When germinated and grown on-board the ISS (International Space Station) plant do not exhibit abnormal structures but they do have altered growth habits and this project aims to investigate the molecular mechanisms that provide the foundation for the altered growth habits observed in orbit. APEX03-2 (Advanced Plant Experiment 03-2) also known as TAGES-ISA (Transgenic Arabidopsis Gene Expression System-Intracellular Signaling Architecture) specifically addresses the growth and molecular changes that occur in Arabidopsis thaliana plants during spaceflight by using molecular and genetic tools and by asking fundamental questions regarding root structure growth and cell wall remodeling may be answered. This investigation advances the fundamental understanding of the molecular biological responses to extraterrestrial environments. This understanding helps to further define the impacts of spaceflight on biological systems to better enable NASA xc3 xaf xc2 xbf xc2 xbds future space exploration goals.

The JAXA MHU-2 mission had two objectives: 1) To increase understanding of effects of spaceflight on the gut environment (microbiota and metabolites) and immune system using multi-omics based analysis; 2) To evaluate whether fructo-oligosaccharides added to the diet as prebiotics improve the gut environment and immune function during spaceflight. Twelve 16-18 week old male C57BL/6J mice were singly housed in the JAXA Habitat Cage Units (HCUs) on the ISS for 30 days. Six flight mice were housed in microgravity while six were exposed to simulated 1g by centrifugation. These two flight groups were further divided in half so that three mice in each group received standard JAXA chow while the other three were fed chow supplemented with fructooligosaccharides (FOS). Mice were returned live and euthanized and dissected <1 day after splashdown. Ground controls (n=6) were asynchronous and housed in HCUs. Vivarium controls (n=6) were asynchronous and housed in standard habitats. Three ground control and three vivarium animals received standard chow while the other three each ground control and vivarium animals received FOS-supplemented chow. Ground and vivarium samples were dissected by a separate dissection team than flight samples. Femoral skin was dissected 30 minutes after euthanasia and snap frozen in liquid nitrogen. Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150). Study Factor Levels: 1)Spaceflight ug Std. Chow: 3; 2)Spaceflight ug FOS: 3; 3) Spaceflight Artificial 1g Std. Chow: 3; 4)Spaceflight Artificial 1g FOS: 3; 5)Ground 1g Std. Chow: 3; 6)Ground 1g FOS: 3; 7)Vivarium 1g Std. Chow: 3; 8)Vivarium 1g FOS: 3

The objective of the Rodent Research-7 mission (RR-7) was to study the impact of the space environment on the gut microbiota of two strains of mice and how any changes in-turn affect the immune system metabolic system and circadian or daily rhythms. To this end ten 11-week-old female C57BL/6J and ten 11-week-old female C3H/HeJ mice were flown to the International Space Station on June 29 2018 on SpaceX-15 and housed in two Rodent Habitats. Samples of food swabs from living surfaces and fecal pellets were collected from each animal before launch and regularly during the mission. The mission also involved extended video collection (48 hr video segments per Habitat) to monitor circadian rhythms and on-orbit mass measurement. After 25 days on-orbit half of the mice of each strain were euthanized on the ISS with Ketamine/Xylazine/Acepromazine and cardiac puncture after which carcasses were segmented in three sections and preserved in RNA later. After 75-76 days the remaining 5 animals from each group were euthanized and processed in the same manner. The 25-day dissected carcasses returned on SpX-15 and the 75-day dissected carcasses returned on SpX-16. In addition to the Flight group three ground control groups were also part of the study: Basal (representing the pre-launch state) Vivarium (standard vivarium housing for the same duration of time as flight) and Ground (same habitat in the International Space Station Environment Simulator ISSES). Twenty mice (10 of each strain) were included in each of these control groups which were euthanized and processed on the same schedule and in the same manner as the flight samples. Dissections for tissues from all experimental groups were completed by the PI groups along with NASA s Biospecimen Sharing Program in February 2019. GeneLab received dorsal skin samples from forty C57BL/6J mice: 10 Basal 5 Ground (25 days) 5 Ground (75 days) 5 Flight (25 days) 5 Flight (75 days) 5 Vivarium (25 days) 5 Vivarium (75 days). GeneLab received dorsal skin samples from forty C3H/HeJ mice: 10 Basal 5 Ground (25 days) 5 Ground (75 days) 5 Flight (25 days) 5 Flight (75 days) 5 Vivarium (25 days) 5 Vivarium (75 days). From these skin samples RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 98 bp).

The JAXA MHU-2 mission had two objectives: 1) To increase understanding of effects of spaceflight on the gut environment (microbiota and metabolites) and immune system using multi-omics based analysis; 2) To evaluate whether fructo-oligosaccharides added to the diet as prebiotics improve the gut environment and immune function during spaceflight. Twelve 16-18 week old male C57BL/6J mice were singly housed in the JAXA Habitat Cage Units (HCUs) on the ISS for 30 days. Six flight mice were housed in microgravity while six were exposed to simulated 1g by centrifugation. These two flight groups were further divided in half so that three mice in each group received standard JAXA chow while the other three were fed chow supplemented with fructooligosaccharides (FOS). Mice were returned live and euthanized and dissected <1 day after splashdown. Ground controls (n=6) were asynchronous and housed in HCUs. Vivarium controls (n=6) were asynchronous and housed in standard habitats. Three ground control and three vivarium animals received standard chow while the other three each ground control and vivarium animals received FOS-supplemented chow. Ground and vivarium samples were dissected by a separate dissection team than flight samples. Dorsal skin was dissected 30 minutes after euthanasia and snap frozen in liquid nitrogen. Total RNA was extracted and sequenced at a target depth of 60 M clusters per sample (ribodepleted paired end 150). Study Factor Levels: 1)Spaceflight ug Std. Chow: 3; 2)Spaceflight ug FOS: 3; 3) Spaceflight Artificial 1g Std. Chow: 3; 4)Spaceflight Artificial 1g FOS: 3; 5)Ground 1g Std. Chow: 3; 6)Ground 1g FOS: 3; 7)Vivarium 1g Std. Chow: 3; 8)Vivarium 1g FOS: 3.

The root apex is an important section of the plant root involved in environmental sensing and cellular development. Analyzing the gene profile of root apex in diverse environments is important and challenging especially when the samples are limiting and precious such as in spaceflight. The feasibility of using tiny root sections for transcriptome analysis was examined in this study. To understand the gene expression profiles of the root apex Arabidopsis thaliana Col-0 roots were sectioned into Zone-I (0.5 mm root cap and meristematic zone) and Zone-II (1.5 mm transition elongation and growth terminating zone). Gene expression was analyzed using microarray and RNA seq. Both the techniques arrays and RNA-Seq identified 4180 common genes as differentially expressed (with > two-fold changes) between the zones. In addition 771 unique genes and 19 novel TARs were identified by RNA-Seq as differentially expressed which were not detected in the arrays. Single root tip zones can be used for full transcriptome analysis; further the root apex zones are functionally very distinct from each other. RNA-Seq provided novel information about the transcripts compared to the arrays. These data will help optimize transcriptome techniques for dealing with small rare samples.

Traveling to nearby extraterrestrial objects having a reduced gravity level (partial gravity) compared to Earth's gravity is becoming a realistic objective for space agencies. The use of plants as part of life support systems will require a better understanding of the interactions among plant growth responses including tropisms, under partial gravity conditions. Here, we present results from our latest space experiments on the ISS, in which seeds of Arabidopsis thaliana were germinated, and seedlings grew for six days under different gravity levels, namely micro-g, several intermediate partial-g levels, and 1g, and were subjected to irradiation with blue light for the last 48 hours. RNA was extracted from 20 samples for subsequent RNAseq analysis. Transcriptomic analysis was performed using the HISAT2-Stringtie-DESeq pipeline. Differentially expressed genes were further characterized for global responses using the GEDI tool, gene networks and for Gene Ontology (GO) enrichment.

The objective of the Rodent Research-5 (RR-5) study was to evaluate bone loss in mice during spaceflight and to determine if treatment with a modified version of NEL-like molecule-1 (NELL-1) can reduce or prevent bone loss that would otherwise occur during spaceflight. To this end a cohort of forty 30-weeks-old female BALB/cAnNTac mice were flown to the ISS and housed in the Rodent Habitat. Six days after launch half of the mice were treated with NELL-1 (10 mg/kg in 0.3 ml PBS) while the other half were treated with vehicle control (0.3 ml PBS). Fourteen days after launch animals were again treated with NELL-1 or vehicle control as before except that all animals were also injected with the bone marker calcein green (20 mg/kg in 0.1 ml). Injections of vehicle NELL-1 and bone markers were intraperitoneal. After all forty mice on orbit received two treatments; ten control mice and ten experimental mice were randomly selected for live animal return (LAR). At approximately 30 days after launch the twenty LAR mice were transported live back to Earth. Animals were allowed to recover for 30 days in standard habitats before euthanasia via intraperitoneal injection with ketamine/xylazine. During the recovery the animals received another two treatments. GeneLab received RNA later preserved femoral skin from nine live animal return and ten matching ground control mice. These were from the vehicle control animals only. RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

The objective of the Rodent Research-5 (RR-5) study was to evaluate bone loss in mice during spaceflight and to determine if treatment with a modified version of NEL-like molecule-1 (NELL-1) can reduce or prevent bone loss that would otherwise occur during spaceflight. To this end a cohort of forty 30-weeks-old female BALB/cAnNTac mice were flown to the ISS and housed in the Rodent Habitat. Six days after launch half of the mice were treated with NELL-1 (10 mg/kg in 0.3 ml PBS) while the other half were treated with vehicle control (0.3 mls PBS). Fourteen days after launch animals were again treated with NELL-1 or vehicle control as before except that all animals were also injected with the bone marker calcein green (20 mg/kg in 0.1 ml). Injections of vehicle NELL-1 and bone markers were intraperitoneal. After all forty mice on orbit received two treatments; ten control mice and ten experimental mice were randomly selected for live animal return (LAR). At approximately 30 days after launch the twenty LAR mice were transported live back to Earth. Animals were allowed to recover for 30 days in standard habitats before euthanasia via intraperitoneal injection with ketamine/xylazine. During the recovery the animals received another two treatments. GeneLab received RNA later preserved dorsal skin from ten live animal return and ten matching ground control mice. These were from the vehicle control animals only. RNA was extracted libraries generated (stranded ribodepleted) and sequenced (target 60 M clusters at PE 150 bp).

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.