The objective of the Rodent Research-9 (RR-9) mission was to use mice to understand the molecular basis of phenomena that affect astronauts during long-duration spaceflight particularly visual impairment and joint tissue degradation. To this end a flight group (FLT) of 10-week-old male C57BL/6J mice was launched from Kennedy Space Center (KSC) on 8/14/2017 and housed in Rodent Habitats on the ISS for 33 days before being returned alive to Earth. After splashdown in the Pacific Ocean the animals were transported to Loma Linda University (LLU) for testing euthanasia and dissection on 9/18/2018. A Basal Control (BSL) was housed in standard cages at Kennedy Space Center (KSC) and euthanized one day after launch of the FLT animals (8/15/2017). Ground Control (GC) and Vivarium Control (VIV) studies were planned to commence at KSC approximately one-week after the conclusion of the flight experiments. However all the GC and VIV mouse studies at KSC had to be cancelled due to Hurricane Irma and potential adverse effects on the animal housing facility. The GC and VIV studies were therefore rescheduled and begun in May 2018. The GC was euthanized and dissected 6/18/2018 - 6/20/2018 while the VIV was euthanized and dissected 6/22/2018 - 6/23/2018. Because this resulted in a different cohort of mice being used for the GC and VIV controls as compared to the flight (FLT) and basal (BSL) groups two cohort controls were included in the study. The first Cohort Control 1 (CC_C1) was from the same cohort as the FLT and BSL animals and was sacrificed and dissected 4 days after the FLT group (9/22/2017). The second Cohort Control 2 (CC_C2) was from the same cohort as the GC and VIV animals and was sacrificed and dissected 2-8 days after the GC and VIV groups (6/24/2018 - 6/26/2018). The CC_C1 and CC_C2 groups were housed in standard cages and fed standard chow in contrast to all other groups which received Rodent Foodbars. To clarify the connections between treatment groups and animal cohorts the following group abbreviations are used in the sample metadata: Flight (FLT_C1); Basal (BSL_C1); Ground Control (GC_C2); Vivarium Control (VIV_C2) Cohort Control 1 (CC_C1); Cohort Control 2 (CC_C2). Fecal pellets were isolated directly from mice during dissection and preserved by flash freezing in liquid nitrogen before stored at -80 C. DNA was then extracted shotgun metagenomic libraries generated and libraries sequenced (target 10 M clusters at PE 250 bp). Metagenomic data was generated from the following groups: Basal Control (n=5) Ground Control (n=5) Vivarium Control (n=5) Cohort Control 1 (n=5) Cohort Control 2 (n=5) Flight (n=5).

Lettuce crops grown in the Veggie payload aboard ISS harvested and returned for chemical microbiological and molecular analysis

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.

To understand the molecular mechanisms affected by spaceflight it is essential to achieve high quality sample preservation on-orbit for downstream gene expression analysis. However sample preservation protocols must also be compatible with available equipment and crew time. NASA s Rodent Research (RR) missions have used various methods for euthanasia carcass preservation and tissue preservation. This study extends the sample preservation study performed by GeneLab in GLDS-49 which examined conditions used for the RR-1 mission to include conditions used for multiple RR missions and is designed to help determine factors which may confound data analysis. To determine whether these various factors affect changes in gene expression this ground-based study generated gene expression profiles measured by RNAseq from the quadriceps of 20-21 week-old female C57BL/6J mice. Multiple interacting factors were investigated: 1) To understand how euthanasia protocols affect gene expression when mouse carcasses are slow frozen mice were euthanized by either euthasol injection ketamine/xylazine injection or CO2 inhalation and carcasses slow frozen on dry-ice mimicking carcass preservation in the MELFI on the ISS. Carcasses were thawed and RNA extracted from quadriceps; 2) To understand how carcass preservation protocols affect gene expression mice were euthanized with euthasol and carcasses preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater following three-way segmentation. Carcasses were thawed and RNA extracted from quadriceps; 3) To understand how tissue preservation protocols affect gene expression mice were euthanized with euthasol and quadriceps immediately dissected and preserved by flash freezing in liquid nitrogen slow freezing on dry ice or immersion in RNAlater.

While it has been shown that astronauts suffer immune disorders after spaceflight the underlying causes are still poorly understood and there are many variables to consider when investigating the immune system in a complex environment. Additionally there is growing evidence that suggests that not only is the immune system being altered but the pathogens that infect the host are significantly influenced by spaceflight and ground-based spaceflight conditions. In this study we demonstrate that Serratia marcescens (strain Db11) was significantly more lethal to Drosophila melanogaster after growth on the International Space Station than ground-based controls but that the host immune system is not significantly altered amongst known immune genes. High-throughput sequencing of wild-type (w1118) adult hosts infected with either space or ground-reared S. marcescens revealed few changes in gene expression with 11 genes significantly differentially expressed (q-values <0.05) and only one gene related to the immune system. This data supports the main findings of the paper which state that both spaceflight and low-shear modeled microgravity conditions increase the virulence of this pathogen independent of the host immune system. This data which shows that there are no significant immune-related changes to the host when infected with space-grown sample compared to ground-grown sample provides further evidence that there are likely phenotypic changes to the pathogen itself that is causing increased virulence in spaceflight and in low-shear modeled microgravity. RNA was extracted in triplicate from 2 pooled adult (2-3 day old female) Drosophila melanogaster (w1118) per treatment with 4 total treatment groups (no injection control sham injection with PBS ground bacteria-injected and space bacteria-injected) with poly(A)+ RNA libraries. Samples were multiplexed and sequenced 100bp paired-end ready were sequenced on one lane of the Illumina HiSeq-4000.

These data were generated from mice that were skeletally unloaded using antiorthostatic suspension (AOS). Mice were either subjected to AOS or tail restraint without unloading for two weeks before injection with saline xc2 xb1tetanus toxoid xc2 xb1Cpg. Mice were unloaded for an additional two weeks before being sacrificed.

The on-going Microbial Observatory Experiments on the International Space Station (ISS) revealed the presence of various microorganisms that may be affected by the distinct environment of the ISS. The low-nutrient environment combined with enhanced irradiation and microgravity may trigger changes in the molecular suit of microorganisms leading to increased virulence and resistance of microbes. Proteomic characterization of two Aspergillus fumigatus strains ISSFT-021 and IF1SW-F4 isolated from HEPA filter debris and cupola surface of the ISS respectively is presented along with a comparison to experimentally established clinical isolates Af293 and CEA10. In-depth analysis highlights variations in the proteome of both ISS-isolated strains when compared to the clinical strains. Proteins up-regulated in ISS isolates were involved in oxidative stress response and carbohydrate and secondary metabolism. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment. Lastly an attempt was made to elucidate plausible causes of the enhanced virulence of both ISS-isolated A. fumigatus strains.

With extended stays aboard the International Space Station (ISS) becoming commonplace there is a need to better understand the effects of microgravity on cardiac function. We utilized human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of microgravity on cell-level cardiac function and gene expression. The hiPSC-CMs were cultured aboard the ISS for 5.5 weeks and their gene expression structure and functions were compared to ground control hiPSC-CMs. Exposure to microgravity on the ISS caused alterations in hiPSC-CM calcium handling. RNA-sequencing analysis demonstrated 2,635 genes were differentially expressed among flight post-flight and ground control samples including genes involved in mitochondrial metabolism. This study represents the first use of hiPSCs to model the effects of spaceflight on human cardiomyocyte structure and function.

Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of NASA we assayed the genomic and metabolomics perturbations in the quadriceps of C57BL/6j male mice that lived on the spaceflight (FLT) or at Ground Control (CTR) for approximately four weeks. Wet weight of quadriceps were significantly reduced in FLT mice. Deep next generation sequencing and untargeted mass spectroscopic assay interrogated the gene-metabolite landscape of same tissues. A majority of top ranked differentially suppressed genes in FLT encode proteins from myosin or troponin family suggesting a sarcomere alteration in space. Significantly enriched gene-metabolite networks were found linked to saromeric integrity immune fitness and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of FLT mitochondrial DNA copy numbers underlined the energy deprivation associated with spaceflight induced stress and this hypothesis was reinforced by the omics analysis that showed inhibited networks related to protein lipid and carbohydrate metabolism and ATP synthesis and hydrolysis. Finally we reported a list of upstream regulators which could be targeted for next generation therapeutic intervention for the betterment of the musculoskeletal system in male mice subjected to chronic disuse.

Rodent hind limb unloading was used as a model for reduced muscle activity and eventual atrophy. After a 10 day period of unloading mice in this study were xe2 x80 x9creloaded xe2 x80 x9d for 3 days and regained use of their hind limbs. We report the application of Next-generation sequencing (NGS) technology for high-throughput profiling of mRNA in soleus muscle of adult (6 mo) and aged (22-24 mo) mice. Our goal was to determine the effects of hind limb unloading and reloading on mRNA profiles in soleus muscle and compare between adult and aged mice. We find that there are distinct response in the profile of fatty acid oxidation TCA cycle ETC oxidative phosphorylation gene expression patterns in response to unloading and reloading. The repsonses are generally simialr between young and old mice.