The general objective of the study was to determine modulation of gene expression by environmental factors with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a Large Diameter Centrifuge and brought to a gravitational force of 3g or 5g for 24 hours. We show that this treatment causes a clear increase of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images and by gene expression studies of selected genes. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.

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

Physical forces greatly influence the growth and function of an organism. Altered gravity can perturb normal development and induce corresponding changes in gene expression. Understanding this relationship between the physical and biological realms is important for NASA s space travel goals. We use combined RNA-Seq and qRT-PCR to profile changes in early Drosophila melanogaster pupae exposed to chronic hypergravity (3 g three times Earth s gravity) to highlight gravity-dependent pathways and gene products. Robust transcriptional response was evident among the pupae developed in a hypergravity environment compared to control. 1,513 genes showed significantly (p < 0.05) altered gene expression in the 3 g samples. These findings were supported with qRT-PCR data. Major biological processes affected include ion transport redox homeostasis immune and humoral stress response proteolysis and cuticle development.

The general objective of the study was to determine modulation of gene expression by environmental factors with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. This experiment was designed as a new type of gravitational experiment which we like to call relative microgravity referring to the fact that the larvae first grow in hyper gravity for 5 days and are then returned to 1g normal gravity for 1 day. Zebrafish embryos were placed on a Large Diameter Centrifuge at 3 hpf brought to a gravitational force of 3 g until 5 dpf. Reference embryos were kept in parallel at 1g (Inc). At 5dpf one batch was left at 3g (3g) one batch was returned to 1g (3g>1g) while a third batch was returned to 1g but left on the axis of the centrifuge (Axe; 3g>Axe). The experiment was repeated 4 times each time with 4 batches of 60 larvae.

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.

Bacterial behavior has been observed to change during spaceflight. Higher final cell counts enhanced biofilm formation increased virulence and reduced susceptibility to antibiotics have been reported to occur for cells cultured in space . Most of these phenomena are theorized as being an indirect effect of an altered extracellular environment where the carbon source uptake is inhibited and excreted acidic byproducts buildup around the cell due to the lack of gravity-driven transport forces. However to date neither spaceflight results ground-based studies physical measurement techniques nor computational approaches have provided sufficient evidence needed to confirm this model. Gene expression data from the Antibiotic Effectiveness in Space (AES-1) experiment however have now allowed us to look into the biomolecular processes behind these observations and showed a systematic activation of glucose starvation and acid resistance genes. These results corroborate the reduced mass transport model proposed to govern bacterial responses to spaceflight. Furthermore the gene expression data suggests that metabolism was stimulated in space which could play a role in causing the observed increase in bacterial cell concentrations in microgravity. Similarly the decrease in extracellular pH may also be involved with the reported increase in virulence in space.

In recent times long-term stay has become a common occurrence in the International Space Station (ISS). However adaptation to the space environment can sometimes pose physiological problems to the astronauts after their return. Therefore it is important to develop healthcare technologies for astronauts. In this study hair an easy-to-obtain sample was identified as the candidate. In order to investigate the genetic changes in human hair during space flight the hair follicles of 10 astronauts were analyzed by DNA microarray and real time q-PCR analyses. Space environment induced gene expression of hair follicles of astronaut was measured 6 differnent times included 2 in flight on orbit. Ten independent experiments were performed on differing astronauts. and the sampling day was differed for each astronaut because of their schedules.

The general objective of the study was to determine modulation of gene expression by environmental factors specifically simulation of microgravity with a special emphasis on bone formation. For this reason the specific period of treatment was chosen between 5-6 days post-fertilization (dpf) when bone formation and calcification are taking place. Zebrafish larvae were placed at 5 dpf into a clinostat (CLINO) for 24 hours which was shown to simulate microgravity by the specific rotational movements it generates. We show that CLINO exposure causes a clear decrease of bone formation as illustrated by cranial skeleton staining of the bone matrix by Alizarin Red by morphometric analysis of the resulting images. Thus a whole genome micro-array experiment was conducted to identify genes that may be involved in the observed effect on bone formation.