NASA s Rodent Research (RR) project is playing a critical role in advancing biomedical research on the physiological effects of space environments. Due to the limited resources for conducting biological experiments aboard the International Space Station (ISS) it is imperative to use crew time efficiently while maximizing high-quality science return. NASA s GeneLab project has as its primary objectives to 1) further increase the value of these experiments using a multi-omics systems biology-based approach and 2) disseminate these data without restrictions to the scientific community. The current investigation assessed viability of RNA DNA and protein extracted from archived RR-1 tissue samples for epigenomic transcriptomic and proteomic assays. During the first RR spaceflight experiment a variety of tissue types were harvested from subjects snap-frozen or RNAlater-preserved and then stored at least a year at -80C after return to Earth. They were then prioritized for this investigation based on likelihood of significant scientific value for spaceflight research. All tissues were made available to GeneLab through the bio-specimen sharing program managed by the Ames Life Science Data Archive and included mouse adrenal glands quadriceps gastrocnemius tibialis anterior extensor digitorum longus soleus eye and kidney. We report here protocols for and results of these tissue extractions and thus the feasibility and value of these kinds of omics analyses. In addition to providing additional opportunities for investigation of spaceflight effects on the mouse transcriptome and proteome in new kinds of tissues our results may also be of value to program managers for the prioritization of ISS crew time for rodent research activities.

Space environment is suspected to generate reactive oxygen species (ROS) and induce oxidative stress in plants however little is known about the gene expression of ROS gene network in plants grown in long-term space flight. RNA-Seq was used to define the large-scale gene expression profiles of Mizuna harvested after 27 days cultivation in the international space station to understand the molecular response and adaptation to space environment.Results: Total reads of transcripts from the Mizuna grown in the international space station as well as on the ground by RNA-Seq using next generation sequencing technology showed 8,258 and 14,170 transcripts up- and down-regulated in the space-grown Mizuna respectively when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated including high expression of 4 hallmarks and preferentially expressed gene associated with ROS-scavenging genes was thioredoxin glutaredoxin and alternative oxidase genes. In the transcription factors of ROS gene network MEKK1-MKK4-MPK3 OXI1-MKK4-MPK3 and OXI1-MPK3 of MAP cascades induction of WRKY22 by MEKK1-MKK4-MPK3 cascade induction of WRKY25 and repression of ZAT7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes which produce ROS.Conclusions: Our large-scale transcriptome analysis demonstrated that the space environment induced oxidative stress and ROS gene network was activated in the space-grown Mizuna some of which were common genes up-regulated by abiotic and biotic stress and were preferentially up-regulated genes by the space environment even though Mizuna grew in the space as well as on the ground showing that plants could acclimate to the space environment by reprograming the expression of ROS gene network.

Microbes interact with humans in complex ways and understanding how they respond to the spaceflight environment is important to the success of future manned spaceflight missions. The BRIC-23 mission was designed to measure the response of Bacillus subtilis and Staphylococcus aureus to the spaceflight environment. This experiment aimed to produce high quality omics data from B. subtilis and S. aureus grown aboard the International Space Station (ISS) to allow comparison to matched ground controls. There were two primary objectives for this experiment: (1) Demonstrate all post-flight processes and operations required for successful completion of GeneLab Reference Missions conducted on ISS and (2) Generate high quality GeneLab Reference Mission omics data sets for two prokaryotic model organisms Bacillus subtilis and Staphylococcus aureus. Freezing Control Experiment: The BRIC hardware has significant thermal inertia thus the freezing rate of samples placed at -80 C is quite slow. This could affect RNA-sequencing proteomic and metabolic data sets. In an effort to understand how slow freezing could affect these data sets a control experiment was designed in which B. subtilis and S. aureus were grown in petri plates and either slow frozen to -80 C at a rate matching the BRIC-23 spaceflight samples or processed immediately to harvest RNA and protein.

RR-1 is a validation flight to evaluate the hardware operational and science capabilities of the Rodent Research Project on the ISS. RNA DNA and protein were purified from liver tissues from RR-1 mice (female C57BL/6J 16wk old at time of launch). From each group two liver samples were collected and frozen immediately after euthanasia. The rest of the liver samples from each group were collected from frozen carcasses dissected post-flight. RNA-Seq whole genome and RNA BS-Seq (bisulfite sequencing) and proteomic expression profiling were performed. xc2 xa0RNA extracted from these tissues was re-sequenced; these data are available as part of GLDS-168 (https://genelab-data.ndc.nasa.gov/genelab/accession/GLDS-168). xc2 xa0

Methods: RNA-seq libraries were prepared using the Illumina TruSeq RNA kit and the TrueSeq method was employed for mRNA enrichment. The libraries were quantified and samples were multiplexed in each lane of the flowcell. Cluster generation was performed and then sequenced on the Illumina HiSeq1000 system. Reads were mapped on the Human Genome Reference and normalized expression table was generated. Results: Among differentially expressed genes 53 of them were up-regulated and 75 were down-regulated. Conclusions: Data demonstrate increased expression of genes associated with growth development and pro-survival in cardiac progenitors cultured under simulated microgravity compared with those cultured under standard gravity. RNA-sequencing analysis was performed to compare global gene expression profiles of cells at differentiation day 8 under simulated microgravity vs. standard gravity.

Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight host factors that could contribute to oral diseases such as caries and periodontitis. However the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study High Aspect Ratio Vessel (HARV) S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles H2O2 resistance and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated >= 2-fold and 94 genes down-regulated >= 2-fold during simulated microgravity HARV growth. These included a number of genes located on extrachromosomal elements as well as genes involved in carbohydrate metabolism translation and stress responses. Collectively these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions. Overall design: Differential gene expression was compared between RNA from S. mutans grown in normal gravity HARVs (n=3 independent cultures) and RNA from S. mutans grown in simulated microgravity HARVs (n=3 independent cultures)

During space flight bone mineral density is decreased by the influence of osteoclast activation which molecular mechanism is expectantly investigated. In the study of medaka bone development we investigated the system of vertebra formation and firstly identified the presence of osteoclasts in medaka. Moreover osteoclast rsorbing activity was affected by hypergravity indicating the possibility that we can investigate the effect of microgravity on osteoclasts in space. To find this effect we examine the alteration of osteoclast activity under microgravity with the histological analysis or the expression analysis by RNA in-situ hybridization. Furthermore since we have succeeded the establishment of the medaka osteoclast-specific transgenic lines we perform the in-vivo imaging analyses for gene expression and cell mobility. Finally to examine the gravity sensing system we employ tooth and bone as the high density organs which are highly sensitive to gravity and perform the histological analysis and the gene expression analysis of such gravity-sensitive tissues at surrounding pharyngeal teeth and supporting bone.

Our study focuses on the hardiest microorganisms inhabiting the ISS in order to assess their diversity and capabilities to resist certain stresses. We specifically selected dust samples from the Russian modules that were obtained 8-10 years ago and stored since then under sealed conditions on Earth. Targeting long-time survivors and spore-forming microorganisms we assessed consequently the cultivable microbial community of these samples in order to obtain model microbial strains that could help to analyze specific adaptation towards environmental stresses such as desiccation and lack of nutrients. In this study we analyzed these microorganisms with respect to their resistance towards thermal stress and exposure to clinically relevant antibiotics. In addition we assessed the bacterial and archaeal community via molecular methods (NGS sequencing) and compared our new data with the previously derived information from the ISS microbiome.

Spaceflight is known to affect immune cell populations. In particular splenic B-cell numbers decrease during spaceflight and in ground-based physiological models. Although antibody isotype changes have been assessed during and after spaceflight an extensive characterization of the impact of spaceflight on antibody composition has not been conducted in mice. Next Generation Sequencing and bioinformatic tools are now available to assess antibody repertoires. We can now identify immunoglobulin gene- segment usage junctional regions and modifications that contribute to specificity and diversity. Due to limitations on the International Space Station alternate sample collection and storage methods must be employed. Our group compared Illumina MiSeq xc2 xae sequencing data from multiple sample preparation methods in normal C57Bl/6J mice to validate that sample preparation and storage would not bias the outcome of antibody repertoire characterization. In this report we also compared sequencing techniques and a bioinformatic workflow on the data output when we assessed the IgH and Ig xce xba variable gene usage. Our bioinformatic workflow has been optimized for Illumina HiSeq xc2 xae and MiSeq xc2 xae datasets and is designed specifically to reduce bias capture the most information from Ig sequences and produce a data set that provides other data mining options.

Technology demonstration of a nanopore sequencer operating aboard the ISS in comparison with the same sequencer on the ground.