Radiation affects tissue and cellular integrity at the level of DNA protein and metabolites of the cell and extracellular space. The effects of radiation are not limited to targeted cells and tissue and radiation induced bystander effects are significant to exposed individuals in accidental or therapeutic situations. These non-targeted effects of radiation have been studied extensively at the low dose range where they appear to have adverse effects on cells and surrounding environments. The requirement of cellular contact and shared fluid media has been established as critical to the bystander effect yet there is not much known about the actual signaling mechanism and its ability to transmit the damaging effect over space and time. Experimental cell types and context within the tissue are also quite important to the nature and extent of this bystander effect and must be considered when drawing parallels at the organismal level. Our approach was to use a genomic level analysis of global mRNA expression in primary lung fibroblast cells to understand the cellular triggers and mechanism of the bystander effect. Gene ontology and pathway analyses suggested that the p53 induced transcriptional response appears muted in bystanders while cytokine and cell signaling mechanisms such as those controlled by NFkB and p38 MAPK are highly active in both populations. We validated a large number of genes that are significantly changed at 4hrs after irradiation in both irradiated and bystander populations. We investigated time course gene expression profiles of cyclooxygenase2 (PTGS2) interleukin 8 (IL8) and BCL2 related protein 2 (BCL2A1) as genes that are involved in cellular signaling via the NFkB pathway which revealed that there is a dramatic response at 0.5hr after irradiation followed by another wave at 4hr in both populations. The induction of interleukins such as cytokine IL8 and chemokine IL6 at the transcriptional level is both early and amplified and if followed by translation and secretion of these proteins could explain the concerted response seen in bystander cells. Our results are the first to show that there is a significant and distinct global response of cellular signaling genes in bystander cells with some genes showing a response as early as 0.5hr after irradiation which implies a fast moving intercellular signal that leads to a concerted response in the irradiated and bystander populations. Keywords: gene expression fold change There are 12 total samples 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C) irradiated (alpha=A) and bystander (B)

The radiation bystander effect is an important component of the overall biological response of tissues and organisms to ionizing radiation. Little is known about the contribution of genome level changes in neighboring bystander cells to tissue and organ stress after irradiation. The timing of these changes is critical in the physiological context and these questions can only be answered by studying signaling and global transcriptomics in a chronological way. Here we present a strategy to identify different biologically important signaling modules that act in concert in the radiation and bystander responses. We used time series gene expression analysis of normal human fibroblast cells measured at 0.5 hour 1 hour 2 hours 4 hours 6 hours and 24 hours after exposure to radiation coupled with a novel clustering method targeted to short time series Feature Based Partitioning around medoids Algorithm (FBPA) to look for genes that were potentially co-regulated. This method uses biologically meaningful features of the expression profile and dimension augmentation to address the analysis of sparse data sets such as ours. We applied FBPA and Short Time series Expression Miner (STEM) to the same datasets and present the results of our comparisons using computational metrics as well as biological enrichment. Enrichment showed that gene expression in irradiated cells fell into broad categories of signal transduction cell cycle/cell death and inflammation/immunity; but only FBPA clustered functions well. In bystander cells the gene expression response was also broadly categorized into functions associated with cell communication and motility signal transduction and inflammation; but neither STEM nor FBPA separated biological functions as well as in irradiated samples. Network analysis revealed that p53 and NF-kappaB were central players in gene expression in both irradiated and bystander gene clusters. Analysis of individual clusters also suggested new regulators of gene expression in the radiation and bystander response that may act at the epigenetic level such as histone deacetylases (HDAC1 and HDAC2) and methylases (KDM5B) that can act as strong transcription repressors. Based on these results we propose a novel time series clustering method FBPA as a powerful approach that can be applied to sparse data sets (including genomic profiling data) where the choice of features selected for clustering and stringent statistical outcome analysis can augment our knowledge of the underlying cellular mechanisms in biological processes. There are 72 total samples 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C) irradiated (alpha=A) and bystander (B) cells were harvested at 0.5 hour 1 hour 2 hours 4 hours 6 hours and 24 hours after treatment

Mitochondrial DNA-depleted human skin fibroblasts (HSF rho0) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes encoding numerous protein classes in NF-kappaB and STAT3 signaling pathways and by decreased activity of the mitochondrial death pathway compared to the parent rho+ HSF. In contrast the extrinsic TRAIL/TRAIL-Receptor-mediated death pathway remained highly active and exogenous TRAIL induced higher levels of apoptosis in rho0 cells compared to rho+ HSF. Global gene expression analysis using microarray and quantitative RT-PCR demonstrated that expression levels of many growth factors and their adaptor proteins (FGF13 HGF IGFBP4 IGFBP6 IGFL2) cytokines (IL6 IL17B IL18 IL19 IL28B) and cytokine receptors (IL1R1 IL21R IL31RA) were substantially decreased after mitochondrial depletion. Some of these genes were targets of NF-kappaB and STAT3 and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation induced expression of several NF-kappaB/STAT3 target genes including IL1A IL1B IL6 PTGS2/COX2 and MMP12 in rho+ HSF but this response was substantially decreased in rho0 HSF. Suppression of the IKK-NF-kappaB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated rho+ HSF. Inhibitory antibodies against IL6 the main activator of JAK2-STAT3 pathway added into the cell media also increased TRAIL-induced apoptosis in rho+ HSF. However NF-kappaB activation was partially lost in mitochondrial DNA-depleted HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-kappaB targets further suppressing IL6-JAK2-STAT3 that in concert with NF-kappaB regulated protection against TRAIL-induced apoptosis. There are 12 total samples 3 biological replicates each of HSF rho+ and rho0 cells that were not irradiated (control=C) or irradiated (alpha=A). Cells were harvested at 4 hours after treatment.

The existence of a radiation bystander effect in which non-irradiated cells respond to signals from irradiated cells is well established. It raises concerns for the interpretation of risks from exposure to low doses of ionizing radiation. Sparse data exists about the bystander signaling mechanisms and the ability to transmit damaging effects both spatially and temporally. To understand early signaling and cellular changes in bystanders we have measured global gene expression 30 minutes after direct and bystander exposure to alpha particle in primary human lung fibroblasts. Gene ontology and pathway analyses suggested that the earliest measured changes at 30 minutes after treatment are in cell structure motility and adhesion categories and a significant number of genes belong to the category of inflammation and cell-to-cell communication. We investigated time course gene expression profiles of matrix metalloproteinases 1 and 3 (MMP1 and MMP3) chemokine ligands 2 3 and 5 (CXCL2 CXCL3 and CXCL5) interleukins 1a 1b 6 and 33 (IL1A IL1B IL6 and IL33) growth differentiation factor 15 (GDF15) and superoxide dismutase2 (SOD2) by real time quantitative PCR. These encode proteins involved in cellular signaling via the NFkappaB pathway and time course of mRNA levels revealed an increased response at 30 minutes after irradiation followed by another wave at 4 to 6 hours. We also investigated protein modifications in the AKT-GSK-3 signaling pathway and found that in irradiated cells AKT and GSK3beta are hyper-phosphorylated at 30 minutes and this effect is maintained until 4 hours after exposure. In bystanders there is a similar response with a delay of 30 minutes. In irradiated cells inactivated GSK3beta led to decreased phosphorylation of beta-catenin. Our results are the first to show that the radiation induced bystander signal can induce a widespread gene expression response as early as 30 minutes after exposure and that these changes are accompanied by protein modification of signaling modules such as AKT and GSK3beta. There are 12 total samples 4 corresponding biological replicates of IMR90 cells that were not irradiated (control=C) irradiated (alpha=A) and bystander (B) cells were harvested 0.5 hr after treatment

Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions two spaceflight-analogue culture systems i.e. the rotating wall vessel (RWV) and the random position machine (RPM) were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG) revealed a regulatory role for AlgU (RpoE). Specifically P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore cultivation in LSMMG increased heat and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.

Martian regolith (unconsolidated surface material) is a potential medium for plant growth in bioregenerative life support systems during manned missions on Mars. However hydrated magnesium sulfate mineral levels in the regolith of Mars can reach as high as 10 wt% and would be expected to be highly inhibitory to plant growth. A global approach was used to identify novel genes with potential to enhance tolerance to high MgSO4 stress. The early Arabidopsis root transcriptome response to elevated concentrations of magnesium sulfate was characterized in col-0 and also between col-0 and the mutant line cax1-1 - a mutant relatively tolerant of high levels of MgSO4-7H2O in soil solution. After 3 weeks of growth under hydroponic conditions Arabidopsis thaliana col-0 roots were exposed to a basic nutrient solution (0.25 g/L MES 1/16x MS pH 5.7) with an additional 2.08 mM magnesium sulfate (total Ca:Mg ratio = 1:15) for 45 min. 90 min. or 180 min. while a col-0 control set was exposed to the basic nutrient solution without additional magnesium sulfate for 45 minutes. Arabidopsis thaliana cax1-1 roots were exposed to the basic nutrient solution with additional magnesium sulfate for 180 min. only. Four replicate containers were harvested for the control and each of the treatment sets resulting in a total of 20 samples. Gene expression of the col-0 sets exposed to magnesium sulfate treatment for 45 min. 90 min. or 180 min. was compared to gene expression of the col-0 control set. Gene expression of the cax1-1 set exposed to magnesium sulfate treatment for 180 min. was compared to gene expression of the col-0 set exposed to magnesium sulfate treatment for 180 minutes.

In March 2006 murine Bone Marrow Stromal Cells (BMSC) were flown in the Soyuz 12S to the International Space Station to investigate the effects of microgravity on their osteogenic potential in a three-dimensional environment. BMSC were grown in porous bioceramic Skelite disks (dia 9 mm x T 1.2 mm). The constructs were exposed to microgravity for ca. 8 days then fixed for RNA extraction. While the flight experiment was performed in fully automated hardware inside the KUBIK incubator one group of control samples were incubated inside manually operated hardwares (flight control) and the other control group was incubated under routine laboratory conditions (lab control). The altered gene expression profile was analyzed by Mouse Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. Each one of the 28853 genes is represented on the array by approximately 26 probes spread across the full length of the gene providing a more complete and more accurate picture of gene expression than the 3 based expression array design. A few days of microgravity were sufficient to determinate at least at the molecular level an effect in the BMSC; this response expressed a stress condition able to determinate consequences on several compartments and cellular functions. In particular it seems to promote a gene expression known to be associated with neurogenic activity (e.g. axon guidance) perhaps promoting the BMSC capability to be committed in that direction. The osteo-induction by dexamethasone-based medium due to the short duration of stimulation did not have the possibility to manifest itself at the phenotypic level but only partially at the molecular level.

Bystander mechanisms that originate in the areas surrounding a tissue damage presumably play an important role participating in wound healing and tissue remodeling. Thus identification and characterization of bystander mechanisms will help to development of new treatments of patients with a radiation exposure. In the present study we irradiated 3-dimensional tissue model of human epidermis Epi-200 (Mat-Tek Ashland MA) with 2.5 Gy protons. By exposing only a thin strip across the center of the EPI-200 tissue we have been able to measure global gene expression responses in directly irradiated and bystander cells located at 0.125-0.375 0.375-0.625 0.625-875 mm from the irradiation line. The data were analyzed using BRB-Array Tools (NIH) and further gene ontology analysis and network analysis was performed with Panther (Applied Biosystems) and IPA (Ingenuity) accordingly. Significantly responding genes were identified at all distances and included sets common to both direct and bystander responses. False discovery rate in bystander samples did not exceed 20% (p=0.001) and was sufficiently low in the samples obtained after the whole tissue exposure (0.06-1.16%). Analysis of the fragments cut at the same distance revealed 52 54 and 88 differentially expressed genes. These gene lists overlapped each other had from 3 to 12 genes in common including CLED2 S100A7A. Samples obtained after the whole tissue exposure discovered 949 differentially expressed genes. Moreover the performed gene ontology analysis showed there overrepresentation of TP53 pathway (pathways p=2.04E-02) a common marker of direct irradiation response and also overrepresentation of the following groups of genes: signal transduction (p=4.52E-04) cell communication (p=1.24E-04) and cell cycle in the category of biological processes; DNA helicase activity (p=2.54E-07) receptor binding (p=6.19E-04) calcium ion binding proteins (p=2.57E-03) as the molecular functions. Differentially expresses genes of bystander samples had few categories in common such as cell communication (p=2.36E-03) and signal transduction (p=2.42E-03) among the biological processes and receptor activity (p=4.54E-03) among the molecular functions. Categories specific for the bystander samples included G-protein coupled receptors (p=7.24E-03) and ligand-gated ion channels (p=4.16E-03) suggesting a role of external stimulation and ion trafficking in bystander mechanisms. Radiation induced gene expression in 3-dimensional tissue model Epi-200 was measured in 16 hours after exposure to 2.5 Gy of protons. Four independent experiments were performed for the samples collected at different distances from the irradiation line (125-375 375-625 and 625-875 micrometers) using three tissue fragments per a data point. Moreover three sets of whole tissue irradited samples were also generated for 0 and 2.5 Gy (6 samples total) and used for comparison of bystander and direct responses.

Using diamagnetic levitation we have exposed A. thaliana in vitro callus cultures to five environments with different levels of effective gravity (from levitation i.e. simulated mg* to 2g*) and magnetic fields (10.1 to 16.5 Tesla) and we have compared the results with those of similar experiments done in a Random Position Machine (simulated micro g) and a Large Diameter Centrifuge (2g) free of high magnetic fields. Microarray analysis indicates that there are changes in overall gene expression of the cultured cells exposed to these unusual environments but also that gravitational and magnetic field produce synergic variations in the steady state of the transcriptional profile of A. thaliana. Significant changes in the expression of structural abiotic stress and secondary metabolism genes were observed into the magnet field. These results confirm that the strong magnetic field both at micro g* or 2g* has a significant effect on the expression of these genes but subtle gravitational effects are still observable. These subtle responses to microgravity environments are opposite to the ones observed in a hypergravity one. seven-condition experiment MM2D Arabidopsis culture callus control vs. Treatment (altered gravity simulation GBF). Three GBF were used (LDC (2g) + control RPM (mg) + control and Magnet (mg* 0.1g* 1g* 1.9g* 2g*) + control). Biological replicates: 3 replicates in all conditions and controls except 1.9g* (2 replicates)

The spaceflight experiment was carried out using male C57BL/10J mice (8 weeks old at launch). Wild type mice (n=3) were launched by Space Shuttle Discovery and housed on the International Space Station (ISS) for 91 days. They returned to the Earth by Space Shuttle Atlantis. But only one mouse returned to the Earth alive. Whole brain was sampled from the mouse killed by inhalation of carbon dioxide at the Life Sciences Support Facility of Kennedy Space Center within 3-4 hours after landing. After the spaceflight experiment the on-ground experiment was also carried out at the Advanced Biotechnology Center in Genova Italy. A mouse with the same species sex and age was housed in mice drawer system (MDS) which was utilized for the spaceflight (SF) mice for 3 months as the ground control (GC). Another mouse was housed in normal vivarium cage as the laboratory control (LC). Amount of food and water supplementation and environmental conditions were simulated as the flight group. After 3 months brain was sampled from one mouse in group GC and LC respectively. Comprehensive analyses of gene expression were performed in the right brain. Total of 4,000 genes were analyzed. The expression levels of 60 genes significantly changed in response to SF compared with LC and/or GC. The 15 and 16 genes were up- (> 2 folds) and down-regulated (< 0.5 folds) respectively following SF vs. GC. The levels of 58 genes were significantly altered by housing in MDS in space and/or on the ground. Forty seven and 11 genes were significantly up- and down-regulated vs. LC. Twenty seven out of these genes responded to caging in MDS both in space and on the ground. Further 31 genes were influenced by housing in MDS on the Earth. Responses of the characteristics of brain to long-term gravitational unloading were investigated in mice.