The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS and launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of thymus from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=9) ISS Terminal (n=10); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=9). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

Our study aims to comprehensively understand effects induced by the space environment on mammals. To achieve this aim we analyze the male mice housed under environments as the artificial gravity and the microgravity (space environment) in Japanese Experiment Module JEM) of the International Space Station (ISS) on orbit for 35 days. After recovered these mice on the ground transcriptome analysis by next-generation sequencing technology is performed about spleen to examine alteration of gene expression in the space.

Microarray Analysis of Space-flown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors. We used microarrays to detail the gene expression of space-flown thymic tissue and identified distinct classes of up-regulated genes during this process. We report here microarray gene expression analysis in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5 fold or greater change. When these data were averaged (n=4) we identified 12 genes that were significantly up- or down-regulated by at least 1.5 fold after spaceflight (p < 0.05). Together these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress glucocorticoid receptor metabolism and T cell signaling activity. These data explain in part the reported systemic compromise of the immune system after exposure to the microgravity of space.

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

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 were 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). Upon dissection livers were preserved in liquid nitrogen and stored at -80 C before RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed and even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation. CARA Seed Lines and Planting: Three seed lines Wild-Type Wassilewskija (Ws) Columbia-0 (Col-0) and Col-0 PhyD (phyD) Mutants were tested for viability sterility and ability to maintain dormancy before the launch. Tested batches of seeds were planted on phytagel plates as one genotype per plate for gene expression analysis in replicates of three. One set was planted for the flight and one for ground control. The plates were wrapped such that every surface of the plate was covered by two layers of Duvetyn Black-Out cloth (Seattle Fabrics) (Sng et al 2014). The plates were stored 4 xb0 C until launch and was then launched in a cold-stow bag to maintain the plates at 4 xb0 C until integration and activation on the ISS. On Orbit Operations and harvest: The dormant plates were activated on station by removing the Black-Out cloth wrapping 12 days after launch. The plates were then placed on a fabric that was mounted in the US Laboratory module on the wall adjoining the MELFI freezer and secured using Velcro. The plants were allowed to grow on orbit for 11 days; some in the ambient light of ISS and some in the dark. The dark-grown plates were first activated by exposing the seeds to light for 4 hours and then re-wrapped in Black-Out cloth for the duration of the growth period. A corresponding set of seedlings were grown as ground control in KSC. At 11 days seedlings were photographed harvested into KFT containing RNAlater solutions and returned for post-flight analysis. All plates were harvested into KFTs with their counterpart (e.g. Light 1 was harvested with Dark 1). Once the plants were placed in the KFTs the KFT was actuated with RNAlater to preserve the sample. At 24 hours post-harvest KFTs were then transferred to MELFI the -32 xb0C freezer. Following SpaceX-3 splashdown in the Pacific Ocean the KFTs transferred to the Cold Stowage charter plane at

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).

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018,. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis Facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of colon from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=10) ISS Terminal (n= 9); Ground Controls: LAR GC (N=8) ISS Terminal GC (N=9) LAR Baseline (n=9) ISS Terminal Baseline (n=9). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

The objective of the Rodent Research-6 (RR-6) study was to evaluate muscle atrophy in mice during spaceflight and to test the efficacy of a novel therapeutic to mitigate muscle wasting. The experiment involved an implantable subcutaneous nanochannel delivery system (nDS; between scapula) which delivered the drug formoterol (FMT; a selective Beta-2 adrenoceptor agonist) over the course of time. To this end a cohort of forty 32-weeks-old female C57BL/6NTac mice were either sham operated or implanted with vehicle or treatment-filled nDS launched in two Transporters (20 mice per Transporter) on SpaceX-13 on December 15 2017. They were transferred to Rodent Habitats onboard the International Space Station (ISS) and maintained in microgravity for 29 days (N=20 Live Animal Return [LAR]) or >50 days (N=20 ISS Terminal). After 29 days the 20 LAR animals were returned live to back to Earth on January 13 2018,. After splashdown the animals were ambulatory on-ground for ~4 days until all subjects were processed during one day of dissections. There were two Baseline groups of animals sacrificed (LAR Baseline & FLT Baseline; N=20; 40 animals; ~36 weeks old) at Kennedy Space Center (KSC; 12/9/17). A Ground Control group mimicked the Flight LAR group which was housed at KSC then shipped alive to Novartis Facilities where both the LAR and LAR Ground Control groups were processed (~41 weeks old; 1/16/18). All were anesthetized with isoflurane blood samples were obtained by closed-chest cardiac puncture and the animals were euthanized by exsanguination and thoracotomy. The 20 ISS Terminal mice were anesthetized via intraperitoneal injection of ketamine/xylazine/acepromazine over the course of a four days of dissections (2/6/18 until 2/9/18; 53-56 days after launch; 44 weeks old at time of on-orbit dissections). Blood samples and euthanasia were conducted the same as LAR and Baseline. Following blood draw and hind limb dissection the ISS-terminal animal carcasses were wrapped in aluminum foil placed in a ziploc bag and placed in storage at -80C or colder until return. The ISS-terminal Ground Controls (at KSC) followed the same euthanasia timeline methods and preservation. The final processing of frozen ISS-terminal frozen ISS-terminal Ground Controls and frozen 0-day FLT baseline animals were completed at Houston Methodist Research Institute in Houston TX (5/21/18 until 5/24/18). GeneLab received samples of spleen from only sham treated animals (no drug treated animals) from the following groups Flight: LAR (n=9) ISS Terminal (n= 9); Ground Controls: LAR GC (N=9) ISS Terminal GC (N=10) LAR Baseline (n=10) ISS Terminal Baseline (n=6). Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150).

Spaceflight results in a number of adaptations to skeletal muscle including atrophy and shifts towards faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations microarray expression analysis was performed on gastrocnemius from mice flown on the STS-108 shuttle flight (11 days 19 hours) versus mice maintained on earth for the same period. Additionally to identify changes that were due to unloading and reloading microarray analyses were conducted on calf muscle from ground-based mice subjected to hindlimb suspension (12 days) and mice subjected to hindlimb suspension plus a brief period of reloading (3.5 hours) to simulate the time between landing and sacrifice of the spaceflight mice.