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

Harsh environmental conditions including microgravity and radiation during prolonged spaceflights are known to alter hepatic metabolism. Our studies have focused on the analysis of possible changes in metabolic pathways in livers of mice which experienced 30 days of spaceflight with and without an additional re-adaption period of 7 days compared to control mice on Earth. Utilizing shotgun mass spectrometry and label-free quantification we performed proteomic profiling to investigate mice livers from the spaceflight project xe2 x80 x9cBion-M 1 xe2 x80 x9d. No significant alterations in protein levels were observed between control mice liver and spaceflight mice which is possibly caused by insufficient fold change detection combined with high variances within the groups. In contrast our results show that more than a third of the quantified protein levels are altered comparing the liver proteome of mice with and without re-adaption time after their spaceflight. Proteins related to amino acid metabolism showed higher levels after re-adaption which may indicate higher rates of gluconeogenesis. Members of the peroxisome proliferator-activated receptor pathway reconstitute their level after 7 days due to a decrease in fold change which indicates decreased signs of non-alcoholic fatty liver disease. Moreover bile acid secretion regenerates on Earth due to reconstitution of related transmembrane proteins and elevated levels of the drug-metabolising enzymes belonging to the CYP superfamily decrease 7 days after the spaceflight. Thus our study demonstrates reconstitution of pharmacological response and early signs of non-alcoholic fatty liver disease recover within 7 days whereas glucose uptake should be monitored due to alterations in gluconeogenesis.

Using a novel flood source construct we performed a direct comparison of genome-wide gene expression regulations resulting from exposure of primary human prostate fibroblast cultures to acute (10cGy and 200cGy) and longer-term chronic (1.0-2.45cGy cumulative over 24hr) exposures.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen Candida albicans grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 454 genes compared to synchronous ground controls which represented 8.4% of the analyzed ORFs. Spaceflight-cultured C. albicans induced genes involved in cell aggregation (similar to flocculation) which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to more normal bipolar budding patterns for ground samples in accordance with the gene expression data. Furthermore genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight including induction of ABC transporters and members of the major facilitator family downregulation of ergosterol-encoding genes and upregulation of genes involved in oxidative stress resistance. Finally downregulation of genes involved in the actin cytoskeleton was observed. Interestingly the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested as this regulator is involved in random budding cell aggregation actin cytoskeleton and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype no increased virulence in a murine intraperitoneal (i.p.) infection model was observed. This study represents an important basis for the assessment of the risk that commensal flora could play during spaceflight missions. Furthermore since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process insights gained from this study could identify novel infectious disease mechanisms with downstream benefits for the general public. Cells were grown for 24 hours on the space shuttle or as ground-based controls preserved in RNALater and stored at -80C. Four samples of each flight- and ground-based controls were harvested for microarray analysis. GAP is Group Activation Pack and each GAP contains 8 FPAs. The numbers represent the # assigned to the particular GAP and the number assigned to the specific FPA (1-8) within the indicated GAP. The same hardware is used for the flight samples and the ground samples.

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 DSB response 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.

One of the most likely risks astronauts on long duration space missions face is exposure to ionizing radiation associated with highly energetic and charged heavy (HZE) particles. Since access to medical expertise on such a mission is limited at best early diagnosis and mitigation of such exposure is critical. In order to accurately determine the dosage within 1 hour post-exposure dose-dependent biomarkers are needed. Therefore we performed a dose-course transcriptional analysis for radiation exposure at 0 0.3 1.5 and 3.0 Gy with corresponding time point at 1 hour (hr) post-exposure using Affymetrix GeneChip Human Gene 1.0 ST v1 Array chips. The analysis of our data suggests a set of sensitive genetic biomarkers specific to each radiation level as well as generic radiation response biomarkers. Upregulated biomarkers can then be used within lab-on-a-chip (LOC) systems to detect exposure to ionizing radiation. A total of sixteen human samples representing radiation exposure at levels 0 Gy 0.3 Gy 1.5 Gy and 3.0 Gy at time point 1 hour (hr) post-exposure were constructed. Blood samples were extracted from four human volunteers and were irradiated. Leukocytes were extracted and gene expression was measured. Samples for all four volunteers were measured at 1 hr for all four dose levels resulting in four replicates at each dose level. Thus a total of 4 samples at each of the four radiation levels were sampled yielding the total of 16 samples.

Densely ionizing radiation is a major component of the space radiation environment and has potentially greater carcinogenic effect compared to sparsely ionizing radiation that is prevalent in the terrestrial environment. It is unknown to what extent the irradiated microenvironment contributes to the differential carcinogenic potential of densely ionizing radiation. To address this gap 10-week old BALB/c mice were irradiated with 100 cGy sparsely ionizing g-radiation or 10 30 or 80 cGy of densely ionizing 350 MeV/amu Si particles and transplanted 3 days later with syngeneic Trp53 null mammary fragments. Tumor appearance was monitored for 600 days. Tumors arising in Si-particle irradiated mice had a shorter median time to appearance grew faster and were more likely to metastasize. Most tumors arising in sham-irradiated mice were ER-positive pseudo-glandular and contained both basal keratin 14 and luminal keratin 8/18 cells (designated K14/18) while most tumors arising in irradiated hosts were K8/18 positive (designated K18) and ER negative. Comparison of K18 vs K14/18 tumor expression profiles showed that genes increased in K18 tumors were associated with ERBB2 and KRAS while decreased genes overlapped with those down regulated in metastasis and by loss of E-cadherin. Consistent with this K18 tumors grew faster than K14/18 tumors and more mice with K18 tumors developed lung metastases compared to mice with K14/18 tumors. However K18 tumors arising in Si-particle irradiated mice grew even faster and were more metastatic compared to control mice. A K18 Si-irradiated host profile was enriched in genes involved in mammary stem cells stroma and Notch signaling. Thus systemic responses to densely ionizing radiation enriches for a ER-negative K18-positive tumor whose biology is more aggressive compared to similar tumors arising in non-irradiated hosts. Key Words: ionizing radiation; breast cancer; heavy ion radiation;initiation; promotion 3 different dose of Si were used. Total RNA was extracted from mammary tumors derived from transplantations of non-irradiated p53null mammary fragments into irradiated hosts. We analyzed a total of 45 Trp53-null tumors: 18 from sham-irradiated hosts 9 from 10 cGy Si-irradiated hosts 10 from 30 cGy Si-irradiated hosts and 8 from irradiated hosts.

Salmonella transcription profiles were obtained from samples flown on space shuttle mission STS-115 and compared to profiles from Salmonella grown under identical conditions on the ground. Keywords: stress response transcriptional profile Triplicate experimental samples were hybridized to slides that contain three identical Salmonella ORF arrays. Each hybridization was performed with Cy3 labeled total RNA and Cy5 labeled gDNA as control.

Microgravity alters the immune response to in vitro LPS assault engineered in spaceflight: A multi-omics study Microgravity can facilitate creation of a potent environment for opportunistic infection by augmenting virulence and suppressing the host defense. Presumably extraterrestrial infection may trigger potentially novel bionetworks different from the terrestrial equivalent which could only be probed by investigating the host-pathogen relationship with minimum terrestrial bias. Towards this objective we strategically engineered a cell culture module equipped with a feedback controlled semi-automated platform to expose human endothelial cells to lipopolysaccharide (LPS). The assay was carried out in the STS-135 space shuttle and a concurrent ground study constituted the baseline. Transcriptomic investigation revealed an immune blunting in microgravity; Lbp MyD88 and MD-2 failed to encode proteins responsible for early LPS uptake. Longer exposure results implied that there was a delayed response potentially ineffectual in preventing pathogens from opportunistically modulating the infection network. Lack of recruitment of growth factors and a debilitated apoptosome supported this potential explanation. Certain cytokines such as IL-6 and IL-8 surged in response to LPS insult in microgravity. Contrasting expressions of B2M TIMP-1 and VEGRs suggested impaired pro-survival adaptation and healing mechanisms. The susceptibility of oxidative stress and immune regulation to microgravity compelled further investigation of the respective microRNA modulators such as miR-200a and miR-146b. These miRNAs were expressed differently in response to LPS assaults in different gravitational limits. In conclusion despite a serious drawback attributed to the small sample size we delineated some of the important aspects of the extraterrestrial etiology; more comprehensive follow up studies are warranted. Present study though compromised by the small sample size was able to shade lights on several aspects of immunological responses to the endotoxic assault mediated by uG. Implementing the host-pathogen interactions in the spaceflight and subsequently lysing the cells onboard presented the critical distinguishing features of the present study from the past reports. We identified the CCM of Tissue Genesis Inc. HI as the suitable hardware system to carry out the experiment in the spaceflight. CCM is an automated feedback controlled module that can concurrently support 24 bioreactors following protocols exclusively programmed for individual bioreactor. For this experiment we use samples EA41 EA 47 EA45 and EA155 that were exposed to LPS for 4 hours. Samples EA123 EA165 EA127 EA126 were exposed to LPS for 8Hrs. Samples EA33 EA 125 EA79 and EA 39 were controls in this experiment.

Upon weightlessness and microgravity deleterious effects on the neurosensory and neurovestibular systems haematological changes and deconditioning of musculoskeletal cardiovascular and cardiopulmonary functions have been reported. In particular loss of muscle mass and strength are triggered by weightlessness in humans during space flights that is similarly observed as a result of physical inactivity conditions and aging on Earth. However skeletal muscle tissue is of paramount importance for health maintenance (e.g. being essential to locomotion heat production and metabolism). To better prevent or eventually treat microgravity-induced muscle atrophy its underlying mechanisms have first to be characterized in detail. Using cutting-edge quantitative proteomics the aim of the present study was therefore to get an in depth view of the molecular regulations triggered by space conditions in skeletal muscles of mice during the 30-day flight of the BION-M1 biosatellite. As muscles differing in their fiber type composition appear to respond differently to microgravity (see above) we characterized here the differential response of the soleus extensor digitorum longus and vastus lateralis muscles.