The purpose of the present study was to evaluate damage in brain and eye in a ground-based model for spaceflight which includes prolonged unloading and low-dose radiation. Low-dose/Low-dose-rate (LDR) gamma-radiation using 57Co plates (0.04 Gy) was delivered whole-body to mature 6-month old female C57BL/6 mice to simulate the radiation component. Anti-orthostatic tail suspension was used to model the unloading fluid shift and physiological stress aspects of the microgravity component. Mice were hindlimb suspended and/or irradiated for 21 days. Tissues were collected at 7 days 1 4 and 9 months following simulated microgravity. Herein we proposed to use omics-based molecular phenotyping approach for identification and characterization of genomic signatures in multiple organ system associated with low-dose radiation and simulated microgravity.

The purpose of the present study was to evaluate damage in brain and eye in a ground-based model for spaceflight which includes prolonged unloading and low-dose radiation. Low-dose/Low-dose-rate (LDR) gamma-radiation using 57Co plates (0.04 Gy) was delivered whole-body to mature 6-month old female C57BL/6 mice to simulate the radiation component. Anti-orthostatic tail suspension was used to model the unloading fluid shift and physiological stress aspects of the microgravity component. Mice were hindlimb suspended and/or irradiated for 21 days. Tissues were collected at 7 days 1 and 4 months following simulated microgravity. Herein we proposed to use omics-based molecular phenotyping approach for identification and characterization of genomic signatures in multiple organ system associated with low-dose radiation and simulated microgravity.

Whole seedlings of wild type (4d) and atm mutants (4d) have been analyzed after a gamma ray irradiation of 0.75h 1.5h 3h & 5h (time course). Roots of wt (4d) atm (3d) and atr (4d) mutants have been analyzed after a 1h irradiation. Ataxia Telangiectasia Mutated (ATM) encodes a large protein with a phosphatidylinositol 3-kinase (PI3K)-like domain at the C terminus (reviewed by Rotman and Shiloh 1998). PI3K-related proteins make up a large family of Ser-Thr protein kinases numerous members of which are involved in the regulation of cell cycle progression responses to DNA damage and the maintenance of genomic stability (Hoekstra 1997). AtATM plays an essential role in meiosis and in the somatic response to DNA damage in plants similar to the function of ATM in mammals and other eukaryotes. Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation transmitting DNA damage signals to downstream effectors of cell-cycle progression.

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.

Background: mRNA interactions with each other and other signaling molecules define different biological pathways and functions. Researchers have been investigating various tools to analyze these types of interactions. In particular gene co-expression network methods have proved useful in finding and analyzing these molecular interactions. Many different analytical pipelines to identify these interactions networks have been proposed with the aim of identifying an optimal partition of the network where the individual modules are neither too small to make any general inference or too large to be biologically interpretable. Results: In this study we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline uses WGCNA a widely used software to perform different aspects of gene co-expression network analysis and modularity maximization algorithm to analyze novel RNA-Seq data to understand the effects of low-dose 56Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results along with experimental validation show that using WGCNA combined with Modularity provide a more biologically interpretable network in our dataset. Our pipeline showed better performance than the existing clustering algorithm in WGCNA in finding modules and identified a module with mitochondrial subunits that are supported by mitochondrial complex assay. Conclusions: We present a pipeline that can reduce the problem of parameter selection with the existing algorithm in WGCNA for comparable RNA-Seq datasets which may assist in future research to discover novel mRNA interactions and their downstream molecular effects. C57BL16 males were placed into 2 treatment groups and received the following irradiation treatments at Brookhaven National Laboratories (Long Island NY): 600 MeV/n 56Fe (0.2 Gy) and no irradiation. Left liver lobes were collected at 30 60 120 270 and 360 days post-irradiation flash frozen and stored at -80 xc2 xb0C until they could be processed for RNA-Seq. Livers were sampled by taking two 40-micron thick slices using a cryotome at -20 xc2 xb0C. This allowed multiple sampling of the tissue without the tissue going through multiple freeze/thaw cycles. Total RNA was isolated from the liver slices using RNAqueousTM Total RNA Isolation Kit (ThermoFisher Scientific Waltham MA) and rRNA was removed via Ribo-ZeroTM rRNA Removal Kit (Illumina San Diego CA) prior to library preparation with the Illumina TruSeq RNA Library kit. Samples were sequenced in a paired-end 50 base format on an Illumina HiSeq 1500. Reads were aligned to the mouse GRCm38 reference genome using the STAR alignment program version 2.5.3a with the recommended ENCODE options. The -quantMode GeneCounts option was used to obtain read counts per gene based on the Gencode release M14 annotation file. Total number of reads used in analysis varies between 23-35 millions of reads.

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.

The purpose of this study was to evaluate transcriptional changes in mouse spleens using a ground-based model for spaceflight. This model includes prolonged unloading and low-dose irradiation. Low-dose-rate gamma-radiation was delivered to 6-month old female C57BL/6J mice using 57Co plates (0.04 Gy) to simulate the radiation environment of spaceflight. Anti-orthostatic tail suspension was used to model the unloading fluid shift and physiological stress aspects of the microgravity component of spaceflight. Mice were hindlimb suspended and/or irradiated for 21 days. Mice were euthanized and spleens collected 7 days following treatment. RNA sequencing data was generated to assess transcriptional changes in these spleens.

The purpose of this study was to evaluate transcriptional changes in mouse skin using a ground-based model for spaceflight. This model includes prolonged unloading and low-dose irradiation. Low-dose-rate gamma-radiation was delivered to 6-month old female C57BL/6J mice using 57Co plates (0.04 Gy) to simulate the radiation environment of spaceflight. Anti-orthostatic tail suspension was used to model the unloading fluid shift and physiological stress aspects of the microgravity component of spaceflight. Mice were hindlimb suspended and/or irradiated for 21 days. Mice were euthanized and dorsal skin was collected 7 days following treatment. RNA sequencing data was generated to assess transcriptional changes in these skin samples.

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)

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach our previous NASA work has shown both TGF xce xb2 signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We investigated this hypothesis both in vivo and in vitro and test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach was used to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR) we used the ions energy and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To simulate high-dose radiation exposure due to solar particle events (SPE) we used an acute dose of SPE simulated beam at 1Gy which has energies ranging from 50MeV to 150MeV. C57BL/6 wild-type mice were utilized for irradiation with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions will also be irradiated. To expand on the circulating miRNA signature determined from our preliminary data we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing on serum (before IR and at time of sacrifice) liver heart and muscle tissue for all radiation groups revealed the key circulating miRNA signature (consisting of multiple miRNAs) impacting disease risk. Collectively understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation.