradiation ex vivo with graded doses of gamma rays. Results have been used in building and testing classifiers to predict exposure dose for use in radiological triage and also provide insight into immune cell responses. Results were compared with those from earlier times and from patients exposed in vivo. Peripheral blood from 5 healthy donors was exposed ex vivo to 0. 0.5 2 5 or 8 Gy gamma-rays and gene expression was analyzed up to 48 hours after exposure.

Proton irradiation is touted for its improved tumor targeting due to the physical advantages of ion beams for radiotherapy. Recent studies from our laboratory have shown that in addition to targeting advantages proton irradiation can inhibit angiogenic and immune factors and thereby modulate tumor progression. High-energy protons also constitute a principal component of the galactic cosmic rays to which astronauts are exposed. Increased understanding of the biological effects of proton exposure would thus contribute to both improved cancer therapy and carcinogenesis risk assessment for space travel. In addition age plays a major role in tumor incidence and is a critical consideration for estimating cancer risk. We investigated the effects of host age and proton exposure on tumor progression. Tumor lag time and growth dynamics were tracked following injection of murine Lewis lung carcinoma (LLC) cells into young (68 day) versus old (736 day) mice with or without coincident irradiation. Tumor progression was suppressed in old compared to young mice. Differences in progression were further modulated by proton irradiation (1GeV) with increased inhibition evident in old mice. Through global transcriptome analysis TGFB1 and TGFB2 were determined to be key players that contributed to the tumor dynamics observed. These findings point to older hosts providing decreased systemic tumor support which can be further inhibited by proton irradiation. Overall design: For genome-wide expression profiling of tumor tissue Mouse WG-6 BeadArray chips (Illumina San Diego CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion Austin TX) and labeled from all replicate biological samples for each condition. For tumor replicates thirty tumor samples from adolescent and thirty tumor samples from old mice for a total of 60 tumor samples were used. All replicate samples were run individually. For each age group ten tumor samples had received proton irradiation while twenty tumor samples were from unirradiated mice (as described above). Total RNA was isolated and purified using TRIzol (Invitrogen) and quantified using an Agilent Bioanalyzer. Samples were deemed suitable for amplification and hybridization if they had 28s/18s = 2:1 RIN >7. Total RNA of 500ng per sample was amplified using AmbionTotalPrep and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. In GenomeStudio data was background subtracted and rank invariant normalization was applied. Data was imported into MultiExperiment Viewer MeV for statistical analysis. The statistically significant genes were determined using MeV by applying a one-way ANOVA analysis with standard Bonferroni correction with a FDR <0.05 that resulted in a list of significant genes. Average gene expression signals <10 were filtered out due to signal being

Age plays a major role in tumor incidence and is an important consideration when modeling the carcinogenesis process or estimating cancer risks. Epidemiological data show that from adolescence through middle age cancer incidence increases with age. This effect is commonly attributed to a lifetime accumulation of cellular particularly DNA damage. However during middle-age the incidence begins to decelerate and for many tumor sites it actually decreases at sufficiently advanced ages. We investigated if the observed deceleration and potential decrease in incidence could be attributed to a decreased capacity of older hosts to support tumor progression and whether HZE (high atomic number (Z) high energy (E)) radiation differentially modulates tumor progression in young versus middle-age hosts issues relevant to estimating carcinogenesis risk for astronauts. Lewis lung carcinoma (LLC) cells were injected into syngeneic mice (143 and 551 days old) which were then subject to whole-body 56Fe irradiation (1GeV/amu). Three findings emerged: 1) among unirradiated animals substantial inhibition of tumor progression and significantly decreased tumor growth rates were seen for middle-aged mice compared to young mice; 2) whole-body 56Fe irradiation (1GeV/amu) inhibited tumor progression in both young and in middle-aged mice (with greater suppression seen in case of young animals) with little effect on tumor growth rates; and 3) 56Fe irradiation (1GeV/amu) suppressed tumor progression in young mice to a degree not significantly different than transiting from young to middle-aged. Thus 56Fe irradiation (1GeV/amu) acted similar to aging with respect to tumor progression. We further investigated the molecular underpinnings driving the radiation modulation of tumor dynamics in young and middle-aged mice. Through global gene expression analysis the key players FASN AKT1 and the CXCL12/CXCR4 complex were determined to be contributory. In sum these findings demonstrate a reduced capacity of middle-aged hosts to support the progression phase of carcinogenesis and identify molecular factors contributory to HZE radiation modulation of tumor progression as a function of age. For genome-wide expression profiling of tumor tissue Mouse WG-6 BeadArray chips (Illumina San Diego CA) were used. Total RNA was amplified with the Ambion Illumina TotalPrep Amplification Kit (Ambion Austin TX) and labeled from all replicate biological samples for each condition. The number of tumor sample replicates used from each condition is as follows: 10 samples from young unirradiated mice 8 samples from young irradiated mice 7 samples from middle-aged unirradiated mice 5 samples from middle-aged irradiated mice. Total RNA was isolated and purified using Trizol (Invitrogen) or RNeasy (Qiagen) quantified and qualified using Agilent Bioanalyzer (Agilent) and samples were deemed suitable for amplification and hybridization if they had O.D. 260/280 = 1.7 - 2.1 28s/18s = 2:1 RIN (RNA integrity number) >7. Total RNA of 500ng per sample was amplified using Ambion TotalPrep (Ambion) and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C the chips were washed and then scanned using the Illumina iScan (Illumina) and the data were analyzed using GenomeStudio (Illumina). Data were first analyzed for gene expression and then culled for present genes (genes that meet the criteria of detection p-value < 0.05). Expression above background was included in an expressed genes working data set for further analyses. Rank variant normalization was applied to the data before extensive analysis. Differential gene expression analysis was used to compare to the reference group young unirradiated mice and genes were then evaluated and validated.