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Investigated medical kits / drugs

Medicine Name	Medicine Category	Study ID	Study Title	Investigation Class	Investigation Abstract
Monorden	Experimental Drug	SLID-300	The effects of space flight on the production of monorden by Humicola fuscoatra WC5157 in solid-state fermentation	Fungi	The effect of space flight on the production of the antibiotic monorden on two types of agar media, T8 and PG, by Humicola fuscoatra WC5157 was examined on board the US Space Shuttle mission STS-77 in May 1996. Paired space-flight and ground control samples were prepared using identical hardware, protocol, media, and inoculum. Inoculation occurred simultaneously for both groups 2.5 after launch. The flight and ground samples were allowed to grow for the entire 10-day mission in a dark, thermally controlled (22 degrees C) environment. Post-flight HPLC analysis of the flight and ground sample extracts indicated that the production of monorden by H. fuscoatra WC5157 in the flight samples was higher than in the ground samples in both agar media. In the T8 medium, the production of monorden in the flight and ground samples was 11.6 +/- 3.5 micrograms and 8.9 +/- 1.1 micrograms respectively (30% increase). In the PG medium, the production of monorden in the flight and ground samples was 23.8 +/- 3.3 micrograms and 8.2 +/- 2.2 micrograms respectively (190% increase). The production of monorden in the flight and ground control samples was confirmed by HPLC-MS analysis.
Actinomycin D	Therapeutic Drug	SLID-303	The effect of space flight on the production of actinomycin D by Streptomyces plicatus	Bacteria	The effect of space flight on production of the antibiotic actinomycin D by Streptomyces plicatus WC56452 was examined onboard the US Space Shuttle mission STS-80. Paired space flight and ground control samples were similarly prepared using identical hardware, media, and inoculum. The cultures were grown in defined and complex media under dark, anaerobic, thermally controlled (20 degrees C) conditions with samples fixed after 7 and 12 days in orbit, and viable residuals maintained through landing at 17 days, 15 h. Postflight analyses indicated that space flight had reduced the colony-forming unit (CFU) per milliliter count of S. plicatus and increased the specific productivity (pg CFU(-1)) of actinomycin D. The antibiotic compound itself was not affected, but its production time course was altered in space. Viable flight samples also maintained their sporulation ability when plated on agar medium postflight, while the residual ground controls did not sporulate.
Actinomycin D	Therapeutic Drug	SLID-305	Microbial antibiotic production aboard the International Space Station	Bacteria	Previous studies examining metabolic characteristics of bacterial cultures have mostly suggested that reduced gravity is advantageous for microbial growth. As a consequence, the question of whether space flight would similarly enhance secondary metabolite production was raised. Results from three prior space shuttle experiments indicated that antibiotic production was stimulated in space for two different microbial systems, albeit under suboptimal growth conditions. The goal of this latest experiment was to determine whether the enhanced productivity would also occur with better growth conditions and over longer durations of weightlessness. Microbial antibiotic production was examined onboard the International Space Station during the 72-day 8A increment. Findings of increased productivity of actinomycin D by Streptomyces plicatus in space corroborated with previous findings for the early sample points (days 8 and 12); however, the flight production levels were lower than the matched ground control samples for the remainder of the mission. The overall goal of this research program is to elucidate the specific mechanisms responsible for the initial stimulation of productivity in space and translate this knowledge into methods for improving efficiency of commercial production facilities on Earth.
Kanglemycin C	Bioactive Molecule	SLID-306	Preliminary report on the biological effects of space flight on the producing strain of a new immunosuppressant, Kanglemycin C	Bacteria	Kanglemycin C (K-C) is a new immunosuppressant isolated from the culture broth of Nocardia mediterranei var. kanglensis 1747-64. To improve the productivity of K-C and to study the biological effects of space flight on its producing strain, spores from five K-C producing strains (U-10, U-15, U-7, M-13, γ-33) mutated from the wild strain N. mediterranei var. kanglensis 1747-64 were carried into space by an unmanned spaceship, “Shenzhou III” (Divine Vessel III) on March 25, 2002. Comparatively, the strain U-7 was the highest K-C producing strain among the above five starting strains when cultivated in 500-ml Erlenmeyer flasks. After a 6 day and 18 h flight, the treated spores went through serial screening processes to screen for high-yield K-C mutant strains, using thin layer chromatography and high performance liquid chromatography (HPLC). The K-C yield produced by one mutant strain, designated as F-16, derived from the starting strain U-7 was increased by up to 200% when compared to that produced by the starting strain U-7 in 500-ml Erlenmeyer flasks after careful postflight HPLC analysis. Another mutant strain, designated as F-210, derived from the starting strain M-13 showed reduced productivity of K-C as well as exhibited changes in some morphological and physiological characteristics. For example, the broth color of the strain F-210 changed from yellow to purple after 96 h of culture, but that of the ground control strain M-13 remained yellow. Similarly, the mycelium morphological change from filamentous to coccoid of F-210 occurred later than that of ground control M-13. Examination of the survivability of postflight spores indicated that exposure to radiation, during the 162 h of space flight, plays a critical role in the survival rates of spores such that spores exposed to strong radiation exhibited lower survival rates than spores exposed to weak radiation.
2-keto-L-gulonic acid	Bioactive Molecule	SLID-317	Spaceflight-induced enhancement of 2-keto-L-gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis	Bacteria	Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l−1 h−1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m3 fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.
Pyranonigrin A	Bioactive Molecule	SLID-359	Metabolomic Analysis of Aspergillus niger Isolated From the International Space Station Reveals Enhanced Production Levels of the Antioxidant Pyranonigrin A	Fungi	Secondary metabolite (SM) production in Aspergillus niger JSC-093350089, isolated from the International Space Station (ISS), is reported, along with a comparison to the experimentally established strain ATCC 1015. The analysis revealed enhanced production levels of naphtho-γ-pyrones and therapeutically relevant SMs, including bicoumanigrin A, aurasperones A and B, and the antioxidant pyranonigrin A. Genetic variants that may be responsible for increased SM production levels in JSC-093350089 were identified. These findings include INDELs within the predicted promoter region of flbA, which encodes a developmental regulator that modulates pyranonigrin A production via regulation of Fum21. The pyranonigrin A biosynthetic gene cluster was confirmed in A. niger, which revealed the involvement of a previously undescribed gene, pyrE, in its biosynthesis. UVC sensitivity assays enabled characterization of pyranonigrin A as a UV resistance agent in the ISS isolate.
Natamycin	Therapeutic Drug	SLID-364	Space-flight Mutation of Streptomyces gilvosporeus for Enhancing Natamycin Production	Bacteria	Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the survival ratio was 43.1% and 3.0%, respectively. An improved mutant producing natamycin, S. gilvosporeus LK-45, was screened, which showed natamycin productivity of 1420mg · L−1. A mutant resistant to 2-deoxy glucose, S. gilvosporeus LK-119, was further obtained using a rational screening procedure. The natamycin productivity of 1940mg · L−1 was achieved when glucose was used as the carbon source.
Acetaminophen	Therapeutic Drug	SLID-370	Pharmacokinetics of acetaminophen administered in tablets and capsules under long-term space flight conditions	Therapeutic drug	The pharmacokinetics of acetaminophen in two medicinal forms, tablets and capsules, have been studied in healthy volunteers after single peroral administration at a dose of 499 mg under usual living conditions and during long-term space flight. The rate of drug absorption from tablets decreases significantly whereas the relative bioavailability increases substantially under microgravity conditions (compared with usual conditions). For the encapsulated medicinal form, the time of absorption decreases statistically reliably and the half-elimination time, the average retention time, and the distribution volume increase considerably whereas the bioavailability changes insignificantly.
Phenytoin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Ciprofloxacin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Clotrimazole	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Fluconazole	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Furosemide	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Ibuprofen	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Lidocaine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Metoprolol succinate	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Metronidazole	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Promethazine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Risedronic acid	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Sulfamethoxazole	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Temazepam	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Trimethoprim	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Epinephrine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Levothyroxine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Dextroamphetamine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Triamcinolone	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Amoxicillin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Cefadroxil	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Atorvastatin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Sertraline	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Imipenem	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Levofloxacin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Silver sulfadiazine	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Mupirocin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Azithromycin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Clavulanate	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Vitamin B12	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Cilastatin	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Acyclovir	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Progestin/estrogen	Therapeutic Drug	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Multivitamin	Health Supplement	SLID-372	Stability of vitamin B complex in multivitamin and multimineral supplement tablets after space flight	Health Supplement	The effect of storage in space on the stability of vitamin B complex in two commercial vitamin tablets was examined. Multiple vitamin samples returned after storage on the space shuttle and International Space Station (ISS) along with two ground control and three positive control groups were included in the study. Content of vitamin B(3) in the tablets and in vitro dissolution rate were determined using a modified high performance liquid chromatographic assay from USP/NF 2010. Results indicate that vitamin B(3) in one of the brands tested (#2) may be subject to marginal degradation after storage on ISS for 4 months as indicated by the chromatograms for all six tablets showing a split peak appearing as a notch at the peak tip. Chromatograms were not different for ground and flight samples for Brand #1 suggesting that this may be more suitable for use in space.
Melatonin	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Acetaminophen	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Aspirin	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Ibuprofen	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Meclizine	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Promethazine	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Zaleplon	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Zolpidem	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Scopolamine	Therapeutic Drug	SLID-373	Medication use by U.S. crewmembers on the International Space Station	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Melatonin	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Acetaminophen	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Aspirin	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Ibuprofen	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Loperamide	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Loratadine	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Modafinil	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Zolpidem	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Pseudoephedrine	Therapeutic Drug	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Acetaminophen	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Aspirin	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Diphenhydramine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Lidocaine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Meperidine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Procainamide	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Proparacaine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Secobarbital	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Tetrahydrozoline	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Dextroamphetamine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Ampicillin	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Cyclizine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Pseudoephedrine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Dextropropoxyphene	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Diphenoxylate	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Atropine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Scopolamine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Triprolidine	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Simethicone	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Bacitracin	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Methylcellulose	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Tetracycline	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Band-Aids Adhesive bandage	Medical Supply	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Compress bandages	Medical Supply	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Antibiotic	Therapeutic Drug	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Multivitamin	Health Supplement	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Skin cream	Medical Supply	SLID-375	Principles of Clinical Medicine for Space Flight	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Melatonin	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Quetiapine	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Temazepam	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Zaleplon	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Zolpidem	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Eszopiclone	Therapeutic Drug	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Ciprofloxacin	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Loperamide	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Ondansetron	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Prochlorperazine	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Dexamethasone	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Silver sulfadiazine	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Dolasetron	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Granisetron	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Palonosetron	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Cephalosporin	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Corticosteroid	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Macrolides	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Normal saline	Medical Supply	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Parenteral opioid analgesics	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Penicillins	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Recombinant human granulocyte macrophage colony stimulating factor (GM-CSF)	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Recombinant human granulocyte colony stimulating factor (G-CSF)	Therapeutic Drug	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Sterile gauze	Medical Supply	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Acetaminophen	Therapeutic Drug	SLID-378	Physiological, pharmacokinetic, and pharmacodynamic changes in space	Therapeutic drug	The objectives of this investigation were to 1) determine changes in GI motility during space flight using a noninvasive lactulose breath-hydrogen test, 2) determine absorption, bioavailability, and elimination of acetaminophen during space flight, 3) determine hepatic metabolic activity during space flight by measuring the clearance of antipyrine after an oral dose, and 4) correlate functional changes in the GI tract and liver with the absorption and metabolism of acetaminophen. (Both acetaminophen and antipyrine are substances used to reduce fever and relieve pain.). [Conclusion]: The limited inflight data collected to date, although indicating trends of GI and hepatic function changes during flight, are inadequate to characterize the degree and magnitude of such changes. The mechanisms underlying these changes are difficult to identify because of the complexity of these changes as well as the large number of variables that may influence disposition profiles and kinetic parameters during flight. Further investigation is required to generate information that will be useful for the development of pharmaceutical and nutritional countermeasures for microgravity-induced deconditioning.
Antipyrine	Therapeutic Drug	SLID-378	Physiological, pharmacokinetic, and pharmacodynamic changes in space	Therapeutic drug	The objectives of this investigation were to 1) determine changes in GI motility during space flight using a noninvasive lactulose breath-hydrogen test, 2) determine absorption, bioavailability, and elimination of acetaminophen during space flight, 3) determine hepatic metabolic activity during space flight by measuring the clearance of antipyrine after an oral dose, and 4) correlate functional changes in the GI tract and liver with the absorption and metabolism of acetaminophen. (Both acetaminophen and antipyrine are substances used to reduce fever and relieve pain.). [Conclusion]: The limited inflight data collected to date, although indicating trends of GI and hepatic function changes during flight, are inadequate to characterize the degree and magnitude of such changes. The mechanisms underlying these changes are difficult to identify because of the complexity of these changes as well as the large number of variables that may influence disposition profiles and kinetic parameters during flight. Further investigation is required to generate information that will be useful for the development of pharmaceutical and nutritional countermeasures for microgravity-induced deconditioning.
Promethazine	Therapeutic Drug	SLID-379	Bioavailability of Promethazine during Spaceflight	Therapeutic drug	Promethazine (PMZ) is the choice anti-motion sickness medication for treating space motion sickness (SMS) during flight. The side effects associated with PMZ include dizziness, drowsiness, sedation, and impaired psychomotor performance which could impact crew performance and mission operations. Early anecdotal reports from crewmembers indicate that these central nervous system side effects of PMZ are absent or greatly attenuated in microgravity, potentially due to changes in pharmacokinetics (PK) and pharmacodynamics in microgravity. These changes could also affect the therapeutic effectiveness of drugs in general and PMZ, in particular. In this investigation, we examined bioavailability and associated pharmacokinetics of PMZ in astronauts during and after space flight. Methods. Nine astronauts received, per their preference, PMZ (25 or 50 mg as intramuscular injection, oral tablet, or rectal suppository) on flight day one for the treatment of SMS and subsequently collected saliva samples and completed sleepiness scores for 72 h post dose. Thirty days after the astronauts returned to Earth, they repeated the protocol. Bioavailability and PK parameters were calculated and compared between flight and ground. Results. Maximum concentration (Cmax) was lower and time to reach Cmax (tmax) was longer in flight than on the ground. Area under the curve (AUC), a measure of bioavailability, was lower and biological half-life (t1/2) was longer in flight than on the ground. Conclusion. Results indicate that bioavailability of PMZ is reduced during spaceflight. Number of samples, sampling method, and sampling schedule significantly affected PK parameter estimates.
Acetaminophen	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Aspirin	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Caffeine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Diphenhydramine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Lidocaine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Meperidine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Oxymetazoline	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Phenacetin	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Procainamide	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Proparacaine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Secobarbital	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Tetrahydrozoline	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Dextroamphetamine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Ampicillin	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Cyclizine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Pseudoephedrine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Dextropropoxyphene	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Diphenoxylate	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Atropine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Scopolamine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Triprolidine	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Simethicone	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Bacitracin	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Methylcellulose	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Polymyxin B	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Tetracycline	Therapeutic Drug	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Urine collection and transfer devices	Medical Supply	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Band-Aids	Medical Supply	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Compress bandages	Medical Supply	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Multivitamin	Health Supplement	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Skin cream	Medical Supply	SLID-380	Spaceflight Medical Systems	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Dextroamphetamine	Therapeutic Drug	SLID-392	Inflight Salivary Pharmacokinetics of Scopolamine and Dextroamphetamine	Therapeutic drug	n.a.
Scopolamine	Therapeutic Drug	SLID-392	Inflight Salivary Pharmacokinetics of Scopolamine and Dextroamphetamine	Therapeutic drug	n.a.
Acetaminophen	Therapeutic Drug	SLID-393	In-flight Pharmacokinetics of Acetaminophen in Saliva	Therapeutic drug	n.a.
Bupropion	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Melatonin	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Buspirone	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Caffeine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Clonidine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Diazepam	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Diphenhydramine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Valproic acid	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Haloperidol	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Hydroxyzine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Lorazepam	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Mirtazapine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Modafinil	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Prazosin	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Propranolol	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Trazodone	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Venlafaxine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Zaleplon	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Zolpidem	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Paroxetine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Aripiprazole	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Ziprasidone	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Sertraline	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Benzatropine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.
Olanzapine	Therapeutic Drug	SLID-394	A Psychiatric Formulary for Deep Space	Therapeutic drug	n.a.

All investigations involved in a specific flightspace program can be further browsed by clicking "Project Name" in the 2nd column.
Individual Investigations can be accessed by clicking "Study ID" in the 3th column.

Browse space investigations by spaceflight missions

Project Type	Project Name	Study ID	Study Title	Flight Duration	Investigation Class	Investigation Abstract
Spaceflight Study	International Space Station (ISS)	SLID-001	The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight	n.a.	Health impact of long-duration spaceflight (epigenomic, metabolomic, transcriptomic, proteomic, molecular, microbiome changes, and physiological, cognitive changes )	To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress–related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.
Spaceflight Study	n.a.	SLID-002	Eye-Head Coordination in 31 Space Shuttle Astronauts during Visual Target Acquisition	n.a.	Eye-Head coordination changes	Between 1989 and 1995, NASA evaluated how increases in flight duration of up to 17 days affected the health and performance of Space Shuttle astronauts. Thirty-one Space Shuttle pilots participating in 17 space missions were tested at 3 different times before flight and 3 different times after flight, starting within a few hours of return to Earth. The astronauts moved their head and eyes as quickly as possible from the central fixation point to a specified target located 20°, 30°, or 60° off center. Eye movements were measured with electro-oculography (EOG). Head movements were measured with a triaxial rate sensor system mounted on a headband. The mean time to visually acquire the targets immediately after landing was 7–10% (30–34 ms) slower than mean preflight values, but results returned to baseline after 48 hours. This increase in gaze latency was due to a decrease in velocity and amplitude of both the eye saccade and head movement toward the target. Results were similar after all space missions, regardless of length.
Spaceflight Study	Shuttle Missions	SLID-003	Short-duration spaceflight does not prolong QTc intervals in male astronauts	5 - 10 Days	QTc intervals changes	Although ventricular dysrhythmias are not increased during, and QTc intervals are not prolonged after, short-duration (5 to 16 days) spaceflights, QTc intervals have not previously been reported during these shorter flights. Holter monitor recordings, obtained in 11 male astronauts who flew on shuttle missions ranging from 5 to 10 days, showed that QTc intervals did not change significantly 10 days before launch, on 2 separate days of spaceflight, and 2 days after landing. Taken together, these data and our previous report show that QTc interval prolongation occurs sometime between the 9th and 30th days of spaceflight.
Spaceflight Study	Space Transport System-78	SLID-004	Effects of 17-day spaceflight on knee extensor muscle function and size	17 Days	Muscle strength changes	It is generally held that space travelers experience muscle dysfunction and atrophy during exposure to microgravity. However, observations are scarce and reports somewhat inconsistent with regard to the time course, specificity and magnitude of such changes. Hence, we examined four male astronauts (group mean ~43 years, 86 kg and 183 cm) before and after a 17-day spaceflight (Space Transport System-78). Knee extensor muscle function was measured during maximal bilateral voluntary isometric and iso-inertial concentric, and eccentric actions. Cross-sectional area (CSA) of the knee extensor and flexor, and gluteal muscle groups was assessed by means of magnetic resonance imaging. The decrease in strength (P<0.05) across different muscle actions after spaceflight amounted to 10%. Eight ambulatory men, examined on two occasions 20 days apart, showed unchanged (P>0.05) muscle strength. CSA of the knee extensor and gluteal muscles, each decreased (P<0.05) by 8%. Knee flexor muscle CSA showed no significant (P>0.05) change. The magnitude of these changes concord with earlier results from ground-based studies of similar duration. The results of this study, however, do contrast with the findings of no decrease in maximal voluntary ankle plantar flexor force previously reported in the same crew.
Spaceflight Study	Salyut and Mir Orbital Stations	SLID-005	Microbiological status of cosmonauts during orbital spaceflights on Salyut and Mir orbital stations	n.a.	Changes of microflora	The main feature of the human–microorganism eco-system forming in the environment of manned space vehicles is the periodic accumulation of the potential for pathogenicity. This process is characterized by the activation of opportunistic pathogens, representatives of which grow in large number within the system, settle in various niches, and demonstrate expansion, intruding into microbiocenoses of open biotopes of humans who, until contamination, have been clean of these microorganisms. Clones (similar to hospital strains) arising out of these populations exhibit the ability to spread “epidemically” within isolated teams.
Spaceflight Study	Salyut and Mir Orbital Stations	SLID-005	Microbiological status of cosmonauts during orbital spaceflights on Salyut and Mir orbital stations	n.a.	Changes of microflora	The main feature of the human–microorganism eco-system forming in the environment of manned space vehicles is the periodic accumulation of the potential for pathogenicity. This process is characterized by the activation of opportunistic pathogens, representatives of which grow in large number within the system, settle in various niches, and demonstrate expansion, intruding into microbiocenoses of open biotopes of humans who, until contamination, have been clean of these microorganisms. Clones (similar to hospital strains) arising out of these populations exhibit the ability to spread “epidemically” within isolated teams.
Spaceflight Study	n.a.	SLID-006	Protein expression changes caused by spaceflight as measured for 18 Russian cosmonauts	n.a.	Protein expression changes	The effects of spaceflight on human physiology is an increasingly studied field, yet the molecular mechanisms driving physiological changes remain unknown. With that in mind, this study was performed to obtain a deeper understanding of changes to the human proteome during space travel, by quantitating a panel of 125 proteins in the blood plasma of 18 Russian cosmonauts who had conducted long-duration missions to the International Space Station. The panel of labeled prototypic tryptic peptides from these proteins covered a concentration range of more than 5 orders of magnitude in human plasma. Quantitation was achieved by a well-established and highly-regarded targeted mass spectrometry approach involving multiple reaction monitoring in conjunction with stable isotope-labeled standards. Linear discriminant function analysis of the quantitative results revealed three distinct groups of proteins: 1) proteins with post-flight protein concentrations remaining stable, 2) proteins whose concentrations recovered slowly, or 3) proteins whose concentrations recovered rapidly to their pre-flight levels. Using a systems biology approach, nearly all of the reacting proteins could be linked to pathways that regulate the activities of proteases, natural immunity, lipid metabolism, coagulation cascades, or extracellular matrix metabolism.
Spaceflight Study	n.a.	SLID-007	Stress Related Shift Toward Inflammaging in Cosmonauts After Long-Duration Space Flight	Median mission duration 162 Days	Immune function changes	Space flight exerts a specific conglomerate of stressors on humans that can modulate the immune system. The mechanism remains to be elucidated and the consequences for cosmonauts in the long term are unclear. Most of the current research stems from short-term spaceflights as well as pre- and post-flight analyses due to operational limitations. Immune function of 12 cosmonauts participating in a long-duration (>140 days) spaceflight mission was monitored pre-, post-, and on two time-points in-flight. While the classical markers for stress such as cortisol in saliva where not significantly altered, blood concentrations of the endocannabinoid system (ECS) were found to be highly increased in-flight indicating a biological stress response. Moreover, subjects showed a significant rise in white blood cell counts. Neutrophils, monocytes and B cells increased by 50% whereas NK cells dropped by nearly 60% shortly after landing. Analysis of blood smears showed that lymphocyte percentages, though unchanged pre- and post-flight were elevated in-flight. Functional tests on the ground revealed stable cellular glutathione levels, unaltered baseline and stimulated ROS release in neutrophils but an increased shedding of L-selectin post-flight. In vitro stimulation of whole blood samples with fungal antigen showed a highly amplified TNF and IL-1β response. Furthermore, a significant reduction in CD4+CD25+CD27low regulatory T cells was observed post-flight but returned to normal levels after one month. Concomitantly, high in-flight levels of regulatory cytokines TGF-β, IL-10 and IL-1ra dropped rapidly after return to Earth. Finally, we observed a shift in the CD8+ T cell repertoire toward CD8+ memory cells that lasted even one month after return to Earth.
Spaceflight Study	International Space Station (ISS)	SLID-008	Temporal Telomere and DNA Damage Responses in the Space Radiation Environment	1 - Year or 6 - mont Hours	Telomere length dynamics changes, DNA damage responses	Telomeres, repetitive terminal features of chromosomes essential for maintaining genome integrity, shorten with cell division, lifestyle factors and stresses, and environmental exposures, and so they provide a robust biomarker of health, aging, and age-related diseases. We assessed telomere length dynamics (changes over time) in three unrelated astronauts before, during, and after 1-year or 6-month missions aboard the International Space Station (ISS). Similar to our results for National Aeronautics and Space Administration’s (NASA’s) One-Year Mission twin astronaut (Garrett-Bakelman et al., 2019), significantly longer telomeres were observed during spaceflight for two 6-month mission astronauts. Furthermore, telomere length shortened rapidly after return to Earth for all three crewmembers and, overall, telomere length tended to be shorter after spaceflight than before spaceflight. Consistent with chronic exposure to the space radiation environment, signatures of persistent DNA damage responses were also detected, including mitochondrial and oxidative stress, inflammation, and telomeric and chromosomal aberrations, which together provide potential mechanistic insight into spaceflight-specific telomere elongation.
Spaceflight Study	International Space Station (ISS)	SLID-009	Cardiovascular autonomic control after short-duration spaceflights	10 - 11 Days	Heart rate variability, blood pressure variability and baroreflex sensitivity (BRS)	After spaceflight, astronauts sometimes suffer a variable degree of reduced orthostatic tolerance. Although many studies have addressed this problem, many aspects remain unclear. Also, it is unknown how long the cardiovascular system needs to recover from short duration spaceflights. The scope of the present study was to determine a long-term follow-up of cardiovascular control up to 25 days after spaceflight under control conditions in five astronauts using heart rate variability, blood pressure variability and baroreflex sensitivity (BRS) indices. In standing position heart rate after spaceflight was significantly higher compared with pre-flight (R+1: 99 (SD 9) BPM vs L-30: 77 (SD 3) BPM; p<0.001). At the same time high frequency modulation of heart rate was extremely depressed (R+1: 70 (SD 334) ms2 vs L-30: 271 (SD 68) ms2; p<0.01), as was BRS: (R+1: 5 (SD 1) vs L-30: 10 (SD 2) ms/mmHg, p<0.05). These changes had largely recovered after 4 days upon return to Earth. Orthostatic blood pressure control was well maintained from the first day after landing. The decrease in BRS and in vagal heart rate modulation following short-duration spaceflight appear to constitute an adequate autonomic neural response to restored gravity. After 4 days upon return to Earth, vagal heart rate modulation is almost completely recovered to the pre-flight level. The findings of the present study demonstrate that the decrease in vagal heart rate modulation in standing position should not be characterised as some kind of cardiovascular deconditioning, but rather as the normal response to orthostatic stress after spaceflight.
Spaceflight Study	n.a.	SLID-010	Impairments of manual tracking performance during spaceflight: more converging evidence from a 20-day space mission	20 Days	Frequency response analyses of tracking performance	Studies of human performance during spaceflight have consistently revealed degradations of manual tracking performance in space. The present investigation analysed these performance decrements in more detail by applying frequency-response analyses of tracking performance. It was hypothesized that tracking impairments result from two factors: at an early adaptation phase in space they primarily reflect effects of microgravity on human visuo-motor processes, whereas later into the mission they are also caused by impairments of attentional processes induced by cumulative workload and fatigue. In order to investigate this hypothesis, performance of one cosmonaut in a first-order unstable tracking task was repeatedly assessed before, during and after a 20-day space mission. Single-case statistical analyses revealed the following effects: tracking performance declined at the first assessment in space and in two later inflight sessions compared to pre-flight baseline. Whereas the early tracking decrement was mainly due to an increase of the effective time-delay during tracking and accompanied by only minor changes of mood or workload, one of the later inflight impairments was due to an increase of effective time-delay, a decreased tracking gain, and an increase of tracking remnant, and both were associated with considerably higher workload ratings. This pattern of effects supports the two-factor hypothesis.
Spaceflight Study	International Space Station (ISS)	SLID-011	Ocular Counter Rolling in Astronauts After Short- and Long-Duration Spaceflight	113 - 286 Days (mean 165.8 Days)	Ocular counter-rolling (OCR) changes	Ocular counter-rolling (OCR) is a reflex generated by the activation of the gravity sensors in the inner ear that stabilizes gaze and posture during head tilt. We compared the OCR measures that were obtained in 6 astronauts before, during, and after a spaceflight lasting 4-6 days with the OCR measures obtained from 6 astronauts before and after a spaceflight lasting 4-9 months. OCR in the short-duration fliers was measured using the afterimage method during head tilt at 15°, 30°, and 45°. OCR in the long-duration fliers was measured using video-oculography during whole body tilt at 25°. A control group of 7 subjects was used to compare OCR measures during head tilt and whole body tilt. No OCR occurred during head tilt in microgravity, and the response returned to normal within 2 hours of return from short-duration spaceflight. However, the amplitude of OCR was reduced for several days after return from long-duration spaceflight. This decrease in amplitude was not accompanied by changes in the asymmetry of OCR between right and left head tilt. These results indicate that the adaptation of otolith-driven reflexes to microgravity is a long-duration process.
Spaceflight Study	Space Shuttle Mission	SLID-011	Ocular Counter Rolling in Astronauts After Short- and Long-Duration Spaceflight	4 - 6 Days (mean 4.9 Days)	Ocular counter-rolling (OCR) changes	Ocular counter-rolling (OCR) is a reflex generated by the activation of the gravity sensors in the inner ear that stabilizes gaze and posture during head tilt. We compared the OCR measures that were obtained in 6 astronauts before, during, and after a spaceflight lasting 4-6 days with the OCR measures obtained from 6 astronauts before and after a spaceflight lasting 4-9 months. OCR in the short-duration fliers was measured using the afterimage method during head tilt at 15°, 30°, and 45°. OCR in the long-duration fliers was measured using video-oculography during whole body tilt at 25°. A control group of 7 subjects was used to compare OCR measures during head tilt and whole body tilt. No OCR occurred during head tilt in microgravity, and the response returned to normal within 2 hours of return from short-duration spaceflight. However, the amplitude of OCR was reduced for several days after return from long-duration spaceflight. This decrease in amplitude was not accompanied by changes in the asymmetry of OCR between right and left head tilt. These results indicate that the adaptation of otolith-driven reflexes to microgravity is a long-duration process.
Spaceflight Study	Shuttle Missions	SLID-012	Mechanism of spaceflight-induced changes in left ventricular mass	9 - 16 Days	Left ventricular (LV) mass changes	Decrements in left ventricular (LV) mass observed after microgravity exposure have been previously postulated to be a central component of spaceflight-induced cardiovascular deconditioning. In this study, echocardiographic measurements of LV mass in astronauts demonstrated a comparative 9.1% reduction in postflight LV mass that returned to preflight values by the third day of recovery. A ground-based study in normal subjects determined that these pre- to postflight LV mass changes could be reproduced by simple dehydration. Reductions in LV mass observed immediately after spaceflight may be secondary to simple physiologic fluid exchanges.
Spaceflight Study	n.a.	SLID-013	Immune system dysregulation occurs during short duration spaceflight on board the space shuttle	10 - 15 Days	Leukocyte distribution, T cell blastogenesis and cytokine production	Background: Post-flight data suggests immunity is dysregulated immediately following spaceflight, however this data may be influenced by the stress effects of high-G entry and readaptation to terrestrial gravity. It is unknown if immunity is altered during spaceflight. Methods: Blood samples were collected from 19 US Astronauts onboard the Space Shuttle ~24 h prior to landing and returned for terrestrial analysis. Assays consisted of leukocyte distribution, T cell blastogenesis and cytokine production profiles. Results: Most bulk leukocyte subsets (WBC, differential, lymphocyte subsets) were unaltered during spaceflight, but were altered following landing. CD8+ T cell subsets, including cytotoxic, central memory and senescent were altered during spaceflight. T cell early blastogenesis varied by culture mitogen. Functional responses to staphylococcal enterotoxin were reduced during and following spaceflight, whereas response to anti-CD3/28 antibodies was elevated post-flight. The level of virus specific T cells were generally unaltered, however virus specific T cell function was depressed both during and following flight. Plasma levels of IFNα, IFNγ, IL-1β, IL-4, IL-10, IL-12, and TNFα were significantly elevated in-flight, while IL-6 was significantly elevated at R + 0. Cytokine production profiles following mitogenic stimulation were significantly altered both during, and following spaceflight. Specifically, production of IFNγ, IL-17 and IL-10 were reduced, but production of TNFα and IL-8 were elevated during spaceflight. Conclusions: This study indicates that specific parameters among leukocyte distribution, T cell function and cytokine production profiles are altered during flight. These findings distinguish in-flight dysregulation from stress-related alterations observed immediately following landing.
Spaceflight Study	International Space Station (ISS)	SLID-014	Latent virus reactivation in astronauts on the international space station	180 Days	Latent viral reactivation	Reactivation of latent herpes viruses was measured in 23 astronauts (18 male and 5 female) before, during, and after long-duration (up to 180 days) spaceflight onboard the international space station . Twenty age-matched and sex-matched healthy ground-based subjects were included as a control group. Blood, urine, and saliva samples were collected before, during, and after spaceflight. Saliva was analyzed for Epstein–Barr virus, varicella-zoster virus, and herpes simplex virus type 1. Urine was analyzed for cytomegalovirus. One astronaut did not shed any targeted virus in samples collected during the three mission phases. Shedding of Epstein–Barr virus, varicella-zoster virus, and cytomegalovirus was detected in 8 of the 23 astronauts. These viruses reactivated independently of each other. Reactivation of Epstein–Barr virus, varicella-zoster virus, and cytomegalovirus increased in frequency, duration, and amplitude (viral copy numbers) when compared to short duration (10 to 16 days) space shuttle missions. No evidence of reactivation of herpes simplex virus type 1, herpes simplex virus type 2, or human herpes virus 6 was found. The mean diurnal trajectory of salivary cortisol changed significantly during flight as compared to before flight (P = 0.010). There was no statistically significant difference in levels of plasma cortisol or dehydoepiandosterone concentrations among time points before, during, and after flight for these international space station crew members, although observed cortisol levels were lower at the mid and late-flight time points. The data confirm that astronauts undertaking long-duration spaceflight experience both increased latent viral reactivation and changes in diurnal trajectory of salivary cortisol concentrations.
Spaceflight Study	Russian Soyuz Vehicle	SLID-015	Alterations in adaptive immunity persist during long-duration spaceflight	6 Months	Immune system alterations	Background: It is currently unknown whether immune system alterations persist during long-duration spaceflight. In this study various adaptive immune parameters were assessed in astronauts at three intervals during 6-month spaceflight on board the International Space Station (ISS). AIMS: To assess phenotypic and functional immune system alterations in astronauts participating in 6-month orbital spaceflight. Methods: Blood was collected before, during, and after flight from 23 astronauts participating in 6-month ISS expeditions. In-flight samples were returned to Earth within 48 h of collection for immediate analysis. Assays included peripheral leukocyte distribution, T-cell function, virus-specific immunity, and mitogen-stimulated cytokine production profiles. Results: Redistribution of leukocyte subsets occurred during flight, including an elevated white blood cell (WBC) count and alterations in CD8+ T-cell maturation. A reduction in general T-cell function (both CD4+ and CD8+) persisted for the duration of the 6-month spaceflights, with differential responses between mitogens suggesting an activation threshold shift. The percentage of CD4+ T cells capable of producing IL-2 was depressed after landing. Significant reductions in mitogen-stimulated production of IFNγ, IL-10, IL-5, TNFα, and IL-6 persisted during spaceflight. Following lipopolysaccharide (LPS) stimulation, production of IL-10 was reduced, whereas IL-8 production was increased during flight. Conclusions: The data indicated that immune alterations persist during long-duration spaceflight. This phenomenon, in the absence of appropriate countermeasures, has the potential to increase specific clinical risks for crewmembers during exploration-class deep space missions.
Spaceflight Study	US Space Shuttle	SLID-015	Alterations in adaptive immunity persist during long-duration spaceflight	Five had mission durations of > 100 Days, two astronauts had mission durations of < 60 Days	Immune system alterations	Background: It is currently unknown whether immune system alterations persist during long-duration spaceflight. In this study various adaptive immune parameters were assessed in astronauts at three intervals during 6-month spaceflight on board the International Space Station (ISS). AIMS: To assess phenotypic and functional immune system alterations in astronauts participating in 6-month orbital spaceflight. Methods: Blood was collected before, during, and after flight from 23 astronauts participating in 6-month ISS expeditions. In-flight samples were returned to Earth within 48 h of collection for immediate analysis. Assays included peripheral leukocyte distribution, T-cell function, virus-specific immunity, and mitogen-stimulated cytokine production profiles. Results: Redistribution of leukocyte subsets occurred during flight, including an elevated white blood cell (WBC) count and alterations in CD8+ T-cell maturation. A reduction in general T-cell function (both CD4+ and CD8+) persisted for the duration of the 6-month spaceflights, with differential responses between mitogens suggesting an activation threshold shift. The percentage of CD4+ T cells capable of producing IL-2 was depressed after landing. Significant reductions in mitogen-stimulated production of IFNγ, IL-10, IL-5, TNFα, and IL-6 persisted during spaceflight. Following lipopolysaccharide (LPS) stimulation, production of IL-10 was reduced, whereas IL-8 production was increased during flight. Conclusions: The data indicated that immune alterations persist during long-duration spaceflight. This phenomenon, in the absence of appropriate countermeasures, has the potential to increase specific clinical risks for crewmembers during exploration-class deep space missions.
Spaceflight Study	International Space Station (ISS)	SLID-016	Latent viral reactivation is associated with changes in plasma antimicrobial protein concentrations during long-duration spaceflight	6 Months	Plasma antimicrobial proteins (AMPs) changes	Long duration spaceflights are associated with profound dysregulation of the immune system and latent viral reactivations. However, little is known on the impact of long duration spaceflight on innate immunity which raises concerns on crewmembers' ability to fight infections during a mission. The aim of this study was to determine the effects of spaceflight on plasma antimicrobial proteins (AMPs) and how these changes impact latent herpesvirus reactivations. Plasma, saliva and urine samples were obtained from 23 crewmembers before, during and after a 6-month mission on the International Space Station (ISS). Plasma AMP concentrations were determined by ELISA, and saliva Epstein-Barr virus (EBV) and varicella zoster virus (VZV) and urine cytomegalovirus (CMV) DNA levels were quantified by Real-Time PCR. There was a non-significant increase in plasma HNP1-3 and LL-37 during the early and middle stages of the missions, which was significantly associated with changes in viral DNA during and after spaceflight. Plasma HNP1-3 and Lysozyme increased at the late mission stages in astronauts who had exhibited EBV and VZV reactivations during the early flight stages. Following return to Earth and during recovery, HNP1-3 and lysozyme concentrations were associated with EBV and VZV viral DNA levels, reducing the magnitude of viral reactivation. Reductions in plasma LL-37 upon return were associated with greater CMV reactivation. This study shows that biomarkers of innate immunity appeared to be partially restored after 6-months in space and suggests that following adaptation to the space environment, plasma HNP1-3 and lysozyme facilitate the control of EBV and VZV reactivation rate and magnitude in space and upon return on earth. However, the landing-associated decline in plasma LL-37 may enhance the rate of CMV reactivation in astronauts following spaceflight, potentially compromising crewmember health after landing.
Spaceflight Study	International Space Station (ISS)	SLID-017	Optic Nerve Length before and after Spaceflight	109 ± 75 Days	Optic nerve length changes	Purpose: The spaceflight-associated neuro-ocular syndrome (SANS) affects astronauts on missions to the International Space Station (ISS). The SANS has blurred vision and ocular changes as typical features. The objective of this study was to investigate if microgravity can create deformations or movements of the eye or optic nerve, and if such changes could be linked to SANS. Design: Cohort study. Participants: Twenty-two astronauts (age 48 ± 4 years). Methods: The intervention consisted of time in microgravity at the ISS. We co-registered pre- and postspaceflight magnetic resonance imaging (MRI) scans and generated centerline representations of the optic nerve. The coordinates for the optic nerve head (ONH) and optic chiasm (OC) ends of the optic nerve were recorded along with the entire centerline path. Main outcome measures: Optic nerve length, ONH movement, and OC movement after time in microgravity. Results: Optic nerve length increased (0.80 ± 0.74 mm, P < 0.001), primarily reflecting forward ONH displacement (0.63 ± 0.53 mm, P < 0.001). The forward displacement was positively related to mission duration, preflight body weight, and clinical manifestations of SANS. We also detected upward displacement of the OC (0.39 ± 0.50 mm, P = 0.002), indicative of brain movement, but this observation could not be linked to SANS. Conclusions: The spaceflight-induced optic nerve lengthening and anterior movement of the ONH support that SANS is caused by an altered pressure difference between the brain and the eye, leading to a forward push on the posterior of the eye. Body weight is a potential contributing risk factor. Direct assessment of intracranial pressure in space is required to verify the implicated mechanism behind the ocular findings in SANS.
Spaceflight Study	International Space Station (ISS)	SLID-018	Plasma cytokine concentrations indicate that in vivo hormonal regulation of immunity is altered during long-duration spaceflight	6 Months	Plasma cytokine concentration	Aspects of immune system dysregulation associated with long-duration spaceflight have yet to be fully characterized and may represent a clinical risk to crewmembers during deep space missions. Plasma cytokine concentration may serve as an indicator of in vivo physiological changes or immune system mobilization. The plasma concentrations of 22 cytokines were monitored in 28 astronauts during long-duration spaceflight onboard the International Space Station. Blood samples were collected 3 times before flight, 3–5 times during flight (depending on mission duration), at landing, and 30 days after landing. Analysis was performed by bead array immunoassay. With few exceptions, minimal detectable mean plasma concentrations were observed at baseline (launch minus 180) for innate inflammatory cytokines or adaptive regulatory cytokines; however, interleukin (IL)-1ra and several chemokines and growth factors were constitutively present. An increase in the plasma concentration, tumor necrosis factor-α (TNFα), IL-8, IL-1ra, thrombopoietin (Tpo), vascular endothelial growth factor (VEGF), C-C motif chemokine ligand 2 (CCL2), chemokine ligand 4/macrophage inhibitory protein 1b (CCL4), and C-X-C motif chemokine 5/epithelial neutrophil-activating protein 78 (CXCL5) was observed associated with spaceflight. No significant alterations were observed during or following spaceflight for the inflammatory or adaptive/T-regulatory cytokines: IL-1α, IL-1β, IL-2, interferon-gamma (IFN-γ), IL-17, IL-4, IL-5, IL-10, G-CSF, GM-CSF, FGF basic, CCL3, or CCL5. This pattern of cytokine dysregulation suggests multiple physiological adaptations persist during flight, including inflammation, leukocyte recruitment, angiogenesis, and thrombocyte regulation.
Spaceflight Study	n.a.	SLID-019	Spaceflight modulates gene expression in the whole blood of astronauts	6 Months	Cellular stress responses(gene-expression changes)	Astronauts are exposed to a unique combination of stressors during spaceflight, which leads to alterations in their physiology and potentially increases their susceptibility to disease, including infectious diseases. To evaluate the potential impact of the spaceflight environment on the regulation of molecular pathways mediating cellular stress responses, we performed a first-of-its-kind pilot study to assess spaceflight-related gene-expression changes in the whole blood of astronauts. Using an array comprised of 234 well-characterized stress-response genes, we profiled transcriptomic changes in six astronauts (four men and two women) from blood preserved before and immediately following the spaceflight. Differentially regulated transcripts included those important for DNA repair, oxidative stress, and protein folding/degradation, including HSP90AB1, HSP27, GPX1, XRCC1, BAG-1, HHR23A, FAP48, and C-FOS. No gender-specific differences or relationship to number of missions flown was observed. This study provides a first assessment of transcriptomic changes occurring in the whole blood of astronauts in response to spaceflight.
Spaceflight Study	International Space Station (ISS)	SLID-020	Intrinsic cardiovascular autonomic regulatory system of astronauts exposed long-term to microgravity in space: observational study	172.6 ± 14.6 Days	Cardiovascular changes	The fractal scaling of the long-term heart rate variability (HRV) reflects the ‘intrinsic’ autonomic regulatory system. Herein, we examine how microgravity on the ISS affected the power-law scaling β (beta) of astronauts during a long-duration (about 6 months) spaceflight. Ambulatory electrocardiographic (ECG) monitoring was performed on seven healthy astronauts (5 men, 52.0±4.2 years of age) five times: before launch, 24±5 (F01) and 73±5 (F02) days after launch, 15±5 days before return (F03), and after return to Earth. The power-law scaling β was calculated as the slope of the regression line of the power density of the MEM spectrum versus frequency plotted on a log10–log10 scale in the range of 0.0001–0.01 Hz (corresponding to periods of 2.8 h to 1.6 min). β was less negative in space (−0.949±0.061) than on Earth (−1.163±0.075; P<0.025). The difference was more pronounced during the awake than during the rest/sleep span. The circadian amplitude and acrophase (phase of maximum) of β did not differ in space as compared with Earth. An effect of microgravity was detected within 1 month (F01) in space and continued throughout the spaceflight. The intrinsic autonomic regulatory system that protects life under serious environmental conditions on Earth is altered in the microgravity environment, with no change over the 6-month spaceflight. It is thus important to find a way to improve conditions in space and/or in terms of human physiology, not to compromise the intrinsic autonomic regulatory system now that plans are being made to inhabit another planet in the near future.
Spaceflight Study	Mir Space Station; International Space Station;Space Shuttle	SLID-021	Effect of short- and long-duration spaceflight on QTc intervals in healthy astronauts	n.a.	Electrocardiograms or Holter monitor tracings	No abstract available
Spaceflight Study	International Space Station (ISS)	SLID-022	Bisphosphonates as a supplement to exercise to protect bone during long-duration spaceflight	5.5 Months (mean)	Bone changes	Summary We report the results of alendronate ingestion plus exercise in preventing the declines in bone mass and strength and elevated levels of urinary calcium and bone resorption in astronauts during 5.5 months of spaceflight. Introduction This investigation was an international collaboration between NASA and the JAXA space agencies to investigate the potential value of antiresorptive agents to mitigate the well-established bone changes associated with long-duration spaceflight. Methods We report the results from seven International Space Station (ISS) astronauts who spent a mean of 5.5 months on the ISS and who took an oral dose of 70 mg of alendronate weekly starting 3 weeks before flight and continuing throughout the mission. All crewmembers had available for exercise a treadmill, cycle ergometer, and a resistance exercise device. Our assessment included densitometry of multiple bone regions using X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) and assays of biomarkers of bone metabolism. Results In addition to pre- and post-flight measurements, we compared our results to 18 astronauts who flew ISS missions and who exercised using an early model resistance exercise device, called the interim resistance exercise device, and to 11 ISS astronauts who exercised using the newer advanced resistance exercise device (ARED). Our findings indicate that the ARED provided significant attenuation of bone loss compared with the older device although post-flight decreases in the femur neck and hip remained. The combination of the ARED and bisphosphonate attenuated the expected decline in essentially all indices of altered bone physiology during spaceflight including: DXA-determined losses in bone mineral density of the spine, hip, and pelvis, QCT-determined compartmental losses in trabecular and cortical bone mass in the hip, calculated measures of fall and stance computed bone strength of the hip, elevated levels of bone resorption markers, and urinary excretion of calcium.
Spaceflight Study	n.a.	SLID-023	Anti-aging effects of long-term space missions, estimated by heart rate variability	151.3 ± 21.8 Days	Heart rate variability	Reports that aging slows down in space prompted this investigation of anti-aging effects in humans by analyzing astronauts’ heart rate variability (HRV). Ambulatory 48-hour electrocardiograms from 7 astronauts (42.1 ± 6.8 years; 6 men) 20.6 ± 2.7 days (ISS01) and 138.6 ± 21.8 days (ISS02) after launch were divided into 24-hour spans of relative lower or higher magnetic disturbance, based on geomagnetic measures in Tromso, Norway. Magnetic disturbances were significantly higher on disturbed than on quiet days (ISS01: 72.01 ± 33.82 versus 33.96 ± 17.90 nT, P = 0.0307; ISS02: 71.06 ± 51.52 versus 32.53 ± 27.27 nT, P = 0.0308). SDNNIDX was increased on disturbed days (by 5.5% during ISS01, P = 0.0110), as were other HRV indices during ISS02 (SDANN, 12.5%, P = 0.0243; Triangular Index, 8.4%, P = 0.0469; and TF-component, 17.2%, P = 0.0054), suggesting the action of an anti-aging or longevity effect. The effect on TF was stronger during light (12:00–17:00) than during darkness (0:00–05:00) (P = 0.0268). The brain default mode network (DMN) was activated, gauged by increases in the LF-band (9.7%, P = 0.0730) and MF1-band (9.9%, P = 0.0281). Magnetic changes in the magnetosphere can affect and enhance HRV indices in space, involving an anti-aging or longevity effect, probably in association with the brain DMN, in a light-dependent manner and/or with help from the circadian clock.
Spaceflight Study	International Space Station (ISS)	SLID-024	Measurements of jugular, portal, femoral, and calf vein cross-sectional area for the assessment of venous blood redistribution with long duration spaceflight (Vessel Imaging Experiment)	6 Months	Vessel Imaging	Purpose To determine if 6 months in microgravity resulted in significant changes in the major central and peripheral veins indicating a redistribution of venous blood flow. Methods Ten astronauts participated in the study. Jugular vein (JV), portal vein (PV), femoral vein (FV), tibial vein (TibV), and gastrocnemius vein (Gast V) were assessed by echography for the measurement of vessel cross-sectional area. Inflight exams were conducted by astronauts using a volume capture method in which images collected were processed to produce a 3D reconstruction of the vessel which was later analyzed by a trained sonographer. Measurements were conducted pre-flight, at the beginning of the flight (day 15), near the end of the flight (4–5.5 months), and post-flight. Results During the flight, JV, PV, JV/PV ratio, and FV were found significantly increased from pre-flight at 15 days and 4–5.5 months (JV: 178 and 225 %, p < 0.05; PV: 36 and 45 %, p < 0.05; JV/PV ratio: 102 and 120 %, p < 0.05; FV: 124 and 169 %, p < 0.05). Conversely, calf veins decreased at day 15 and at 4–5.5 months (TibV: −45 % and−52 %, p < 0.05; Gast V: −68 and −55 %, p < 0.05). All veins returned to base line conditions 4 days after returning to Earth. Conclusions The increase in JV, PV, and FV cross-sectional area during spaceflight confirmed that there was venous blood pooling in the cephalic, splanchnic, and pelvic regions. Further investigation is needed to determine the consequences of this fluid stagnation on the brain, eye, splanchnic, and pelvic organ morphology and or function.
Spaceflight Study	International Space Station (ISS)	SLID-025	NK cell function is impaired during long-duration spaceflight	340 Days	Changes in natural killer (NK) cell phenotype and function	Maintaining astronaut health during space travel is paramount for further human exploration of the solar system beyond Earth's orbit. Of concern are potential dysregulations in immunity, which could increase the likelihood of cancer and latent viral reactivation. Natural killer (NK) cells are critical effectors of the innate immune system, and their function and phenotype are important to immunosurveillance of nascent tumors and latent viral infections. We compared changes in NK cell phenotype and function in eight crew members who completed an ~6-mo mission to the International Space Station (ISS) with healthy controls who remained on Earth. Assessments were made before (180 and 60 days before launch), during [flight day + 90 days (FD+90) and 1 day before return (R-1)], and after the mission (at R+0, R+18, R+33, and R+66). These samples, plus an additional in-flight sample (FD+180), were collected from a crew member who spent 340 days (~1 yr) on the ISS. NK cell cytotoxic activity (NKCA) against K562 leukemia targets in vitro was reduced by ~50% at FD+90 in ISS crew but not controls. This decrease was more pronounced in "rookie" compared with "veteran" crew members. The ~1-yr mission crew member did not show declines in NKCA against K562 until late in the mission (R-1 and R+0). NK cell numbers, expression of activating and inhibitory receptors, target cell binding, and expression and degranulation of perforin and granzyme B were unaltered with spaceflight. Similarly, when we exposed an immortalized NK cell line (NK-92) to sera collected at different mission time points (before, during, and after flight), there was no effect on NKCA. This is the first study to report impaired NK cell function during long-duration space travel. Countermeasures may be needed to mitigate immune system impairment in exploration class mission crew during long-duration spaceflight missions. NEW & NOTEWORTHY Immune system impairment may inhibit future human space exploration missions to Mars. Natural killer (NK) cells are key components of immunity and vital for tumor surveillance and the prevention of latent virus reactivation. We report that NK cell function is impaired in astronauts during an ~6-mo orbital space mission compared with preflight levels and ground-based controls. Declines in NK cell function were more marked in first-time "rookie" fliers. Countermeasures are needed to preserve NK cell-mediated immunity during spaceflight.
Spaceflight Study	n.a.	SLID-026	Modification of unilateral otolith responses following spaceflight	Median 13.5 Days	Unilateral otolith responses	The aim of the study was to resolve the issue of spaceflight-induced, adaptive modification of the otolith system by measuring unilateral otolith responses in a pre- versus post-flight design. The study represents the first comprehensive approach to examining unilateral otolith function following space flight. Ten astronauts participated in unilateral otolith function tests three times preflight and up to four times after Shuttle flights from landing day through the subsequent 10 days. During unilateral centrifugation, utricular function was examined by the perceptual changes reflected by the subjective visual vertical (SVV) and the otolith-mediated ocular counter-roll, designated as utriculo-ocular response (UOR). Unilateral saccular reflexes were recorded by measurement of collic vestibular evoked myogenic potentials (cVEMP). The findings demonstrate a general increase in interlabyrinth asymmetry of otolith responses on landing day relative to preflight baseline, with subsequent reversal in asymmetry within 2–3 days. Recovery to baseline levels was achieved within 10 days. This fluctuation in asymmetry was consistent for the utricle tests (SVV and UOR) while apparently stronger for SVV. A similar asymmetry was observed during cVEMP testing. In addition, the results provide initial evidence of a dominant labyrinth. The findings require reconsideration of the otolith asymmetry hypothesis; in general, on landing day, the response from one labyrinth was equivalent to preflight values, while the other showed considerable discrepancy. The finding that one otolith response can return to one-g level within hours after re-entry while the other takes considerably longer demonstrates the importance of considering the otolith response as a result of both peripheral and associated central neural processing.
Spaceflight Study	International Space Station (ISS)	SLID-027	Telomere Length Dynamics and DNA Damage Responses Associated with Long-Duration Spaceflight	6 Months or 1 Year	Telomeric adaptive response	Telomere length dynamics and DNA damage responses were assessed before, during, and after one-year or shorter duration missions aboard the International Space Station (ISS) in a comparatively large cohort of astronauts (n = 11). Although generally healthy individuals, astronauts tended to have significantly shorter telomeres and lower telomerase activity than age- and sex-matched ground controls before and after spaceflight. Although telomeres were longer during spaceflight irrespective of mission duration, telomere length shortened rapidly upon return to Earth, and overall astronauts had shorter telomeres after spaceflight than they did before; inter-individual differences were identified. During spaceflight, all crewmembers experienced oxidative stress, which positively correlated with telomere length dynamics. Significantly increased frequencies of chromosomal inversions were observed during and after spaceflight; changes in cell populations were also detected. We propose a telomeric adaptive response to chronic oxidative damage in extreme environments, whereby the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway is transiently activated in normal somatic cells.
Spaceflight Study	International Space Station (ISS)	SLID-028	Reduction in proximal femoral strength due to long-duration spaceflight	4.3 - 6.5 Months	Changes in proximal femoral strength	Loss of bone mass is a well-known medical complication of long-duration spaceflight. However, we do not know how changes in bone density and geometry ultimately combine to affect the strength of the proximal femur as a whole. The goal of this study was to quantify the changes in proximal femoral strength that result from long-duration spaceflight. Pre-and post-flight CT scan-based patient-specific finite element models of the left proximal femur of 13 astronauts who spent 4.3 to 6.5 months on the International Space Station were generated. Loading conditions representing single-limb stance and a fall onto the posterolateral aspect of the greater trochanter were modeled, and proximal femoral strength (FFE) was computed. Mean FFE decreased from 18.2 times body weight (BW) pre-flight to 15.6 BW post-flight for stance loading and from 3.5 BW pre-flight to 3.1 BW post-flight for fall loading. When normalized for flight duration, FFE under stance and fall loading decreased at mean rates of 2.6% (0.6% to 5.0%) per month and 2.0% (0.6% to 3.9%) per month, respectively. These values are notably greater than previously reported reductions in DXA total femoral bone mineral density (0.4 to 1.8% per month). In some subjects, the magnitudes of the reductions in proximal femoral strength were comparable to estimated lifetime losses associated with aging. Although average post-flight proximal femoral strength is greater than forces expected to occur due to falls or normal activities, some subjects have small margins of safety. If proximal femoral strength is not recovered, some crew members may be at increased risk for age-related hip fractures decades after their missions.
Spaceflight Study	Space Shuttle Transport System (STS-78)	SLID-029	Flexor bias of joint position in humans during spaceflight	17 Days	Neuromotor control involving ankle and elbow joint	The ability to estimate ankle and elbow joint position was tested before, during, and after a 17-day spaceflight. Subjects estimated targeted joint angles during isovelocity (IsoV) joint movements with agonist muscle groups either active or relaxed. These movements included elbow extension (EE) and elbow flexion (EF), and plantarflexion (PF) and dorsiflexion (DF) of the ankle. Subjects also estimated these joint positions while moving the dynamometer at their chosen (variable) velocity (VarV) during EE and PF. For IsoV tests, no differences were observed between active and passive movements for either the ankle or elbow. Compared with those of pre-flight test days, estimates of targeted elbow joint angles were ~5° to 15° more flexed in-flight, and returned toward the pre-flight values during recovery. The spaceflight effects for the ankle were inconsistent and less prevalent than those for the elbow. The VarV PF test condition for the 120° target angle at the ankle exhibited ~5° to 7° more DF target angle estimates in-flight compared with those pre- or post-flight. In contrast, during IsoV PF there was a tendency for ankle estimates to be ~2° to 3° more PF after 2–3 days exposure to spaceflight. These data indicate that during spaceflight the perception of elbow extension is greater than actuality, and are consistent with the interpretation that microgravity induced a flexor bias in the estimation of the actual elbow joint position. Moreover, these effects in joint proprioception during spaceflight were observed in individual isolated single-joint movements during tasks in which vestibular function in maintaining posture were minimal.
Spaceflight Study	International Space Station (ISS)	SLID-030	Plasticity of the human IgM repertoire in response to long-term spaceflight	124 - 186 Days	IgM repertoire changes	Immune dysregulation is among the main adverse outcomes of spaceflight. Despite the crucial role of the antibody repertoire in host protection, the effects of spaceflight on the human antibody repertoire are unknown. Consequently, using high-throughput sequencing, we examined the IgM repertoire of five cosmonauts 25 days before launch, after 64 ± 11 and 129 ± 20 days spent on the International Space Station (ISS), and at 1, 7, and 30 days after landing. This is the first study of this kind in humans. Our data revealed that the IgM repertoire of the cosmonauts was different from that of control subjects (n = 4) prior to launch and that two out the five analyzed cosmonauts presented significant changes in their IgM repertoire during the mission. These modifications persisted up to 30 days after landing, likely affected the specificities of IgM binding sites, correlated with changes in the V(D)J recombination process responsible for creating antibody genes, and coincided with a higher stress response. These data confirm that the immune system of approximately half of the astronauts who spent 6 months on the ISS is sensitive to spaceflight conditions, and reveal individual responses indicating that personalized approaches should be implemented during future deep-space exploration missions that will be of unprecedented durations.
Spaceflight Study	International Space Station (ISS)	SLID-031	Cortical reorganization in an astronaut's brain after long-duration spaceflight	169 Days	Alterations in human brain function	To date, hampered physiological function after exposure to microgravity has been primarily attributed to deprived peripheral neuro-sensory systems. For the first time, this study elucidates alterations in human brain function after long-duration spaceflight. More specifically, we found significant differences in resting-state functional connectivity between motor cortex and cerebellum, as well as changes within the default mode network. In addition, the cosmonaut showed changes in the supplementary motor areas during a motor imagery task. These results highlight the underlying neural basis for the observed physiological deconditioning due to spaceflight and are relevant for future interplanetary missions and vestibular patients.
Spaceflight Study	n.a.	SLID-032	Brain Tissue-Volume Changes in Cosmonauts	n.a.	Changes in brain volumes	No abstract available.
Spaceflight Study	International Space Station (ISS)	SLID-033	Spaceflight-Associated Brain White Matter Microstructural Changes and Intracranial Fluid Redistribution	<= 200 Days	Intracranial extracellular free water (FW) shifts and brain white matter microstructural changes	Importance: Spaceflight results in transient balance declines and brain morphologic changes; to our knowledge, the effect on brain white matter as measured by diffusion magnetic resonance imaging (dMRI), after correcting for extracellular fluid shifts, has not been examined. Objective: To map spaceflight-induced intracranial extracellular free water (FW) shifts and to evaluate changes in brain white matter diffusion measures in astronauts. Design, setting and participants: We performed retrospective, longitudinal analyses on dMRI data collected between 2010 and 2015. Of the 26 astronauts' dMRI scans released by the National Aeronautics and Space Administration Lifetime Surveillance of Astronaut Health, 15 had both preflight and postflight dMRI scans and were included in the final analyses. Data were analyzed between 2015 and 2018. Interventions or exposures: Seven astronauts completed a space shuttle mission (≤30 days) and 8 completed a long-duration International Space Station mission (≤200 days). Main outcomes and measures: The dMRI scans were acquired for clinical monitoring; in this retrospective analysis, we analyzed brain FW and white matter diffusion metrics corrected for FW. We also obtained scores from computerized dynamic posturography tests of balance to assess brain-behavior associations. Results: Of the 15 astronauts included, the median (SD) age was 47.2 (1.5) years; 12 were men, and 3 were women. We found a significant, widespread increase in FW volume in the frontal, temporal, and occipital lobes from before spaceflight to after spaceflight. There was also a significant decrease in FW in the posterior aspect of the vertex. All FW changes were significant and ranged from approximately 2.5% to 4.0% across brain regions. We observed white matter changes in the right superior and inferior longitudinal fasciculi, the corticospinal tract, and cerebellar peduncles. All white matter changes were significant and ranged from approximately 0.75% to 1.25%. Spaceflight mission duration was associated with cerebellar white matter change, and white matter changes in the superior longitudinal fasciculus were associated with the balance changes seen in the astronauts from before spaceflight to after spaceflight. Conclusions and relevance: Free water redistribution with spaceflight likely reflects headward fluid shifts occurring in microgravity as well as an upward shift of the brain within the skull. White matter changes were of a greater magnitude than those typically seen during the same period with healthy aging. Future, prospective assessments are required to better understand the recovery time and behavioral consequences of these brain changes.
Spaceflight Study	Space Shuttle Mission	SLID-033	Spaceflight-Associated Brain White Matter Microstructural Changes and Intracranial Fluid Redistribution	<= 30 Days	Intracranial extracellular free water (FW) shifts and brain white matter microstructural changes	Importance: Spaceflight results in transient balance declines and brain morphologic changes; to our knowledge, the effect on brain white matter as measured by diffusion magnetic resonance imaging (dMRI), after correcting for extracellular fluid shifts, has not been examined. Objective: To map spaceflight-induced intracranial extracellular free water (FW) shifts and to evaluate changes in brain white matter diffusion measures in astronauts. Design, setting and participants: We performed retrospective, longitudinal analyses on dMRI data collected between 2010 and 2015. Of the 26 astronauts' dMRI scans released by the National Aeronautics and Space Administration Lifetime Surveillance of Astronaut Health, 15 had both preflight and postflight dMRI scans and were included in the final analyses. Data were analyzed between 2015 and 2018. Interventions or exposures: Seven astronauts completed a space shuttle mission (≤30 days) and 8 completed a long-duration International Space Station mission (≤200 days). Main outcomes and measures: The dMRI scans were acquired for clinical monitoring; in this retrospective analysis, we analyzed brain FW and white matter diffusion metrics corrected for FW. We also obtained scores from computerized dynamic posturography tests of balance to assess brain-behavior associations. Results: Of the 15 astronauts included, the median (SD) age was 47.2 (1.5) years; 12 were men, and 3 were women. We found a significant, widespread increase in FW volume in the frontal, temporal, and occipital lobes from before spaceflight to after spaceflight. There was also a significant decrease in FW in the posterior aspect of the vertex. All FW changes were significant and ranged from approximately 2.5% to 4.0% across brain regions. We observed white matter changes in the right superior and inferior longitudinal fasciculi, the corticospinal tract, and cerebellar peduncles. All white matter changes were significant and ranged from approximately 0.75% to 1.25%. Spaceflight mission duration was associated with cerebellar white matter change, and white matter changes in the superior longitudinal fasciculus were associated with the balance changes seen in the astronauts from before spaceflight to after spaceflight. Conclusions and relevance: Free water redistribution with spaceflight likely reflects headward fluid shifts occurring in microgravity as well as an upward shift of the brain within the skull. White matter changes were of a greater magnitude than those typically seen during the same period with healthy aging. Future, prospective assessments are required to better understand the recovery time and behavioral consequences of these brain changes.
Spaceflight Study	n.a.	SLID-034	Lumbopelvic Muscle Changes Following Long-Duration Spaceflight	1 - Year or 6 - mont Hours	Lumbopelvic Muscle Changes	Long-duration spaceflight has been shown to negatively affect the lumbopelvic muscles of crewmembers. Through analysis of computed tomography scans of crewmembers on 4- to 6-month missions equipped with the interim resistive exercise device, the structural deterioration of the psoas, quadratus lumborum, and paraspinal muscles was assessed. Computed tomography scans of 16 crewmembers were collected before and after long-duration spaceflight. The volume and attenuation of lumbar musculature at the L2 vertebral level were measured. Percent changes in the lumbopelvic muscle volume and attenuation (indicative of myosteatosis, or intermuscular fat infiltration) following spaceflight were calculated. Due to historical studies demonstrating only decreases in the muscles assessed, a one-sample t test was performed to determine if these decreases persist in more recent flight conditions. Crewmembers on interim resistive exercise device-equipped missions experienced an average 9.5% (2.0% SE) decrease in volume and 6.0% (1.5% SE) decrease in attenuation in the quadratus lumborum muscles and an average 5.3% (1.0% SE) decrease in volume and 5.3% (1.6% SE) decrease in attenuation in the paraspinal muscles. Crewmembers experienced no significant changes in psoas muscle volume or attenuation. No significant changes in intermuscular adipose tissue volume or attenuation were found in any muscles. Long-duration spaceflight was associated with preservation of psoas muscle volume and attenuation and significant decreases in quadratus lumborum and paraspinal muscle volume and attenuation.
Spaceflight Study	International Space Station (ISS)	SLID-035	Alterations in hematologic indices during long-duration spaceflight	6 Months	Red blood cell (RBC) and hemoglobin changes	Background: Although a state of anemia is perceived to be associated with spaceflight, to date a peripheral blood hematologic assessment of red blood cell (RBC) indices has not been performed during long-duration space missions. Methods: This investigation collected whole blood samples from astronauts participating in up to 6-months orbital spaceflight, and returned those samples (ambient storage) to Earth for analysis. As samples were always collected near undock of a returning vehicle, the delay from collection to analysis never exceeded 48 h. As a subset of a larger immunologic investigation, a complete blood count was performed. A parallel stability study of the effect of a 48 h delay on these parameters assisted interpretation of the in-flight data. Results: We report that the RBC and hemoglobin were significantly elevated during flight, both parameters deemed stable through the delay of sample return. Although the stability data showed hematocrit to be mildly elevated at +48 h, there was an in-flight increase in hematocrit that was ~3-fold higher in magnitude than the anticipated increase due to the delay in processing. Conclusions: While susceptible to the possible influence of dehydration or plasma volume alterations, these results suggest astronauts do not develop persistent anemia during spaceflight.
Spaceflight Study	Space Transport System (STS-78)	SLID-036	Effects of 17-day spaceflight on electrically evoked torque and cross-sectional area of the human triceps surae	17 Days	Muscle function changes	The effects of spaceflight on triceps surae muscle torque and cross-sectional area (CSA) were investigated on four astronauts using electrically evoked contractions to by-pass neural control. Muscle twitch characteristics, ankle joint angle-twitch torque relation, frequency-torque relation, tetanic torque and fatigability were assessed before, during and after a 17-day Space Shuttle flight (STS-78). Muscle plus bone cross-sectional area (CSAm+b) was evaluated before and after the flight. Whereas no changes in muscle function were observed during the flight, marked alterations were found during the recovery period. Peak twitch (PTw) and tetanic torques at 50 Hz (PT50) continued to fall up to the 8th recovery day (R+8) on which losses in PTw and PT50 were 24.4% (P<0.01) and 22.0% (P<0.01), respectively. The decline in PTw was not joint-angle-specific. Post-flight, especially on R+8, torque decreased at all stimulation frequencies (1, 20, 30 and 50 Hz); however the shape of the frequency-torque curve, normalised for PT50, was not modified. Similarly, no changes in twitch kinetics were observed. Post- flight, an 8% (P<0.01) reduction in CSAm+b was found on R+2. Normalisation of PT50 values for CSAm+b showed a progressive loss in specific torque (PT50/CSAm+b), which was maximal on R+2 (19.5%, P<0.05). Also, fatigability during 2-min intermittent stimulation at 20 Hz increased throughout recovery, reaching a nadir of 16.4% (P<0.01) on R+15. In conclusion, 17 days of spaceflight resulted in significant changes in muscle function during the recovery phase, but not in microgravity. The disproportionate loss of torque compared with that of muscle size suggests the presence of muscle damage due to reloading in 1 g.
Spaceflight Study	International Space Station (ISS)	SLID-037	Long-term changes in the density and structure of the human hip and spine after long-duration spaceflight	Average of 181 Days	Changes in the density and structure of the human hip and spine	To determine the long-term effects of long-duration spaceflight, we measured bone mineral density and bone geometry of International Space Station (ISS) crewmembers using quantitative computed tomography (QCT) before launch, immediately upon their return, one year after return, and 2–4.5 years after return from the ISS. Eight crew members (7 male, 1 female, mean age 45±4 years at start of mission) who spent an average of 181 days (range 161–196 days) aboard the ISS took part in the study. Integral bone mineral density (iBMD), trabecular BMD (tBMD), bone mineral content (BMC), and vertebral cross-sectional area (CSA) were measured in the lumbar spine, and iBMD, tBMD, cortical BMD (cBMD), BMC, CSA, volume, and femoral neck section modulus were measured in the hip. Spine iBMD was 95% of the average preflight value upon return from the ISS and reached its preflight value over the next 2–4.5 years. Spine tBMD was 97% of the average preflight value upon return from the ISS and tended to decrease throughout the course of the study. Vertebral CSA remained essentially unchanged throughout the study. Hip iBMD was 91% of the preflight value upon return from the ISS and was 95% of the preflight value after 2–4.5 years of recovery. Hip tBMD was 88% of the preflight value upon return and recovered to only 93% of the preflight value after 1 year. At the 2- to 4.5-year time point, average tBMD was 88% of the preflight value. During the recovery period the total volume and cortical bone volume in the hip reached values of 114% and 110% of their preflight values, respectively. The combination of age-related bone loss, long-duration spaceflight, and re-adaptation to the 1-g terrestrial environment presumably produced these changes. These long-term data suggest that skeletal changes that occur during long-duration spaceflight persist even after multiple years of recovery. These changes have important implications for the skeletal health of crew members, especially those who make repeat trips to space.
Spaceflight Study	n.a.	SLID-038	Increased core body temperature in astronauts during long-duration space missions	180 Days	Core body temperature and interleukin-1 receptor antagonist changes	Humans’ core body temperature (CBT) is strictly controlled within a narrow range. Various studies dealt with the impact of physical activity, clothing, and environmental factors on CBT regulation under terrestrial conditions. However, the effects of weightlessness on human thermoregulation are not well understood. Specifically, studies, investigating the effects of long-duration spaceflight on CBT at rest and during exercise are clearly lacking. We here show that during exercise CBT rises higher and faster in space than on Earth. Moreover, we observed for the first time a sustained increased astronauts’ CBT also under resting conditions. This increase of about 1 °C developed gradually over 2.5 months and was associated with augmented concentrations of interleukin-1 receptor antagonist, a key anti-inflammatory protein. Since even minor increases in CBT can impair physical and cognitive performance, both findings have a considerable impact on astronauts’ health and well-being during future long-term spaceflights. Moreover, our findings also pinpoint crucial physiological challenges for spacefaring civilizations, and raise questions about the assumption of a thermoregulatory set point in humans, and our evolutionary ability to adapt to climate changes on Earth.
Spaceflight Study	STS-70	SLID-039	The effects of spaceflight on mammary metabolism in pregnant rats	9 Days	Animals	The effects of spaceflight on mammary metabolism of 10 pregnant rats was measured on Day 20 of pregnancy and after parturition. Rats were flown on the space shuttle from Day 11 through Day 20 of pregnancy. After their return to earth, glucose oxidation to carbon dioxide increased 43% (P < 0.05), and incorporation into fatty acids increased 300% (P < 0.005) compared to controls. It is unclear whether the enhanced glucose use is due to spaceflight or a response to landing. Casein mRNA and gross histology were not altered at Day 20 of pregnancy. Six rats gave birth (on Day 22 to 23 of pregnancy) and mammary metabolic activity was measured immediately postpartum. The earlier effects of spaceflight were no longer apparent. There was also no difference in expression of beta-casein mRNA. It is clear from these studies that spaceflight does not impair the normal development of the mammary gland, its ability to use glucose, nor the ability to express mRNA for a major milk protein.
Ground Study	2-D Clinostat SM-X1	SLID-040	Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity	12 Days	Animals	Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.
Spaceflight Study	STS-90	SLID-040	Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity	16 Days	Animals	Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.
Spaceflight Study	STS-131	SLID-041	Effects of spaceflight on the murine mandible: Possible factors mediating skeletal changes in non-weight bearing bones of the head	15 Days	Animals	Spaceflight-induced remodeling of the skull is characterized by greater bone volume, mineral density, and mineral content. To further investigate the effects of spaceflight on other non-weight bearing bones of the head, as well as to gain insight into potential factors mediating the remodeling of the skull, the purpose of the present study was to determine the effects of spaceflight on mandibular bone properties. Female C57BL/6 mice were flown 15d on the STS-131 Space Shuttle mission (n=8) and 13d on the STS-135 mission (n=5) or remained as ground controls (GC). Upon landing, mandibles were collected and analyzed via micro-computed tomography for tissue mineralization, bone volume (BV/TV), and distance from the cemento-enamel junction to the alveolar crest (CEJ-AC). Mandibular mineralization was not different between spaceflight (SF) and GC mice for either the STS-131 or STS-135 missions. Mandibular BV/TV (combined cortical and trabecular bone) was lower in mandibles from SF mice on the STS-131 mission (80.7±0.8%) relative to that of GC (n=8) animals (84.2±1.2%), whereas BV/TV from STS-135 mice was not different from GC animals (n=7). The CEJ-AC distance was shorter in mandibles from STS-131 mice (0.217±0.004mm) compared to GC animals (0.283±0.009mm), indicating an anabolic (or anti-catabolic) effect of spaceflight, while CEJ-AC distance was similar between STS-135 and GC mice. These findings demonstrate that mandibular bones undergo skeletal changes during spaceflight and are susceptible to the effects of weightlessness. However, adaptation of the mandible to spaceflight is dissimilar to that of the cranium, at least in terms of changes in BV/TV.
Spaceflight Study	STS-135	SLID-041	Effects of spaceflight on the murine mandible: Possible factors mediating skeletal changes in non-weight bearing bones of the head	13 Days	Animals	Spaceflight-induced remodeling of the skull is characterized by greater bone volume, mineral density, and mineral content. To further investigate the effects of spaceflight on other non-weight bearing bones of the head, as well as to gain insight into potential factors mediating the remodeling of the skull, the purpose of the present study was to determine the effects of spaceflight on mandibular bone properties. Female C57BL/6 mice were flown 15d on the STS-131 Space Shuttle mission (n=8) and 13d on the STS-135 mission (n=5) or remained as ground controls (GC). Upon landing, mandibles were collected and analyzed via micro-computed tomography for tissue mineralization, bone volume (BV/TV), and distance from the cemento-enamel junction to the alveolar crest (CEJ-AC). Mandibular mineralization was not different between spaceflight (SF) and GC mice for either the STS-131 or STS-135 missions. Mandibular BV/TV (combined cortical and trabecular bone) was lower in mandibles from SF mice on the STS-131 mission (80.7±0.8%) relative to that of GC (n=8) animals (84.2±1.2%), whereas BV/TV from STS-135 mice was not different from GC animals (n=7). The CEJ-AC distance was shorter in mandibles from STS-131 mice (0.217±0.004mm) compared to GC animals (0.283±0.009mm), indicating an anabolic (or anti-catabolic) effect of spaceflight, while CEJ-AC distance was similar between STS-135 and GC mice. These findings demonstrate that mandibular bones undergo skeletal changes during spaceflight and are susceptible to the effects of weightlessness. However, adaptation of the mandible to spaceflight is dissimilar to that of the cranium, at least in terms of changes in BV/TV.
Spaceflight Study	STS-62	SLID-042	Effects of Spaceflight on Bone Microarchitecture in the Axial and Appendicular Skeleton in Growing Ovariectomized Rats	14 Days	Animals	This study investigated the effects of a 14-day spaceflight on bone mass, density and microarchitecture in weight bearing (femur and humerus) and non-weight bearing (2nd lumbar vertebra and calvarium) bones in the context of ovarian hormone insufficiency. 12-week-old Fisher 344 rats were ovariectomized 2 weeks before flight and randomized into one of three groups: 1) baseline (n = 6), 2) ground control (n = 12) or 3) spaceflight (n = 12). Additional ground-based ovary-intact rats provided age-matched reference values at baseline (n = 8) and landing (n = 10). Ovariectomy resulted in bone- and bone compartment-specific deficits in cancellous bone volume fraction. Spaceflight resulted in lower cortical bone accrual in the femur but had no effect on cortical bone in the humerus or calvarium. Cancellous bone volume fraction was lower in flight animals compared to ground control animals in lumbar vertebra and distal femur metaphysis and epiphysis; significant differences were not detected in the distal humerus. Bone loss (compared to baseline controls) in the femur metaphysis was associated with lower trabecular number, whereas trabecular thickness and number were lower in the epiphysis. In summary, the effect of spaceflight on bone microarchitecture in ovariectomized rats was bone-and bone compartment-specific but not strictly related to weight bearing.
Spaceflight Study	STS-135	SLID-043	Spaceflight Activates Lipotoxic Pathways in Mouse Liver	13 Days	Animals	Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease.
Spaceflight Study	STS-62	SLID-044	Spaceflight-induced vertebral bone loss in ovariectomized rats is associated with increased bone marrow adiposity and no change in bone formation	14 Days	Animals	There is often a reciprocal relationship between bone marrow adipocytes and osteoblasts, suggesting that marrow adipose tissue (MAT) antagonizes osteoblast differentiation. MAT is increased in rodents during spaceflight but a causal relationship between MAT and bone loss remains unclear. In the present study, we evaluated the effects of a 14-day spaceflight on bone mass, bone resorption, bone formation, and MAT in lumbar vertebrae of ovariectomized (OVX) rats. Twelve-week-old OVX Fischer 344 rats were randomly assigned to a ground control or flight group. Following flight, histological sections of the second lumbar vertebrae (n=11/group) were stained using a technique that allowed simultaneous quantification of cells and preflight fluorochrome label. Compared with ground controls, rats flown in space had 32% lower cancellous bone area and 306% higher MAT. The increased adiposity was due to an increase in adipocyte number (224%) and size (26%). Mineral apposition rate and osteoblast turnover were unchanged during spaceflight. In contrast, resorption of a preflight fluorochrome and osteoclast-lined bone perimeter were increased (16% and 229%, respectively). The present findings indicate that cancellous bone loss in rat lumbar vertebrae during spaceflight is accompanied by increased bone resorption and MAT but no change in bone formation. These findings do not support the hypothesis that increased MAT during spaceflight reduces osteoblast activity or lifespan. However, in the context of ovarian hormone deficiency, bone formation during spaceflight was insufficient to balance increased resorption, indicating defective coupling. The results are therefore consistent with the hypothesis that during spaceflight mesenchymal stem cells are diverted to adipocytes at the expense of forming osteoblasts.
Spaceflight Study	SpaceX-4	SLID-045	Effects of spaceflight on the immunoglobulin repertoire of unimmunized C57BL/6 mice	21 - 22 Days	Animals	Spaceflight has been shown to suppress the adaptive immune response, altering the distribution and function of lymphocyte populations. B lymphocytes express highly specific and highly diversified receptors, known as immunoglobulins (Ig), that directly bind and neutralize pathogens. Ig diversity is achieved through the enzymatic splicing of gene segments within the genomic DNA of each B cell in a host. The collection of Ig specificities within a host, or Ig repertoire, has been increasingly characterized in both basic research and clinical settings using high-throughput sequencing technology (HTS). We utilized HTS to test the hypothesis that spaceflight affects the B-cell repertoire. To test this hypothesis, we characterized the impact of spaceflight on the unimmunized Ig repertoire of C57BL/6 mice that were flown aboard the International Space Station (ISS) during the Rodent Research One validation flight in comparison to ground controls. Individual gene segment usage was similar between ground control and flight animals, however, gene segment combinations and the junctions in which gene segments combine was varied among animals within and between treatment groups. We also found that spontaneous somatic mutations in the IgH and Igκ gene loci were not increased. These data suggest that space flight did not affect the B cell repertoire of mice flown and housed on the ISS over a short period of time.
Spaceflight Study	BION-M1	SLID-046	Responses to spaceflight of mouse mandibular bone and teeth	30 Days	Animals	Objective: To determine if spaceflight and microgravity affect non-weight bearing bones and development and mineralization of teeth, reasoning that combining an organ and a cellular level approach can lead to greater insights about these effects. Design: Mandibles and incisors of mice flown on the US STS-135 space shuttle mission and the Russian Bion-M1 satellite were studied using micro-computed tomography and immunohistochemistry. Ground controls were mice housed in standard vivarium cages and flight habitats. Results: Incisor length was greater in the 13-day STS-135 flight mice than in either control group. Initial incisor mineralization occurred more posteriorly, and incisor, enamel and dentin volumes and enamel and dentin thicknesses were greater in the 30-day Bion-M1 flight and habitat control mice than in vivarium control mice. Mandibular bone volume (BV) was increased in STS-135 flight and habitat groups and decreased in Bion-M1 flight and habitat groups compared to vivarium controls. No significant histological alterations occurred, but changes were seen in the bone and tooth proteins dentin sialoprotein, amelogenin and the type II regulatory subunit of protein kinase A. The percentage of sclerostin positive osteocytes was greatest in flight mice, and greater in STS-135 flight and habitat control mice than in the corresponding Bion-M1 groups. TRAP staining, representing osteoclastic bone remodeling, differed between the two flights and corresponded with changes in BV. Interpretation of the findings was limited by a small number of flight mice, different sex and ages of the mice in the two missions, and different habitats and diets. Conclusions: Microgravity has measurable effects on mandibular bone physiology and incisor development and mineralization. The results also showed that the habitat had an effect either in flight or ground control samples, as demonstrated by the changes in BV and apparent slowing of incisor eruption. Therefore, developing appropriate habitats is critical for future spaceflight missions.
Spaceflight Study	STS-135	SLID-046	Responses to spaceflight of mouse mandibular bone and teeth	13 Days	Animals	Objective: To determine if spaceflight and microgravity affect non-weight bearing bones and development and mineralization of teeth, reasoning that combining an organ and a cellular level approach can lead to greater insights about these effects. Design: Mandibles and incisors of mice flown on the US STS-135 space shuttle mission and the Russian Bion-M1 satellite were studied using micro-computed tomography and immunohistochemistry. Ground controls were mice housed in standard vivarium cages and flight habitats. Results: Incisor length was greater in the 13-day STS-135 flight mice than in either control group. Initial incisor mineralization occurred more posteriorly, and incisor, enamel and dentin volumes and enamel and dentin thicknesses were greater in the 30-day Bion-M1 flight and habitat control mice than in vivarium control mice. Mandibular bone volume (BV) was increased in STS-135 flight and habitat groups and decreased in Bion-M1 flight and habitat groups compared to vivarium controls. No significant histological alterations occurred, but changes were seen in the bone and tooth proteins dentin sialoprotein, amelogenin and the type II regulatory subunit of protein kinase A. The percentage of sclerostin positive osteocytes was greatest in flight mice, and greater in STS-135 flight and habitat control mice than in the corresponding Bion-M1 groups. TRAP staining, representing osteoclastic bone remodeling, differed between the two flights and corresponded with changes in BV. Interpretation of the findings was limited by a small number of flight mice, different sex and ages of the mice in the two missions, and different habitats and diets. Conclusions: Microgravity has measurable effects on mandibular bone physiology and incisor development and mineralization. The results also showed that the habitat had an effect either in flight or ground control samples, as demonstrated by the changes in BV and apparent slowing of incisor eruption. Therefore, developing appropriate habitats is critical for future spaceflight missions.
Spaceflight Study	RR-1 Mission	SLID-047	Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight	37 Days	Animals	Background: Space environment imposes a range of challenges to mammalian physiology and the gut microbiota, and interactions between the two are thought to be important in mammalian health in space. While previous findings have demonstrated a change in the gut microbial community structure during spaceflight, specific environmental factors that alter the gut microbiome and the functional relevance of the microbiome changes during spaceflight remain elusive. Methods: We profiled the microbiome using 16S rRNA gene amplicon sequencing in fecal samples collected from mice after a 37-day spaceflight onboard the International Space Station. We developed an analytical tool, named STARMAPs (Similarity Test for Accordant and Reproducible Microbiome Abundance Patterns), to compare microbiome changes reported here to other relevant datasets. We also integrated the gut microbiome data with the publically available transcriptomic data in the liver of the same animals for a systems-level analysis. Results: We report an elevated microbiome alpha diversity and an altered microbial community structure that were associated with spaceflight environment. Using STARMAPs, we found the observed microbiome changes shared similarity with data reported in mice flown in a previous space shuttle mission, suggesting reproducibility of the effects of spaceflight on the gut microbiome. However, such changes were not comparable with those induced by space-type radiation in Earth-based studies. We found spaceflight led to significantly altered taxon abundance in one order, one family, five genera, and six species of microbes. This was accompanied by a change in the inferred microbial gene abundance that suggests an altered capacity in energy metabolism. Finally, we identified host genes whose expression in the liver were concordantly altered with the inferred gut microbial gene content, particularly highlighting a relationship between host genes involved in protein metabolism and microbial genes involved in putrescine degradation. Conclusions: These observations shed light on the specific environmental factors that contributed to a robust effect on the gut microbiome during spaceflight with important implications for mammalian metabolism. Our findings represent a key step toward a better understanding the role of the gut microbiome in mammalian health during spaceflight and provide a basis for future efforts to develop microbiota-based countermeasures that mitigate risks to crew health during long-term human space expeditions.
Spaceflight Study	BION-M1	SLID-048	Multiscale effects of spaceflight on murine tendon and bone	30 Days	Animals	Despite a wealth of data on the effects of spaceflight on tendons and bones, little is known about its effects on the interfacial tissue between these two structures, the enthesis. Mice were sent to space on three separate missions: STS-131, STS-135, and Bion-M1 to determine how spaceflight affects the composition, structure, mechanics, and gene expression of the humerus-supraspinatus and calcaneus-Achilles entheses. At the nanoscale, spaceflight resulted in decreased carbonate levels in the bone, likely due to increased remodeling, as suggested by increased expression of genes related to osteoclastogenesis (CatK, Tnfsf11) and mature osteoblasts (Col1, Osc). Tendons showed a shift in collagen fibril size towards smaller diameters that may have resulted from increased expression of genes related to collagen degradation (Mmp3, Mmp13). These nanoscale changes did not result in micro- and milliscale changes to the structure and mechanics of the enthesis. There were no changes in bone volume, trabecular structure, failure load, or stiffness with spaceflight. This lack of tissue-level change may be anatomy based, as extremities may be less sensitive to spaceflight than central locations such as vertebrae, yet results highlight that the tendon enthesis may be robust against negative effects of spaceflight.
Spaceflight Study	STS-131	SLID-048	Multiscale effects of spaceflight on murine tendon and bone	15 Days	Animals	Despite a wealth of data on the effects of spaceflight on tendons and bones, little is known about its effects on the interfacial tissue between these two structures, the enthesis. Mice were sent to space on three separate missions: STS-131, STS-135, and Bion-M1 to determine how spaceflight affects the composition, structure, mechanics, and gene expression of the humerus-supraspinatus and calcaneus-Achilles entheses. At the nanoscale, spaceflight resulted in decreased carbonate levels in the bone, likely due to increased remodeling, as suggested by increased expression of genes related to osteoclastogenesis (CatK, Tnfsf11) and mature osteoblasts (Col1, Osc). Tendons showed a shift in collagen fibril size towards smaller diameters that may have resulted from increased expression of genes related to collagen degradation (Mmp3, Mmp13). These nanoscale changes did not result in micro- and milliscale changes to the structure and mechanics of the enthesis. There were no changes in bone volume, trabecular structure, failure load, or stiffness with spaceflight. This lack of tissue-level change may be anatomy based, as extremities may be less sensitive to spaceflight than central locations such as vertebrae, yet results highlight that the tendon enthesis may be robust against negative effects of spaceflight.
Spaceflight Study	STS-135	SLID-048	Multiscale effects of spaceflight on murine tendon and bone	13 Days	Animals	Despite a wealth of data on the effects of spaceflight on tendons and bones, little is known about its effects on the interfacial tissue between these two structures, the enthesis. Mice were sent to space on three separate missions: STS-131, STS-135, and Bion-M1 to determine how spaceflight affects the composition, structure, mechanics, and gene expression of the humerus-supraspinatus and calcaneus-Achilles entheses. At the nanoscale, spaceflight resulted in decreased carbonate levels in the bone, likely due to increased remodeling, as suggested by increased expression of genes related to osteoclastogenesis (CatK, Tnfsf11) and mature osteoblasts (Col1, Osc). Tendons showed a shift in collagen fibril size towards smaller diameters that may have resulted from increased expression of genes related to collagen degradation (Mmp3, Mmp13). These nanoscale changes did not result in micro- and milliscale changes to the structure and mechanics of the enthesis. There were no changes in bone volume, trabecular structure, failure load, or stiffness with spaceflight. This lack of tissue-level change may be anatomy based, as extremities may be less sensitive to spaceflight than central locations such as vertebrae, yet results highlight that the tendon enthesis may be robust against negative effects of spaceflight.
Spaceflight Study	STS-135	SLID-049	Multi-omics analysis of multiple missions to space reveal a theme of lipid dysregulation in mouse liver	13 Days	Animals	Spaceflight has several detrimental effects on the physiology of astronauts, many of which are recapitulated in rodent models. Mouse studies performed on the Space Shuttle showed disruption of lipid metabolism in liver. However, given that these animals were not sacrificed on-orbit and instead returned live to earth, it is unclear if these disruptions were solely induced by space stressors (e.g. microgravity, space radiation) or in part explained by the stress of return to Earth. In this work we analyzed three liver datasets from two different strains of mice (C57BL/6 (Jackson) & BALB/c (Taconic)) flown aboard the International Space Station (ISS). Notably, these animals were sacrificed on-orbit and exposed to varying spaceflight durations (i.e. 21, 37, and 42 days vs 13 days for the Shuttle mice). Oil Red O (ORO) staining showed abnormal lipid accumulation in all space-flown mice compared to ground controls regardless of strain or exposure duration. Similarly, transcriptomic analysis by RNA-sequencing revealed several pathways that were affected in both strains related to increased lipid metabolism, fatty acid metabolism, lipid and fatty acid processing, lipid catabolic processing, and lipid localization. In addition, key upstream regulators were predicted to be commonly regulated across all conditions including Glucagon (GCG) and Insulin (INS). Moreover, quantitative proteomic analysis showed that a number of lipid related proteins were changed in the livers during spaceflight. Taken together, these data indicate that activation of lipotoxic pathways are the result of space stressors alone and this activation occurs in various genetic backgrounds during spaceflight exposures of weeks to months. If similar responses occur in humans, a prolonged change of these pathways may result in the development of liver disease and should be investigated further.
Spaceflight Study	The Rodent Research 1 (RR1) mission	SLID-049	Multi-omics analysis of multiple missions to space reveal a theme of lipid dysregulation in mouse liver	37 Days	Animals	Spaceflight has several detrimental effects on the physiology of astronauts, many of which are recapitulated in rodent models. Mouse studies performed on the Space Shuttle showed disruption of lipid metabolism in liver. However, given that these animals were not sacrificed on-orbit and instead returned live to earth, it is unclear if these disruptions were solely induced by space stressors (e.g. microgravity, space radiation) or in part explained by the stress of return to Earth. In this work we analyzed three liver datasets from two different strains of mice (C57BL/6 (Jackson) & BALB/c (Taconic)) flown aboard the International Space Station (ISS). Notably, these animals were sacrificed on-orbit and exposed to varying spaceflight durations (i.e. 21, 37, and 42 days vs 13 days for the Shuttle mice). Oil Red O (ORO) staining showed abnormal lipid accumulation in all space-flown mice compared to ground controls regardless of strain or exposure duration. Similarly, transcriptomic analysis by RNA-sequencing revealed several pathways that were affected in both strains related to increased lipid metabolism, fatty acid metabolism, lipid and fatty acid processing, lipid catabolic processing, and lipid localization. In addition, key upstream regulators were predicted to be commonly regulated across all conditions including Glucagon (GCG) and Insulin (INS). Moreover, quantitative proteomic analysis showed that a number of lipid related proteins were changed in the livers during spaceflight. Taken together, these data indicate that activation of lipotoxic pathways are the result of space stressors alone and this activation occurs in various genetic backgrounds during spaceflight exposures of weeks to months. If similar responses occur in humans, a prolonged change of these pathways may result in the development of liver disease and should be investigated further.
Spaceflight Study	The Rodent Research 3 (RR3) mission	SLID-049	Multi-omics analysis of multiple missions to space reveal a theme of lipid dysregulation in mouse liver	42 Days	Animals	Spaceflight has several detrimental effects on the physiology of astronauts, many of which are recapitulated in rodent models. Mouse studies performed on the Space Shuttle showed disruption of lipid metabolism in liver. However, given that these animals were not sacrificed on-orbit and instead returned live to earth, it is unclear if these disruptions were solely induced by space stressors (e.g. microgravity, space radiation) or in part explained by the stress of return to Earth. In this work we analyzed three liver datasets from two different strains of mice (C57BL/6 (Jackson) & BALB/c (Taconic)) flown aboard the International Space Station (ISS). Notably, these animals were sacrificed on-orbit and exposed to varying spaceflight durations (i.e. 21, 37, and 42 days vs 13 days for the Shuttle mice). Oil Red O (ORO) staining showed abnormal lipid accumulation in all space-flown mice compared to ground controls regardless of strain or exposure duration. Similarly, transcriptomic analysis by RNA-sequencing revealed several pathways that were affected in both strains related to increased lipid metabolism, fatty acid metabolism, lipid and fatty acid processing, lipid catabolic processing, and lipid localization. In addition, key upstream regulators were predicted to be commonly regulated across all conditions including Glucagon (GCG) and Insulin (INS). Moreover, quantitative proteomic analysis showed that a number of lipid related proteins were changed in the livers during spaceflight. Taken together, these data indicate that activation of lipotoxic pathways are the result of space stressors alone and this activation occurs in various genetic backgrounds during spaceflight exposures of weeks to months. If similar responses occur in humans, a prolonged change of these pathways may result in the development of liver disease and should be investigated further.
Spaceflight Study	JAXA (MHU-1)	SLID-050	Impact of spaceflight on the murine thymus and mitigation by exposure to artificial gravity during spaceflight	35 Days	Animals	The environment experienced during spaceflight may impact the immune system and the thymus appears to undergo atrophy during spaceflight. However, molecular aspects of this thymic atrophy remain to be elucidated. In this study, we analysed the thymi of mice on board the international space station (ISS) for approximately 1 month. Thymic size was significantly reduced after spaceflight. Notably, exposure of mice to 1 × g using centrifugation cages in the ISS significantly mitigated the reduction in thymic size. Although spaceflight caused thymic atrophy, the global thymic structure was not largely changed. However, RNA sequencing analysis of the thymus showed significantly reduced expression of cell cycle-regulating genes in two independent spaceflight samples. These reductions were partially countered by 1 × g exposure during the space flights. Thus, our data suggest that spaceflight leads to reduced proliferation of thymic cells, thereby reducing the size of the thymus, and exposure to 1 × g might alleviate the impairment of thymus homeostasis induced by spaceflight.
Spaceflight Study	JAXA (MHU-2)	SLID-050	Impact of spaceflight on the murine thymus and mitigation by exposure to artificial gravity during spaceflight	35 Days	Animals	The environment experienced during spaceflight may impact the immune system and the thymus appears to undergo atrophy during spaceflight. However, molecular aspects of this thymic atrophy remain to be elucidated. In this study, we analysed the thymi of mice on board the international space station (ISS) for approximately 1 month. Thymic size was significantly reduced after spaceflight. Notably, exposure of mice to 1 × g using centrifugation cages in the ISS significantly mitigated the reduction in thymic size. Although spaceflight caused thymic atrophy, the global thymic structure was not largely changed. However, RNA sequencing analysis of the thymus showed significantly reduced expression of cell cycle-regulating genes in two independent spaceflight samples. These reductions were partially countered by 1 × g exposure during the space flights. Thus, our data suggest that spaceflight leads to reduced proliferation of thymic cells, thereby reducing the size of the thymus, and exposure to 1 × g might alleviate the impairment of thymus homeostasis induced by spaceflight.
Spaceflight Study	COSMOS 1887	SLID-051	Effects of spaceflight on rat humerus geometry, biomechanics, and biochemistry	12.5 Days	Animals	The effects of a 12.5-day spaceflight (Cosmos 1887 biosatellite) on the geometric, biomechanical, and biochemical characteristics of humeri of male specific pathogen-free rats were examined. Humeri of age-matched basal control, synchronous control, and vivarium control rats were contrasted with the flight bones to examine the influence of growth and space environment on bone development. Lack of humerus longitudinal growth occurred during the 12.5 days in spaceflight. In addition, the normal mid-diaphysial periosteal appositional growth was affected; compared with their controls, the spaceflight humeri had less cortical cross-sectional area, smaller periosteal circumferences, smaller anterior-posterior periosteal diameters, and smaller second moments of area with respect to the bending and nonbending axes. The flexural rigidity of the flight humeri was comparable to that of the younger basal control rats and significantly less than that of the synchronous and vivarium controls; the elastic moduli of all four groups, nonetheless, were not significantly different. Generally, the matrix biochemistry of the mid-diaphysial cross sections showed no differences among groups. Thus, the spaceflight differences in humeral mechanical strength and flexural rigidity were probably a result of the differences in humeral geometry rather than material properties.
Spaceflight Study	Space Shuttle Mission SL-3	SLID-052	Protein and collagen content of rat skeletal muscle following space flight	7 Days	Animals	Biochemical determinations of non-collagenous protein and hydroxyproline were made on rat skeletal muscles following 7 days of space flight aboard the NASA space shuttle mission SL-3. Relative to ground-based controls, the wet weight of each experimental muscle was significantly reduced. This was concomitant with a reduction in non-collagenous protein in the muscles. Protein concentration, however, was reduced only in slow-twitch muscles. The effect of space flight on the concentration and hydroxyproline content was different among the muscles. As a result, the loss of muscle mass in some muscles was the consequence of a reduction in both collagenous and non-collagenous proteins, while in others it was primarily the result of a non-collagenous protein loss.
Spaceflight Study	Spacelab 3	SLID-053	Fragility and composition of growing rat bone after one week in spaceflight	7 Days	Animals	To gain some insight into the early effects of spaceflight on skeletal metabolism, we quantified the major chemical constituents and a noncollagenous protein, osteocalcin, in the third-lumbar vertebrae and humeri from 8-wk-old rats that were part of the 7-day NASA Spacelab 3 flight experiments. The ratio of calcium to hydroxyproline in the humeral diaphysis increased from 8.5 in preflight to 9.8 in ground simulation control and only to 8.9 in flight bones. There was no demonstrable change in the fraction of nonmineralized collagen. Osteocalcin content was reduced in the humerus and vertebra. Reduced accumulation of mineral and osteocalcin with no associated decrease in collagen in flight animals suggests that both mineralization and collagen metabolism are impaired in growing animals during spaceflight within a few days after launch. Strength tests of the humeri of flight rats showed substantial deficits that appeared to be related, not only to the reduced bone mass, but also to the composition and quality of new bone formed.
Spaceflight Study	COSMOS 2229	SLID-054	Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys	12 Days	Animals	During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.
Spaceflight Study	STS-63	SLID-055	Spaceflight Downregulates Antioxidant Defense Systems in Rat Liver	8 Days and 7 Hours	Animals	Liver antioxidant enzyme activities, mRNA abundance, and glutathione (GSH) status were investigated in male Sprague-Dawley rats placed in an enclosure module aboard Space Shuttle STS-63 for 8 d (F, n = 6). F animals were compared to rats housed in an enclosure module on the ground (G, n = 9), which simulated the vibration and temperature conditions associated with launch and flight, and rats kept under conventional ground vivarium conditions in individual cages (V, n = 6). Spaceflight significantly decreased catalase, GSH reductase, and GSH sulfur-transferase activities in the liver (p < .05). Neither enzyme activity nor enzyme protein content of Cu-Zn and Mn superoxide dismutase (SOD) was affected by flight. The relative abundance of mRNA for Cu-Zn SOD and catalase was significantly decreased comparing F with G rats (p < .05). Spaceflight resulted in a dramatic decrease of liver GSH, glutathione disulfide, and total GSH contents (p < .01), which were accompanied by a lower gamma-glutamyl transpeptidase activity (p < .05). F rats showed a 47% (p < .05) increase in liver malondialdehyde concentration compared to G and V rats. Liver protein content was not affected by flight. These results indicate that spaceflight can downregulate antioxidant defense capacity and elicit an oxidative stress in the liver.
Ground Study	Orbital Environmental Simulator	SLID-056	Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat	10 Days	Animals	Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22 degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.
Spaceflight Study	STS-57	SLID-056	Sustained microgravity reduces intrinsic wound healing and growth factor responses in the rat	10 Days	Animals	Spaceflight is known to diminish bone mass and reduce immune function, suggesting that repair of connective tissue might be impaired in a microgravity environment. Fisher 344 rats were used to test wound healing responses in the orbiting Space Shuttle Endeavour by preflight implantation of polyvinyl acetal sponge disks in which pellets were placed to release either platelet-derived growth factor (PDGF-BB), basic fibroblast growth factor (bFGF), or placebo. Control groups on the ground included a matched environment group in similar housing modules and temperature control groups in cages at 22 degreesC and 28 degreesC. After 12 days of implantation and 10 days in orbit, the removed sponges were analyzed for histological and biochemical responses. Growth factor responses were histologically evident after release of PDGF-BB and bFGF in ground controls, whereas only immediate-release bFGF and delayed-release PDGF-BB showed significant responses in microgravity. Biochemical data confirmed that cellularity was increased by both factors in ground sponges; however, this response was significantly blunted in flight sponges (P<0.005, ANOVA), irrespective timing of factor release. Collagen content was 62% lower in sponges from animals with 10 days of microgravity exposure (P<0.01, ANOVA) and further reduced by bFGF. These data suggest that orbital exposure retards the capacity of wounds to heal and respond to exogenous stimuli.
Spaceflight Study	STS-60	SLID-057	Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses	8 Days and 7 Hours	Animals	Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Spaceflight Study	STS-60	SLID-057	Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses	8 Days and 7 Hours	Animals	Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Spaceflight Study	STS-63	SLID-057	Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses	8 Days and 7 Hours	Animals	Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Spaceflight Study	STS-63	SLID-057	Effects of spaceflight and PEG-IL-2 on rat physiological and immunological responses	8 Days and 7 Hours	Animals	Sprague-Dawley rats were subjected to two 8-day spaceflights on the space shuttle. Rats housed in the National Aeronautics and Space Administration's animal enclosure were injected (iv or sc) with pegylated interleukin-2 (PEG-IL-2) or a placebo. We tested the hypothesis that PEG-IL-2 would ameliorate some of the effects of spaceflight. We measured body and organ weights; blood cell differentials; plasma corticosterone; colony-forming units (macrophage and granulocyte macrophage); lymphocyte mitogenic, superantigenic, and interferon-gamma responses; bone marrow cell and peritoneal macrophage cytokine secretion; and bone strength and mass. Few immunological parameters were affected by spaceflight. However, some spaceflight effects were observed in each flight. Specifically, peritoneal macrophage spontaneous secretion of tumor necrosis factor-alpha occurred in the first but not in the second flight. A significant monocytopenia and lymphocytopenia were detected in the second but not in the first flight. The second mission produced bone changes more consistent with past spaceflight investigations. PEG-IL-2 did not appear to be beneficial; however, this was mostly due to the lack of spaceflight effects. These studies reflect the difficulty in reproducing experimental models by using current space shuttle conditions.
Spaceflight Study	STS-58	SLID-058	Urinary excretion of LH and testosterone from male rats during exposure to increased gravity: post-spaceflight and centrifugation	14 Days	Animals	A dissociation between plasma luteinizing hormone (LH) and testosterone (T) appears to exist during exposure to altered gravity. The pulsatile nature of LH release and the diurnal variability of T secretion may mask or bias the effects of altered gravity on the pituitary-gonadal axis when analyzing plasma concentrations. Therefore, we examined the relationship between the excretion of urinary LH and T in male Sprague-Dawley rats during exposure to increased gravity upon return to Earth following a 14-day spaceflight (n = 6) and by 12 days of centrifugation at 2g (n = 8). Excreted LH and T were elevated on the first 3 days postflight. Excreted T was elevated between Days 1 and 8 of centrifugation; however, excreted LH was reduced on Days 2 and 3 compared with control animals. Excreted LH and T were significantly correlated (R = 0.731 and 0.706, respectively) in postspaceflight and centrifuged animals. Correlation curves had similar slopes (0.0213 and 0.023, respectively), but different y-intercepts (-1.43 and 3.32, respectively). The sustained increase in excreted T during centrifugation suggests that the pituitary-gonadal axis in postspaceflight animals may adapt quicker to increased gravity. The upward shift in the correlation curve exhibited by the centrifuged animals suggests that the sensitivity of LH-induced T release is increased in these animals. The previous dissociation between plasma LH and T during altered gravity was not observed in the present study in which excreted LH and T were measured.
Spaceflight Study	Endeavour (OV-105)	SLID-059	The effects of 10 days of spaceflight on the shuttle Endeavor on predominantly fast-twitch muscles in the rat	10 Days	Animals	The primary purpose of this investigation was to determine the effects of microgravity on muscle fibers of the predominantly fast-twitch muscles in the rat. Cross sectional area and myosin heavy chain (MHC) composition were assessed in order to establish the acute effects of microgravity associated with spaceflight. The extensor digitorum longus (EDL) and gastrocnemius muscles were removed from 12 male Fisher 344 rats which had undergone 10 days of spaceflight aboard the space shuttle Endeavor and from 12 age- and weight-matched control animals. Both groups of animals received similar amounts of food and water and were synchronized for photoperiods, environmental temperature, and humidity. Significant (P < 0.05) reductions in muscle fiber size were observed in the gastrocnemius (fiber types I, IIA, IIDB, and IIB) and EDL (fiber type IIB) muscles after spaceflight. Significant MHC isoform transformations also resulted during this brief period of microgravity exposure with a significant decrease in MHC IId isoform in the EDL muscle. A significant decrease was also observed in the MHC IId isoform in the superficial (white) component of the gastrocnemius muscle after spaceflight, although no alterations in MHC profile were demonstrated in the deep (red) component of this muscle. These findings highlight the rapid plasticity of skeletal muscle during short-term spaceflight. If such pronounced adaptations to spaceflight also occur in humans, then astronauts are likely to suffer severe decrements in skeletal muscle performance with long-term space flight and upon return to earth after both short- and long-term missions. Thus, countermeasures aimed at slowing or even preventing muscle fiber atrophy are warranted.
Spaceflight Study	STS-90	SLID-060	Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway	16 Days	Animals	To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.
Ground Study	Tail Suspension Hindlimb Unloading Model	SLID-060	Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway	16 Days	Animals	To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.
Spaceflight Study	STS-90	SLID-061	Skeletal muscle gene expression in space-flown rats	16 Days	Animals	Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.
Ground Study	Tail Suspension Hindlimb Unloading Model	SLID-061	Skeletal muscle gene expression in space-flown rats	16 Days	Animals	Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.
Spaceflight Study	Spacelab Life Sciences 2 (SLS-2)	SLID-062	Effects of spaceflight on myosin heavy-chain content, fibre morphology and succinate dehydrogenase activity in rat diaphragm	14 Days	Animals	The present study examined the effect of 14 days of exposure to microgravity during the Spacelab Life Sciences-2 (SLS-2) space shuttle mission on the myosin heavy-chain (MHC) content, fibre size and type distributions and metabolic properties of rat diaphragm. Five adult male Sprague-Dawley rats were exposed to 14 days of microgravity (SF, spaceflight) and compared to five ground-based controls (C). Immunohistochemical analyses using isoform-specific anti-MHC monoclonal antibodies revealed that 14 days of SF did not alter the proportions of type-I, -IIA, -IID/X or -IIB fibres within the crural, sternal or lateral costal regions of the diaphragm; the electrophoretically quantified MHC-isoform contents also remained unchanged. In contrast, the medial gastrocnemius (MG) and tibialis anterior (TA) muscles displayed slow-to-fast fibre type transitions: within the MG the proportion of type-IID/X fibres was reduced by 59% ( P<0.04) and corresponded to a 51% increase ( P<0.03) in type-IIB fibres. Within the TA, the sum of type-IID/X+IIB fibres was elevated by 24% ( P<0.02) at the expense of the slower type-IIA fibres, which decreased by 33% ( P<0.04). Electrophoretic analyses yielded qualitatively similar patterns of transformation. SF did not induce atrophic changes within the diaphragm, MG or TA. Succinate dehydrogenase activity remained unchanged in the crural diaphragm ( P>0.96) but was 34% lower ( P<0.0001) in the TA. We conclude that 14 days of SF did not alter structural or metabolic factors that are known to underlie functional properties of the diaphragm. The findings of the present study show that 14 days of SF does not induce deleterious adaptive changes in the rat diaphragm that occur in hindlimb muscles.
Spaceflight Study	Spacelab Life Sciences 1 (SLS-1)	SLID-063	Comparison of cell body size and oxidative enzyme activity in motoneurons between the cervical and lumbar segments in the rat spinal cord after spaceflight and recovery	9 Days	Animals	The cell body sizes and succinate dehydrogenase (SDH) activities of motoneurons in the dorsolateral region of the ventral horn at the cervical and lumbar segments in the rat spinal cord were determined following 9 days of spaceflight with or without 10 days of recovery on Earth. The motoneurons were divided into three types based on their cell body sizes; small-, medium-, and large-sized motoneurons. In control rats, there was no difference in the cell body size or SDH activity of small- and large-sized motoneurons between the cervical and lumbar segments. The SDH activity of medium-sized motoneurons in control rats was higher in the lumbar segment than in the cervical segment, while the cell body sizes of medium-sized motoneurons were identical. The SDH activity of medium-sized motoneurons in the lumbar segment decreased to a level similar to that in the cervical segment of control rats following spaceflight. In addition, the decreased SDH activity of medium-sized motoneurons persisted for at least 10 days of recovery on Earth. It is concluded that spaceflight selectively affects the SDH activity of medium-sized motoneurons in the lumbar segment of the spinal cord, which presumably innervate skeletal muscles having an antigravity function.
Ground Study	Tail Suspension Hindlimb Unloading Model	SLID-064	Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat	14 Days	Animals	Long-term spaceflight induces hypokinesia and hypodynamia, which, along microgravity per se, result in a number of significant physiological alterations, such as muscle atrophy, force reduction, insulin resistance, substrate use shift from fats to carbohydrates, and bone loss. Each of these adaptations could turn to serious health deterioration during the long-term spaceflight needed for planetary exploration. We hypothesized that resveratrol (RES), a natural polyphenol, could be used as a nutritional countermeasure to prevent muscle metabolic and bone adaptations to 15 d of rat hindlimb unloading. RES treatment maintained a net protein balance, soleus muscle mass, and soleus muscle maximal force contraction. RES also fully maintained soleus mitochondrial capacity to oxidize palmitoyl-carnitine and reversed the decrease of the glutathione vs. glutathione disulfide ratio, a biomarker of oxidative stress. At the molecular level, the protein content of Sirt-1 and COXIV in soleus muscle was also preserved. RES further protected whole-body insulin sensitivity and lipid trafficking and oxidation, and this was likely associated with the maintained expression of FAT/CD36, CPT-1, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in muscle. Finally, chronic RES supplementation maintained the bone mineral density and strength of the femur. For the first time, we report a simple countermeasure that prevents the deleterious adaptations of the major physiological functions affected by mechanical unloading. RES could thus be envisaged as a nutritional countermeasure for spaceflight but remains to be tested in humans.
Ground Study	2g Centrifuge	SLID-065	Observing the mouse thyroid sphingomyelin under space conditions: a case study from the MDS mission in comparison with hypergravity conditions	91 Days	Animals	This is a case report of apparent thyroid structural and functional alteration in a single mouse subjected to low Earth orbit spaceflight for 91 days. Histological examination of the thyroid gland revealed an increase in the average follicle size compared to that of three control animals and three animals exposed to hypergravity (2g) conditions. Immunoblotting analysis detected an increase in two thyroid gland enzymes, sphingomyelinase and sphingomyelin-synthase1. In addition, sphingomyelinase, an enzyme confined to the cell nucleus in the control animals, was found in the mouse exposed to hypogravity to be homogeneously distributed throughout the cell bodies. It represents the first animal observation of the influence of weightlessness on sphingomyelin metabolism.
Spaceflight Study	STS-128	SLID-065	Observing the mouse thyroid sphingomyelin under space conditions: a case study from the MDS mission in comparison with hypergravity conditions	91 Days	Animals	This is a case report of apparent thyroid structural and functional alteration in a single mouse subjected to low Earth orbit spaceflight for 91 days. Histological examination of the thyroid gland revealed an increase in the average follicle size compared to that of three control animals and three animals exposed to hypergravity (2g) conditions. Immunoblotting analysis detected an increase in two thyroid gland enzymes, sphingomyelinase and sphingomyelin-synthase1. In addition, sphingomyelinase, an enzyme confined to the cell nucleus in the control animals, was found in the mouse exposed to hypogravity to be homogeneously distributed throughout the cell bodies. It represents the first animal observation of the influence of weightlessness on sphingomyelin metabolism.
Spaceflight Study	BION-M1	SLID-066	Effect of actual long-term spaceflight on BDNF, TrkB, p75, BAX and BCL-XL genes expression in mouse brain regions	30 Days	Animals	Mice of C57BL/6J strain were exposed to 1-month spaceflight on Russian biosatellite Bion-M1 to determine the effect of long-term actual spaceflight on the expression of genes involved in the processes of neurogenesis and apoptosis. Specifically, we focused on the genes encoding proapoptotic factor BAX, antiapoptotic factor BCL-XL, brain-derived neurotrophic factor (BDNF) and BDNF receptors TrkB and p75. Spaceflight reduced the expression of the antiapoptotic BCL-XL gene in the striatum and hypothalamus, but increased it in the hippocampus. To estimate environmental stress contribution into spaceflight effects we analyzed spaceflight-responsive genes in mice housed for 1 month on Earth in the same shuttle cabins that were used for spaceflight, and in mice of the laboratory control group. It was shown that 1-month shuttle cabin housing decreased BCL-XL gene expression in the striatum but failed to alter BCL-XL mRNA levels in the hippocampus or hypothalamus. Spaceflight failed to alter the expression of the proapoptotic BAX gene in all investigated brain structures, although the insignificant increase of the BAX mRNA level in the hippocampus of spaceflight mice was found. At the same time, shuttle cabin housing produced insignificant decrease in BAX gene expression in the hippocampus. In contrast to the BCL-XL gene, genes encoding BAX, BDNF as well as TrkB and p75 receptors did not respond to 30-day spaceflight. Thus, long-term spaceflight (1) did not affect the expression of genes encoding BDNF as well as TrkB and p75 receptors, (2) produced dysregulation in genetic control of the neuronal apoptosis, (3) implicated BCL-XL as the risk factor for spaceflight-induced behavioral abnormalities.
Spaceflight Study	BION-M1	SLID-067	Spaceflight Effects on Cytochrome P450 Content in Mouse Liver	30 Days	Animals	Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.
Ground Study	Tail Suspension Hindlimb Unloading Model	SLID-068	The effect of long-term hindlimb unloading on the expression of risk neurogenes encoding elements of serotonin-, dopaminergic systems and apoptosis; comparison with the effect of actual spaceflight on mouse brain	30 Days	Animals	The study of spaceflight effects on the brain is technically complex concern; complicated by the problem of applying an adequate ground model. The most-widely used experimental model to study the effect of microgravity is the tail-suspension hindlimb unloading model; however, its compliance with the effect of actual spaceflight on the brain is still unclear. We evaluated the effect of one month hindlimb unloading on the expression of genes related to the brain neuroplasticity-brain neutotrophic factors (Gdnf, Cdnf), apoptotic factors (Bcl-xl, Bax), serotonin- and dopaminergic systems (5-HT2A, Maoa, Maob, Th, D1r, Comt), and compared the results with the data obtained on mice that spent one month in spaceflight on Russian biosatellite Bion-M1. No effect of hindlimb unloading was observed on the expression of most genes, which were considered as risk neurogenes for long-term actual spaceflight. The opposite effect of hindlimb unloading and spaceflight was found on the level of mRNA of D1 dopamine receptor and catechol-O-methyltransferase in the striatum. At the same time, the expression of Maob in the midbrain decreased, and the expression of Bcl-xl genes increased in the hippocampus, which corresponds to the effect of spaceflight. However, the hindlimb unloading model failed to reproduce the majority of effects of long-term spaceflight on serotonin-, dopaminergic systems and some apoptotic factors.
Spaceflight Study	International Space Station (ISS)	SLID-069	Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months	288 Days	Animals	If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 °C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.
Spaceflight Study	BION-M1	SLID-070	One-month spaceflight compromises the bone microstructure, tissue-level mechanical properties, osteocyte survival and lacunae volume in mature mice skeletons	30 Days	Animals	The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (−64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape (synchrotron radiation analyses), and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties (i.e., hardness and modulus) were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair.
Spaceflight Study	BION-M1	SLID-071	Thirty days of spaceflight does not alter murine calvariae structure despite increased Sost expression	30 Days	Animals	Previously our laboratory documented increases in calvaria bone volume and thickness in mice exposed to 15 days of spaceflight aboard the NASA Shuttle mission STS-131. However, the tissues were not processed for gene expression studies to determine what bone formation pathways might contribute to these structural adaptations. Therefore, this study was designed to investigate both the structural and molecular changes in mice calvariae after a longer duration of spaceflight. The primary purpose was to determine the calvaria bone volume and thickness of mice exposed to 30 days of spaceflight using micro-computed tomography for comparison with our previous findings. Because sclerostin, the secreted glycoprotein of the Sost gene, is a potent inhibitor of bone formation, our second aim was to quantify Sost mRNA expression using quantitative PCR. Calvariae were obtained from six mice aboard the Russian 30-day Bion-M1 biosatellite and seven ground controls. In mice exposed to 30 days of spaceflight, calvaria bone structure was not significantly different from that of their controls (bone volume was about 5% lower in spaceflight mice, p = 0.534). However, Sost mRNA expression was 16-fold (16.4 ± 0.4, p < 0.001) greater in the spaceflight group than that in the ground control group. Therefore, bone formation may have been suppressed in mice exposed to 30 days of spaceflight. Genetic responsiveness (e.g. sex or strain of animals) or in-flight environmental conditions other than microgravity (e.g. pCO2 levels) may have elicited different bone adaptations in STS-131 and Bion-M1 mice. Although structural results were not significant, this study provides biochemical evidence that calvaria mechanotransduction pathways may be altered during spaceflight, which could reflect vascular and interstitial fluid adaptations in non-weight bearing bones. Future studies are warranted to elucidate the processes that mediate these effects and the factors responsible for discordant calvaria bone adaptations between STS-131 and Bion-M1 mice.
Spaceflight Study	BION-M1	SLID-072	Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight	30 Days	Animals	Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45+MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.
Ground Study	NASA Space Radiation Laboratory (NSRL)	SLID-073	Space radiation triggers persistent stress response, increases senescent signaling, and decreases cell migration in mouse intestine	7 Days; 60 Days; 12 Months	Animals	Proliferative gastrointestinal (GI) tissue is radiation-sensitive, and heavy-ion space radiation with its high-linear energy transfer (high-LET) and higher damaging potential than low-LET γ-rays is predicted to compromise astronauts' GI function. However, much uncertainty remains in our understanding of how heavy ions affect coordinated epithelial cell migration and extrusion, which are essential for GI homeostasis. Here we show using mouse small intestine as a model and BrdU pulse labeling that cell migration along the crypt-villus axis is persistently decreased after a low dose of heavy-ion 56Fe radiation relative to control and γ-rays. Wnt/β-catenin and its downstream EphrinB/EphB signaling are key to intestinal epithelial cell (IEC) proliferation and positioning during migration, and both are up-regulated after 56Fe radiation. Conversely, factors involved in cell polarity and adhesion and cell-extracellular matrix interactions were persistently down-regulated after 56Fe irradiation-potentially altering cytoskeletal remodeling and cell extrusion. 56Fe radiation triggered a time-dependent increase in γH2AX foci and senescent cells but without a noticeable increase in apoptosis. Some senescent cells acquired the senescence-associated secretory phenotype, and this was accompanied by increased IEC proliferation, implying a role for progrowth inflammatory factors. Collectively, this study demonstrates a unique phenomenon of heavy-ion radiation-induced persistently delayed IEC migration involving chronic sublethal genotoxic and oncogenic stress-induced altered cytoskeletal dynamics, which were seen even a year later. When considered along with changes in barrier function and nutrient absorption factors as well as increased intestinal tumorigenesis, our in vivo data raise a serious concern for long-duration deep-space manned missions.
Spaceflight Study	BION-M1	SLID-074	Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth	30 Days	Animals	Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronauts' health and safety. However, consequences on B-cell development and responses are still under-investigated. To fill this gap, we used advanced proteomics analysis of femur bone and marrow to compare mice flown for 1 mo on board the BION-M1 biosatellite, followed or not by 1 wk of recovery on Earth, to control mice kept on Earth. Our data revealed an adverse effect on B lymphopoiesis 1 wk after landing. This phenomenon was associated with a 41% reduction of B cells in the spleen. These reductions may contribute to explain increased susceptibility to infection even if our data suggest that flown animals can mount a humoral immune response. Future studies should investigate the quality/efficiency of produced antibodies and whether longer missions worsen these immune alterations.-Tascher, G., Gerbaix, M., Maes, P., Chazarin, B., Ghislin, S., Antropova, E., Vassilieva, G., Ouzren-Zarhloul, N., Gauquelin-Koch, G., Vico, L., Frippiat, J.-P., Bertile, F. Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.
Spaceflight Study	SpaceX Falcon 9	SLID-075	Down-regulation of GATA1-dependent erythrocyte-related genes in the spleens of mice exposed to a space travel	35 Days	Animals	Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 g exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes.
Spaceflight Study	SpaceX Mission CRS-12	SLID-076	Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations	35 Days	Animals	The health risks associated with spaceflight-induced ocular structural and functional damage has become a recent concern for NASA. The goal of the present study was to characterize the effects of spaceflight and reentry to 1 g on the structure and integrity of the retina and blood-retinal barrier (BRB) in the eye. To investigate possible mechanisms, changes in protein expression profiles were examined in mouse ocular tissue after spaceflight. Ten week old male C57BL/6 mice were launched to the International Space Station (ISS) on Space-X 12 at the Kennedy Space Center (KSC) on August, 2017. After a 35-day mission, mice were returned to Earth alive. Within 38 +/- 4 hours of splashdown, mice were euthanized and ocular tissues were collected for analysis. Ground control (GC) and vivarium control mice were maintained on Earth in flight hardware or normal vivarium cages respectively. Repeated intraocular pressure (IOP) measurements were performed before the flight launch and re-measured before the mice were euthanized after splashdown. IOP was significantly lower in post-flight measurements compared to that of pre-flight (14.4-19.3 mmHg vs 16.3-20.3 mmHg) (p < 0.05) for the left eye. Flight group had significant apoptosis in the retina and retinal vascular endothelial cells compared to control groups (p < 0.05). Immunohistochemical analysis of the retina revealed that an increased expression of aquaporin-4 (AQP-4) in the flight mice compared to controls gave strong indication of disturbance of BRB integrity. There were also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BRB-related tight junction protein, Zonula occludens-1 (ZO-1). Proteomic analysis showed that many key proteins and pathways responsible for cell death, cell cycle, immune response, mitochondrial function and metabolic stress were significantly altered in the flight mice compared to ground control animals. These data indicate a complex cellular response that may alter retina structure and BRB integrity following long-term spaceflight.
Ground Study	Tailor-made Centrifuge	SLID-077	Hypergravity disrupts murine intestinal microbiota	21 Days	Animals	During spaceflight, organisms are subjected to various physical stressors including modification of gravity (G) that, associated with lifestyle, could lead to impaired immunity, intestinal dysbiosis and thus potentially predispose astronauts to illness. Whether space travel affects microbiota homeostasis has not been thoroughly investigated. The aim of this study was to evaluate changes in intestinal microbiota and mucosa in a ground-based murine model consisting in a 21-days confinement of mice in a centrifuge running at 2 or 3G. Results revealed an increased α-diversity and a significant change in intracaecal β-diversity observed only at 3G, with profiles characterized by a decrease of the Firmicutes/Bacteroidetes ratio. Compared to 1G microbiota, 12.1% of the taxa were significantly impacted in 3G microbiota, most of them (78%) being enriched. This study shows a G-level-dependent disruption of intracaecal microbiota, without alteration of mucosal integrity. These first data reinforce those recently obtained with in-flight experimentations or microgravity models, and emphasize the critical need for further studies exploring the impact of spaceflight on intestinal microbiota in order to optimize long-term space travel conditions.
Spaceflight Study	SpaceX Mission CRS-12	SLID-078	Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina	35 Days	Animals	Extended spaceflight has been shown to adversely affect astronaut visual acuity. The purpose of this study was to determine whether spaceflight alters gene expression profiles and induces oxidative damage in the retina. Ten week old adult C57BL/6 male mice were flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth under identical environmental conditions. Within 38 (+/−4) hours after splashdown, mice ocular tissues were collected for analysis. RNA sequencing detected 600 differentially expressed genes (DEGs) in murine spaceflight retinas, which were enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Twelve DEGs were associated with retinitis pigmentosa, characterized by dystrophy of the photoreceptor layer rods and cones. Differentially expressed transcription factors indicated changes in chromatin structure, offering clues to the observed phenotypic changes. Immunofluorescence assays showed degradation of cone photoreceptors and increased retinal oxidative stress. Total retinal, retinal pigment epithelium, and choroid layer thickness were significantly lower after spaceflight. These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment.
Spaceflight Study	SpaceX-9	SLID-079	Male mice, caged in the International Space Station for 35 days, sire healthy offspring	35 Days	Animals	The effect on the reproductive system and fertility of living in a space environment remains unclear. Here, we caged 12 male mice under artificial gravity (≈1 gravity) (AG) or microgravity (MG) in the International Space Station (ISS) for 35 days, and characterized the male reproductive organs (testes, epididymides, and accessory glands) after their return to earth. Mice caged on earth during the 35 days served as a "ground" control (GC). Only a decrease in accessory gland weight was detected in AG and MG males; however, none of the reproductive organs showed any overt microscopic defects or changes in gene expression as determined by RNA-seq. The cauda epididymal spermatozoa from AG and MG mice could fertilize oocytes in vitro at comparable levels as GC males. When the fertilized eggs were transferred into pseudo-pregnant females, there was no significant difference in pups delivered (pups/transferred eggs) among GC, AG, and MG spermatozoa. In addition, the growth rates and fecundity of the obtained pups were comparable among all groups. We conclude that short-term stays in outer space do not cause overt defects in the physiological function of male reproductive organs, sperm function, and offspring viability.
Spaceflight Study	BION-M1	SLID-080	Effects of spaceflight on the mouse submandibular gland	30 Days	Animals	Objective: This study was conducted to determine if the morphology and biochemistry of the mouse submandibular gland is affected by microgravity and the spaceflight environment. Design: Tissues from female mice flown on the US space shuttle missions Space Transportation System (STS)-131 and STS-135 for 15 and 13 d, respectively, and from male mice flown on the 30 d Russian Bion-M1 biosatellite, were examined using transmission electron microscopy and light and electron microscopic immunohistochemistry. Results: In contrast to the parotid gland, morphologic changes were not apparent in the submandibular gland. No significant changes in protein expression, as assessed by quantitative immunogold labeling, occurred in female mice flown for 13-15 d. In male mice, however, increased labeling for salivary androgen binding protein alpha (in acinar cell secretory granules), and epidermal growth factor and nerve growth factor (in granular convoluted duct cell granules) was seen after 30 d in space. Conclusion: These results indicate that spaceflight alters secretory protein expression in the submandibular gland and suggest that the sex of the animals and the length of the flight may affect the response. These findings also show that individual salivary glands respond differently to spaceflight. Saliva contains proteins secreted from salivary glands and is easily collected, therefore is a useful biofluid for general medical analyses and in particular for monitoring the physiology and health of astronauts.
Spaceflight Study	STS-131	SLID-080	Effects of spaceflight on the mouse submandibular gland	15 Days	Animals	Objective: This study was conducted to determine if the morphology and biochemistry of the mouse submandibular gland is affected by microgravity and the spaceflight environment. Design: Tissues from female mice flown on the US space shuttle missions Space Transportation System (STS)-131 and STS-135 for 15 and 13 d, respectively, and from male mice flown on the 30 d Russian Bion-M1 biosatellite, were examined using transmission electron microscopy and light and electron microscopic immunohistochemistry. Results: In contrast to the parotid gland, morphologic changes were not apparent in the submandibular gland. No significant changes in protein expression, as assessed by quantitative immunogold labeling, occurred in female mice flown for 13-15 d. In male mice, however, increased labeling for salivary androgen binding protein alpha (in acinar cell secretory granules), and epidermal growth factor and nerve growth factor (in granular convoluted duct cell granules) was seen after 30 d in space. Conclusion: These results indicate that spaceflight alters secretory protein expression in the submandibular gland and suggest that the sex of the animals and the length of the flight may affect the response. These findings also show that individual salivary glands respond differently to spaceflight. Saliva contains proteins secreted from salivary glands and is easily collected, therefore is a useful biofluid for general medical analyses and in particular for monitoring the physiology and health of astronauts.
Spaceflight Study	STS-135	SLID-080	Effects of spaceflight on the mouse submandibular gland	13 Days	Animals	Objective: This study was conducted to determine if the morphology and biochemistry of the mouse submandibular gland is affected by microgravity and the spaceflight environment. Design: Tissues from female mice flown on the US space shuttle missions Space Transportation System (STS)-131 and STS-135 for 15 and 13 d, respectively, and from male mice flown on the 30 d Russian Bion-M1 biosatellite, were examined using transmission electron microscopy and light and electron microscopic immunohistochemistry. Results: In contrast to the parotid gland, morphologic changes were not apparent in the submandibular gland. No significant changes in protein expression, as assessed by quantitative immunogold labeling, occurred in female mice flown for 13-15 d. In male mice, however, increased labeling for salivary androgen binding protein alpha (in acinar cell secretory granules), and epidermal growth factor and nerve growth factor (in granular convoluted duct cell granules) was seen after 30 d in space. Conclusion: These results indicate that spaceflight alters secretory protein expression in the submandibular gland and suggest that the sex of the animals and the length of the flight may affect the response. These findings also show that individual salivary glands respond differently to spaceflight. Saliva contains proteins secreted from salivary glands and is easily collected, therefore is a useful biofluid for general medical analyses and in particular for monitoring the physiology and health of astronauts.
Spaceflight Study	SpaceX-4	SLID-081	Validation of a New Rodent Experimental System to Investigate Consequences of Long Duration Space Habitation	37 Days	Animals	Animal models are useful for exploring the health consequences of prolonged spaceflight. Capabilities were developed to perform experiments in low earth orbit with on-board sample recovery, thereby avoiding complications caused by return to Earth. For NASA's Rodent Research-1 mission, female mice (ten 32 wk C57BL/6NTac; ten 16 wk C57BL/6J) were launched on an unmanned vehicle, then resided on the International Space Station for 21/22d or 37d in microgravity. Mice were euthanized on-orbit, livers and spleens dissected, and remaining tissues frozen in situ for later analyses. Mice appeared healthy by daily video health checks and body, adrenal, and spleen weights of 37d-flight (FLT) mice did not differ from ground controls housed in flight hardware (GC), while thymus weights were 35% greater in FLT than GC. Mice exposed to 37d of spaceflight displayed elevated liver mass (33%) and select enzyme activities compared to GC, whereas 21/22d-FLT mice did not. FLT mice appeared more physically active than respective GC while soleus muscle showed expected atrophy. RNA and enzyme activity levels in tissues recovered on-orbit were of acceptable quality. Thus, this system establishes a new capability for conducting long-duration experiments in space, enables sample recovery on-orbit, and avoids triggering standard indices of chronic stress.
Spaceflight Study	SpaceX Mission CRS-16	SLID-082	Spaceflight affects neuronal morphology and alters transcellular degradation of neuronal debris in adult Caenorhabditis elegans	5 Days	Animals	Extended space travel is a goal of government space agencies and private companies. However, spaceflight poses risks to human health, and the effects on the nervous system have to be better characterized. Here, we exploited the unique experimental advantages of the nematode Caenorhabditis elegans to explore how spaceflight affects adult neurons in vivo. We found that animals that lived 5 days of adulthood on the International Space Station exhibited hyperbranching in PVD and touch receptor neurons. We also found that, in the presence of a neuronal proteotoxic stress, spaceflight promotes a remarkable accumulation of neuronal-derived waste in the surrounding tissues, suggesting an impaired transcellular degradation of debris released from neurons. Our data reveal that spaceflight can significantly affect adult neuronal morphology and clearance of neuronal trash, highlighting the need to carefully assess the risks of long-duration spaceflight on the nervous system and to develop adequate countermeasures for safe space exploration.
Spaceflight Study	STS-41	SLID-083	Effects of a Four-day Spaceflight and Recombinant Human Growth Hormone on Cancellous Bone Microarchitecture in Femoral Head of Rapidly Growing Male Rats	4 Days	Animals	Spaceflight results in reduced bone accrual and muscle atrophy in growing rodents. Some studies suggest that the detrimental effects of spaceflight are due, in part, to impaired growth hormone (GH) signaling. An experiment flown aboard STS-41 (October 6–10, 1990) evaluated the efficacy of recombinant human growth hormone (rhGH) in ameliorating the detrimental effects of spaceflight on the musculoskeletal system in male Sprague Dawley rats. The rats were 39 days old at launch and sacrificed following the 4–day flight. Ground controls (n=11/treatment group) and flight animals (n=8/treatment group) were treated with rhGH or excipient delivered using osmotic pumps implanted subcutaneously one day prior to launch. For the present examination, cancellous bone in the femoral head was evaluated using X-ray microtomography (microcomputed tomography), a technology not available when the study was performed. Spaceflight resulted in lower cancellous bone volume fraction, connectivity density, trabecular thickness and trabecular number, and higher trabecular separation. rhGH had no independent effect on cancellous bone architecture and there were no spaceflight by rhGH interactions. These findings suggest that a short interval of microgravity during rapidgrowthwassufficienttoreduceaccrualofcancellousboneandalterbonemicroarchitecture at an important weight bearing skeletal site. Additionally, increasing growth hormone levels was ineffective in preventing cancellous osteopenia in flight animals and did not increase cancellous bone volume fraction in ground controls.
Spaceflight Study	International Space Station (ISS)	SLID-084	Persistent deterioration of visuospatial performance in spaceflight	Half-year	Long-term effects of spaceflight	Although human adaptation to spaceflight has been studied for decades, little is known about its long-term effects on brain and behavior. The present study investigated visuospatial performance and associated electrophysiological responses in astronauts before, during, and after an approximately half-year long mission to the International Space Station. Here we report findings demonstrating that cognitive performance can suffer marked decrements during spaceflight. Astronauts were slower and more error-prone on orbit than on Earth, while event-related brain potentials reflected diminished attentional resources. Our study is the first to provide evidence for impaired performance during both the initial (~ 8 days) and later (~ 50 days) stages of spaceflight, without any signs of adaptation. Results indicate restricted adaptability to spaceflight conditions and calls for new research prior to deep space explorations.
Spaceflight Study	SpaceX-4	SLID-085	Behavior of mice aboard the International Space Station	37 Days	Animals	Interest in space habitation has grown dramatically with planning underway for the first human transit to Mars. Despite a robust history of domestic and international spaceflight research, understanding behavioral adaptation to the space environment for extended durations is scant. Here we report the first detailed behavioral analysis of mice flown in the NASA Rodent Habitat on the International Space Station (ISS). Following 4-day transit from Earth to ISS, video images were acquired on orbit from 16- and 32-week-old female mice. Spaceflown mice engaged in a full range of species-typical behaviors. Physical activity was greater in younger flight mice as compared to identically-housed ground controls, and followed the circadian cycle. Within 7-10 days after launch, younger (but not older), mice began to exhibit distinctive circling or 'race-tracking' behavior that evolved into coordinated group activity. Organized group circling behavior unique to spaceflight may represent stereotyped motor behavior, rewarding effects of physical exercise, or vestibular sensation produced via self-motion. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach yields a useful analog for better understanding human responses to spaceflight, providing the opportunity to assess how physical movement influences responses to microgravity.
Spaceflight Study	COSMOS 1514	SLID-086	Thermoregulatory responses of Rhesus monkeys during spaceflight	5 Days	Animals	This study examines the activity, axillary temperature (T(ax)), and ankle skin temperature (Tsk) of two male Rhesus monkeys exposed to microgravity in space. The animals were flown on a Soviet biosatellite mission (COSMOS 1514). Measurements on the flight animals, as well as synchronous flight controls, were performed in the Soviet Union. Additional control studies were performed in the United States to examine the possible role of metabolic heat production in the T(ax) response observed during the spaceflight. All monkeys were exposed to a 24-h light-dark cycle (LD 16:8) throughout these studies. During weightlessness, T(ax) in both flight animals was lower than on earth. The largest difference (0.75 degree C) occurred during the night. There was a reduction in mean heart rate and Tsk during flight. This suggests a reduction in both heat loss and metabolic rate during spaceflight. Although the circadian rhythms in all variables were present during flight, some differences were noted. For example, the amplitude of the rhythms in Tsk and activity were attenuated. Furthermore, the T(ax) and activity rhythms did not have precise 24.0 hour periods and may have been externally desynchronized from the 24-h LD cycle. These data suggest a weakening of the coupling between the internal circadian pacemaker and the external LD synchronizer.
Spaceflight Study	Spacelab Life Sciences 1 (SLS-1)	SLID-087	A spaceflight study of synaptic plasticity in adult rat vestibular maculas	9 Days	Animals	Behavioral signs of vestibular perturbation in altered gravity have not been well correlated with structural modifications in neurovestibular centers. This ultrastructural research investigated synaptic plasticity in hair cells of adult rat utricular maculas exposed to microgravity for nine days on a space shuttle. The hypothesis was that synaptic plasticity would be more evident in type II hair cells because they are part of a distributed modifying macular circuitry. All rats were shared with other investigators and were subjected to treatments unrelated to this experiment. Maculas were obtained from flight and control rats after shuttle return (R + 0) and nine days post-flight (R + 9). R + 9 rats had chromodacryorrhea, a sign of acute stress. Tissues were prepared for ultrastructural study by conventional methods. Ribbon synapses were counted in fifty serial sections from medial utricular macular regions of three rats of each flight and control group. Counts in fifty additional consecutive sections from one sample in each group established method reliability. All synapses were photographed and located to specific cells on mosaics of entire sections. Pooled data were analyzed statistically. Flown rats showed abnormal posture and movement at R + 0. They had statistically significant increases in total ribbon synapses and in sphere-like ribbons in both kinds of hair cells; in type II cells, pairs of synapses nearly doubled and clusters of 3 to 6 synapses increased twelve-fold. At R + 9, behavioral signs were normal. However, synapse counts remained high in both kinds of hair cells of flight maculas and were elevated in control type II cells. Only counts in type I cells showed statistically significant differences at R + 9. High synaptic counts at R + 9 may have resulted from stress due to experimental treatments. The results nevertheless demonstrate that adult maculas retain the potential for synaptic plasticity. Type II cells exhibited more synaptic plasticity, but space flight induced synaptic plasticity in type I cells.
Spaceflight Study	COSMOS 2229	SLID-088	Effects of spaceflight on bone mineralization in the rhesus monkey	12 Days	Animals	We combined dual-photon absorptiometry, iliac crest histomorphometry, and backscattered electrons analysis to characterize bone mineralization effects of a spaceflight on young monkeys. Two 4- to 5-kg male rhesus monkeys (Macaca mulatta) were flown during a 11.5-day spaceflight that took place onboard Cosmos 2229 biosatellite (Bion 10). Vivarium (n = 4) and Earth-based chair (n = 4) control situations were studied for comparison. Flight monkeys exhibited lower values of iliac cancellous bone volume, associated with nonsignificantly thinner trabeculae. Bone mineralization rate and the proportion of trabecular bone surface involved in mineralization processes were found markedly reduced after spaceflight. Analysis of embedded sections by backscattered electrons imaging showed a nonsignificant shift to lower mineralization in the flight biopsies vs. postflight mock-up biopsies. These results were in accordance with dual-photon absorptiometry evaluations showing a tendency for decreased bone mineral content during flight and recovery thereafter. The ground simulation experiment performed on the same monkeys more than 1 mo after landing suggests that the observed effects were specifically related to spaceflight and that the animals had only partially recovered. Additional animals on future flights will be required to confirm these findings.
Spaceflight Study	Space Transport System-58	SLID-089	Effects of 14 days of spaceflight and nine days of recovery on cell body size and succinate dehydrogenase activity of rat dorsal root ganglion neurons	14 Days	Animals	The cross-sectional areas and succinate dehydrogenase activities of L5 dorsal root ganglion neurons in rats were determined after 14 days of spaceflight and after nine days of recovery. The mean and distribution of the cross-sectional areas were similar to age-matched, ground-based controls for both the spaceflight and for the spaceflight plus recovery groups. The mean succinate dehydrogenase activity was significantly lower in spaceflight compared to aged-matched control rats, whereas the mean succinate dehydrogenase activity was similar in age-matched control and spaceflight plus recovery rats. The mean succinate dehydrogenase activity of neurons with cross-sectional areas between 1000 and 2000 microns2 was lower (between 7 and 10%) in both the spaceflight and the spaceflight plus recovery groups compared to the appropriate control groups. The reduction in the oxidative capacity of a subpopulation of sensory neurons having relatively large cross-sectional areas immediately following spaceflight and the sustained depression for nine days after returning to 1 g suggest that the 0 g environment induced significant alterations in proprioceptive function.
Spaceflight Study	Bion-11 Biosatellite	SLID-090	Spaceflight affects bone formation in rhesus monkeys: a histological and cell culture study	14 Days	Animals	Using analyses of iliac crest cell and tissue, back-scattered electron imaging, and biochemical techniques, we characterized the effects of a 14-day spaceflight (Bion 11) on bone structure and bone formation in two 3- to 4-yr-old male rhesus monkeys compared with eight age-matched Earth-control monkeys. We found that postflight bone volume was 35% lower than preflight values in flight monkeys. This was associated with reduced osteoid (−40%) and mineralizing (−32%) surfaces and decreased bone formation rate (−53%). Moreover, flight monkeys exhibited trends to lower values of mineralization profile in iliac bone (back-scattered electron imaging) and to decreased osteocalcin serum levels (P = 0.08). The initial number of trabecular bone cells yielded in cultures did not differ in flight and control animals before or after the flight. However, osteoblastic cell proliferation was markedly lower in postflight vs. preflight at 9 and 14 days of culture in one flight monkey. This study suggests that a 14-day spaceflight reduces iliac bone formation, osteoblastic activity, and/or recruitment in young rhesus monkeys, resulting in decreased trabecular bone volume.
Spaceflight Study	STS-118	SLID-091	Effects of spaceflight on innate immune function and antioxidant gene expression	13 Days	Animals	Spaceflight conditions have a significant impact on a number of physiological functions due to psychological stress, radiation, and reduced gravity. To explore the effect of the flight environment on immunity, C57BL/6NTac mice were flown on a 13-day space shuttle mission (STS-118). In response to flight, animals had a reduction in liver, spleen, and thymus masses compared with ground (GRD) controls (P < 0.005). Splenic lymphocyte, monocyte/macrophage, and granulocyte counts were significantly reduced in the flight (FLT) mice (P < 0.05). Although spontaneous blastogenesis of splenocytes in FLT mice was increased, response to lipopolysaccharide (LPS), a B-cell mitogen derived from Escherichia coli, was decreased compared with GRD mice (P < 0.05). Secretion of IL-6 and IL-10, but not TNF-α, by LPS-stimulated splenocytes was increased in FLT mice (P < 0.05). Finally, many of the genes responsible for scavenging reactive oxygen species were upregulated after flight. These data indicate that exposure to the spaceflight environment can increase anti-inflammatory mechanisms and change the ex vivo response to LPS, a bacterial product associated with septic shock and a prominent Th1 response.
Spaceflight Study	STS-128	SLID-092	Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days	14 Days	Animals	Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occured only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.
Spaceflight Study	STS-131	SLID-093	Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice	15 Days	Animals	Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)-131 (n=11), STS-133 (n=6), and STS-135 (n=3) shuttle missions and respective ground-based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2-12 cmH(2)O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor-3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor-mediated intracellular Ca(2+) release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.
Spaceflight Study	STS-133	SLID-093	Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice	13 Days	Animals	Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)-131 (n=11), STS-133 (n=6), and STS-135 (n=3) shuttle missions and respective ground-based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2-12 cmH(2)O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor-3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor-mediated intracellular Ca(2+) release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.
Spaceflight Study	STS-135	SLID-093	Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice	13 Days	Animals	Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)-131 (n=11), STS-133 (n=6), and STS-135 (n=3) shuttle missions and respective ground-based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2-12 cmH(2)O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor-3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor-mediated intracellular Ca(2+) release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.
Spaceflight Study	STS-135	SLID-094	Spaceflight-induced alterations in cerebral artery vasoconstrictor, mechanical, and structural properties: implications for elevated cerebral perfusion and intracranial pressure	13 Days	Animals	Evidence indicates that cerebral blood flow is both increased and diminished in astronauts on return to Earth. Data from ground-based animal models simulating the effects of microgravity have shown that decrements in cerebral perfusion are associated with enhanced vasoconstriction and structural remodeling of cerebral arteries. Based on these results, the purpose of this study was to test the hypothesis that 13 d of spaceflight [Space Transportation System (STS)-135 shuttle mission] enhances myogenic vasoconstriction, increases medial wall thickness, and elicits no change in the mechanical properties of mouse cerebral arteries. Basilar and posterior communicating arteries (PCAs) were isolated from 9-wk-old female C57BL/6 mice for in vitro vascular and mechanical testing. Contrary to that hypothesized, myogenic vasoconstrictor responses were lower and vascular distensibility greater in arteries from spaceflight group (SF) mice (n=7) relative to ground-based control group (GC) mice (n=12). Basilar artery maximal diameter was greater in SF mice (SF: 236±9 μm and GC: 215±5 μm) with no difference in medial wall thickness (SF: 12.4±1.6 μm; GC: 12.2±1.2 μm). Stiffness of the PCA, as characterized via nanoindentation, was lower in SF mice (SF: 3.4±0.3 N/m; GC: 5.4±0.8 N/m). Collectively, spaceflight-induced reductions in myogenic vasoconstriction and stiffness and increases in maximal diameter of cerebral arteries signify that elevations in brain blood flow may occur during spaceflight. Such changes in cerebral vascular control of perfusion could contribute to increases in intracranial pressure and an associated impairment of visual acuity in astronauts during spaceflight.
Spaceflight Study	BION-M1	SLID-095	Risk neurogenes for long-term spaceflight: dopamine and serotonin brain system	30 Days	Animals	Mice were exposed to 1 month of spaceflight on Russian biosatellite BION-M1 to determine its effect on the expression of key genes in the brain dopamine (DA) and serotonin (5-HT) systems. Spaceflight decreased the expression of crucial genes involved in DA synthesis and degradation, as well as the D1 receptor. However, spaceflight failed to alter the expression of tryptophan hydroxylase-2, 5-HT transporter, 5-HT1A, and 5-HT3 receptor genes, though it reduced 5-HT2A receptor gene expression in the hypothalamus. We revealed risk DA and 5-HT neurogenes for long-term spaceflight for the first time, as well as microgravity-responsive genes (tyrosine hydroxylase, catechol-O-methyltransferase, and D1 receptor in the nigrostriatal system; D1 and 5-HT2A receptors in the hypothalamus; and monoamine oxidase A (MAO A) in the frontal cortex). Decreased genetic control of the DA system may contribute to the spaceflight-induced locomotor impairment and dyskinesia described for both humans and rats.
Spaceflight Study	BION-M1	SLID-096	Spaceflight on the Bion-M1 biosatellite alters cerebral artery vasomotor and mechanical properties in mice	30 Days	Animals	Conditions during spaceflight, such as the loss of the head-to-foot gravity vector, are thought to potentially alter cerebral blood flow and vascular resistance. The purpose of the present study was to determine the effects of long-term spaceflight on the functional, mechanical, and structural properties of cerebral arteries. Male C57BL/6N mice were flown 30 days in a Bion-M1 biosatellite. Basilar arteries isolated from spaceflight (SF) (n = 6), habitat control (HC) (n = 6), and vivarium control (VC) (n = 16) mice were used for in vitro functional and mechanical testing and histological structural analysis. The results demonstrate that vasoconstriction elicited through a voltage-gated Ca2+ mechanism (30–80 mM KCl) and thromboxane A2 receptors (10−8 − 3 × 10−5 M U46619) are lower in cerebral arteries from SF mice. Inhibition of Rho-kinase activity (1 μM Y27632) abolished group differences in U46619-evoked contractions. Endothelium-dependent vasodilation elicited by acetylcholine (10 μM, 2 μM U46619 preconstriction) was virtually absent in cerebral arteries from SF mice. The pressure-diameter relation was lower in arteries from SF mice relative to that in HC mice, which was not related to differences in the extracellular matrix protein elastin or collagen content or the elastin/collagen ratio in the basilar arteries. Diameter, medial wall thickness, and medial cross-sectional area of unpressurized basilar arteries were not different among groups. These results suggest that the microgravity-induced attenuation of both vasoconstrictor and vasodilator properties may limit the range of vascular control of cerebral perfusion or impair the distribution of brain blood flow during periods of stress.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-097	Effects of microgravity on DNA damage response in Caenorhabditis elegans during Shenzhou-8 spaceflight	17 Days	Animals	Purpose: Space radiations and microgravity both could cause DNA damage in cells, but the effects of microgravity on DNA damage response to space radiations are still controversial. Materials and methods: A mRNA microarray and microRNA micro- array in dauer larvae of Caenorhabditis elegans (C. elegans) that endured spaceflight environment and space radiations environment during 16.5-day Shenzhou-8 space mission was performed. Results: Twice as many transcripts significantly altered in the spaceflight environment than space radiations alone. The majority of alterations were related to protein amino acid dephosphorylation and histidine metabolic and catabolic processes. From about 900 genes related to DNA damage response, 38 differentially expressed genes were extracted; most of them differentially expressed under spaceflight environment but not space radiations, although the identical directions of alteration were observed in both cases. cel-miR-81, cel- miR-82, cel-miR-124 and cel-miR-795 were predicted to regulate DNA damage response through four different anti-correlated genes. Conclusions: Evidence was provided that, in the presence of space radiations, microgravity probably enhanced the DNA damage response in C. elegans by integrating the transcriptome and microRNome.
Spaceflight Study	STS-131	SLID-098	Spaceflight impairs antigen-specific tolerance induction in vivo and increases inflammatory cytokines	15 Days	Animals	The health risks of a dysregulated immune response during spaceflight are important to understand as plans emerge for humans to embark on long-term space travel to Mars. In this first-of-its-kind study, we used adoptive transfer of T-cell receptor transgenic OT-II CD4 T cells to track an in vivo antigen-specific immune response that was induced during the course of spaceflight. Experimental mice destined for spaceflight and mice that remained on the ground received transferred OT-II cells and cognate peptide stimulation with ovalbumin (OVA) 323-339 plus the inflammatory adjuvant, monophosphoryl lipid A. Control mice in both flight and ground cohorts received monophosphoryl lipid A alone without additional OVA stimulation. Numbers of OT-II cells in flight mice treated with OVA were significantly increased by 2-fold compared with ground mice treated with OVA, suggesting that tolerance induction was impaired by spaceflight. Production of proinflammatory cytokines were significantly increased in flight compared with ground mice, including a 5-fold increase in IFN-γ and a 10-fold increase in IL-17. This study is the first to show that immune tolerance may be impaired in spaceflight, leading to excessive inflammatory responses.
Spaceflight Study	STS-108;UF-1	SLID-099	Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice	11.8 Days	Animals	Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.
Spaceflight Study	STS-108;UF-1	SLID-099	Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice	11.8 Days	Animals	Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.
Spaceflight Study	BION-M1	SLID-100	BION-M 1: First continuous blood pressure monitoring in mice during a 30-day spaceflight	30 Days	Animals	Animals are an essential component of space exploration and have been used to demonstrate that weightlessness does not disrupt essential physiological functions. They can also contribute to space research as models of weightlessness-induced changes in humans. Animal research was an integral component of the 30-day automated Russian biosatellite Bion-M 1 space mission. The aim of the hemodynamic experiment was to estimate cardiovascular function in mice, a species roughly 3000 times smaller than humans, during prolonged spaceflight and post-flight recovery, particularly, to investigate if mice display signs of cardiovascular deconditioning. For the first time, heart rate (HR) and blood pressure (BP) were continuously monitored using implantable telemetry during spaceflight and recovery. Decreased HR and unchanged BP were observed during launch, whereas both HR and BP dropped dramatically during descent. During spaceflight, BP did not change from pre-flight values. However, HR increased, particularly during periods of activity. HR remained elevated after spaceflight and was accompanied by increased levels of exercise-induced tachycardia. Loss of three of the five mice during the flight as a result of the hardware malfunction (unrelated to the telemetry system) and thus the limited sample number constitute the major limitation of the study. For the first time BP and HR were continuously monitored in mice during the 30-day spaceflight and 7-days of post-flight recovery. Cardiovascular deconditioning in these tiny quadruped mammals was reminiscent of that in humans. Therefore, the loss of hydrostatic pressure in space, which is thought to be the initiating event for human cardiovascular adaptation in microgravity, might be of less importance than other physiological mechanisms. Further experiments with larger number of mice are needed to confirm these findings.
Ground Study	Chronic Unpredictable Mild Stressors (CUMS)	SLID-101	Socioenvironmental stressors encountered during spaceflight partially affect the murine TCR-β repertoire and increase its self-reactivity	21 Days	Animals	Spaceflights are known to affect the immune system. In a previous study, we demonstrated that hypergravity exposure during murine development modified 85% of the T-cell receptor (TCR)-β repertoire. In this study, we investigated whether socioenvironmental stressors encountered during space missions affect T lymphopoiesis and the TCR-β repertoire. To address this question, pregnant mice were subjected throughout gestation to chronic unpredictable mild stressors (CUMS), a model used to mimic socioenvironmental stresses encountered during space missions. Then, newborn T lymphopoiesis and the TCR-β repertoire were studied by flow cytometry and high-throughput sequencing, respectively. No change in thymocyte maturation or TCR expression were noted. TCR-β repertoire analysis revealed that 75% of neonate TCR-β sequences resulted from the expression of 3 variable (V)β segments and that this core repertoire was not affected by CUMS. However, the minor repertoire, representing 25% of the global repertoire, was sensitive to CUMS exposure. We also showed that the variable (diversity) joining [V(D)J] recombination process was unlikely to be affected. Finally, we noted that the CUMS neonatal minor repertoire was more self-reactive than the one of control pups. These findings show that socioenvironmental stressors such as those encountered during space missions affect a fraction (25%) of the TCR-β repertoire and that these stressors could increase self-reactivity.-Fonte, C., Kaminski, S., Vanet, A., Lanfumey, L., Cohen-Salmon, C., Ghislin, S., Frippiat, J.-P. Socioenvironmental stressors encountered during spaceflight partially affect the murine TCR-β repertoire and increase its self-reactivity.
Spaceflight Study	BION-M1	SLID-102	Articular cartilage and sternal fibrocartilage respond differently to extended microgravity	30 Days	Animals	The effects of spaceflight on cartilaginous structure are largely unknown. To address this deficiency, articular cartilage (AC) and sternal cartilage (SC) from mice exposed to 30 days of microgravity on the BION-M1 craft were investigated for pathological changes. The flight AC showed some evidence of degradation at the tissue level with loss of proteoglycan staining and a reduction in mRNA expression of mechano-responsive and structural cartilage matrix proteins compared to non-flight controls. These data suggest that degradative changes are underway in the AC extracellular matrix exposed to microgravity. In contrast, there was no evidence of cartilage breakdown in SC flight samples and the gene expression profile was distinct from that of AC with a reduction in metalloproteinase gene transcription. Since the two cartilages respond differently to microgravity we propose that each is tuned to the biomechanical environments in which they are normally maintained. That is, the differences between magnitude of normal terrestrial loading and the unloading of microgravity dictates the tissue response. Weight-bearing articular cartilage, but not minimally loaded sternal fibrocartilage, is negatively affected by the unloading of microgravity. We speculate that the maintenance of physiological loading on AC during spaceflight will minimize AC damage.
Ground Study	n.a.	SLID-103	Hypergravity and microgravity exhibited reversal effects on the bone and muscle mass in mice	14 Days	Animals	Spaceflight is known to induce severe systemic bone loss and muscle atrophy of astronauts due to the circumstances of microgravity. We examined the influence of artificially produced 2G hypergravity on mice for bone and muscle mass with newly developed centrifuge device. We also analyzed the effects of microgravity (mostly 0G) and artificial produced 1G in ISS (international space station) on mouse bone mass. Experiment on the ground, the bone mass of humerus, femur and tibia was measured using micro-computed tomography (μCT), and the all bone mass was significantly increased in 2G compared with 1G control. In tibial bone, the mRNA expression of bone formation related genes such as Osx and Bmp2 was elevated. The volume of triceps surae muscle was also increased in 2G compared with 1G control, and the mRNA expression of myogenic factors such as Myod and Myh1 was elevated by 2G. On the other hand, microgravity in ISS significantly induced the loss of bone mass on humerus and tibia, compared with artificial 1G induced by centrifugation. Here, we firstly report that bone and muscle mass are regulated by the gravity with loaded force in both of positive and negative on the ground and in the space.
Spaceflight Study	SpaceX-12	SLID-103	Hypergravity and microgravity exhibited reversal effects on the bone and muscle mass in mice	34 Days	Animals	Spaceflight is known to induce severe systemic bone loss and muscle atrophy of astronauts due to the circumstances of microgravity. We examined the influence of artificially produced 2G hypergravity on mice for bone and muscle mass with newly developed centrifuge device. We also analyzed the effects of microgravity (mostly 0G) and artificial produced 1G in ISS (international space station) on mouse bone mass. Experiment on the ground, the bone mass of humerus, femur and tibia was measured using micro-computed tomography (μCT), and the all bone mass was significantly increased in 2G compared with 1G control. In tibial bone, the mRNA expression of bone formation related genes such as Osx and Bmp2 was elevated. The volume of triceps surae muscle was also increased in 2G compared with 1G control, and the mRNA expression of myogenic factors such as Myod and Myh1 was elevated by 2G. On the other hand, microgravity in ISS significantly induced the loss of bone mass on humerus and tibia, compared with artificial 1G induced by centrifugation. Here, we firstly report that bone and muscle mass are regulated by the gravity with loaded force in both of positive and negative on the ground and in the space.
Ground Study	Hindlimb Immobilization	SLID-104	Skeletal muscle in MuRF1 null mice is not spared in low-gravity conditions, indicating atrophy proceeds by unique mechanisms in space	21 Days	Animals	Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth’s mesosphere.
Spaceflight Study	International Space Station (ISS)	SLID-104	Skeletal muscle in MuRF1 null mice is not spared in low-gravity conditions, indicating atrophy proceeds by unique mechanisms in space	21 Days	Animals	Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth’s mesosphere.
Spaceflight Study	SpaceX Mission CRS-10	SLID-105	The effects of spaceflight and fracture healing on distant skeletal sites	28 Days	Animals	Spaceflight results in reduced mechanical loading of the skeleton, which leads to dramatic bone loss. Low bone mass is associated with increased fracture risk, and this combination may compromise future, long-term, spaceflight missions. Here, we examined the systemic effects of spaceflight and fracture surgery/healing on several non-injured bones within the axial and appendicular skeleton. Forty C57BL/6, male mice were randomized into the following groups: (1) Sham surgery mice housed on the earth (Ground + Sham); (2) Femoral segmental bone defect surgery mice housed on the earth (Ground + Surgery); (3) Sham surgery mice housed in spaceflight (Flight + Sham); and (4) Femoral segmental bone defect surgery mice housed in spaceflight (Flight + Surgery). Mice were 9 weeks old at the time of launch and were euthanized approximately 4 weeks after launch. Micro-computed tomography (μCT) was used to evaluate standard bone parameters in the tibia, humerus, sternebra, vertebrae, ribs, calvarium, mandible, and incisor. One intriguing finding was that both spaceflight and surgery resulted in virtually identical losses in tibial trabecular bone volume fraction, BV/TV (24–28% reduction). Another important finding was that surgery markedly changed tibial cortical bone geometry. Understanding how spaceflight, surgery, and their combination impact non-injured bones will improve treatment strategies for astronauts and terrestrial humans alike.
Spaceflight Study	SpaceX Mission CRS-10	SLID-106	Skeletal adaptations in young male mice after 4 weeks aboard the International Space Station	28 Days	Animals	Gravity has an important role in both the development and maintenance of bone mass. This is most evident in the rapid and intense bone loss observed in both humans and animals exposed to extended periods of microgravity in spaceflight. Here, cohabitating 9-week-old male C57BL/6 mice resided in spaceflight for ~4 weeks. A skeletal survey of these mice was compared to both habitat matched ground controls to determine the effects of microgravity and baseline samples in order to determine the effects of skeletal maturation on the resulting phenotype. We hypothesized that weight-bearing bones would experience an accelerated loss of bone mass compared to non-weight-bearing bones, and that spaceflight would also inhibit skeletal maturation in male mice. As expected, spaceflight had major negative effects on trabecular bone mass of the following weight-bearing bones: femur, tibia, and vertebrae. Interestingly, as opposed to the bone loss traditionally characterized for most weight-bearing skeletal compartments, the effects of spaceflight on the ribs and sternum resembled a failure to accumulate bone mass. Our study further adds to the insight that gravity has site-specific influences on the skeleton.
Ground Study	n.a.	SLID-107	Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee	14 Days	Animals	Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.
Spaceflight Study	CRS-19	SLID-108	Dynamics of entomopathogenic nematode foraging and infectivity in microgravity	n.a.	Animals	Microgravity is a unique environment to elucidate host-parasite biology. Entomopathogenic nematodes (EPNs), model parasites, kill host insects with mutualistic bacteria and provide environmentally friendly pest control. It is unknown how microgravity affects a multistep insect invasion by parasites with mutualistic bacteria. EPNs respond directionally to electromagnetic cues and their sinusoidal locomotion is affected by various physical factors. Therefore, we expected microgravity to impact EPN functionality. Microgravity experiments during space flight on the International Space Station (ISS) indicated that EPNs successfully emerged from consumed insect host cadavers, moved through soil, found and infected bait insects in a manner equivalent to Earth controls. However, nematodes that developed entirely in space, from the egg stage, died upon return to Earth, unlike controls in microgravity and on Earth. This agricultural biocontrol experiment in space gives insight to long-term space flight for symbiotic organisms, parasite biology, and the potential for sustainable crop protection in space.
Spaceflight Study	SpaceX-12	SLID-109	Spaceflight induces oxidative damage to blood-brain barrier integrity in a mouse model	35 Days	Animals	Many factors contribute to the health risks encountered by astronauts on missions outside Earth's atmosphere. Spaceflight-induced potential adverse neurovascular damage and late neurodegeneration are a chief concern. The goal of the present study was to characterize the effects of spaceflight on oxidative damage in the mouse brain and its impact on blood-brain barrier (BBB) integrity. Ten-week-old male C57BL/6 mice were launched to the International Space Station (ISS) for 35 days as part of Space-X 12 mission. Ground control (GC) mice were maintained on Earth in flight hardware cages. Within 38 ± 4 hours after returning from the ISS, mice were euthanized and brain tissues were collected for analysis. Quantitative assessment of brain tissue demonstrated that spaceflight caused an up to 2.2-fold increase in apoptosis in the hippocampus compared to the control group. Immunohistochemical analysis of the mouse brain revealed an increased expression of aquaporin4 (AQP4) in the flight hippocampus compared to the controls. There was also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BBB-related tight junction protein, Zonula occludens-1 (ZO-1). These results indicate a disturbance of BBB integrity. Quantitative proteomic analysis showed significant alterations in pathways responsible for neurovascular integrity, mitochondrial function, neuronal structure, protein/organelle transport, and metabolism in the brain after spaceflight. Changes in pathways associated with adhesion and molecular remodeling were also documented. These data indicate that long-term spaceflight may have pathological and functional consequences associated with neurovascular damage and late neurodegeneration.
Spaceflight Study	BION-M1	SLID-110	Bioinformatic Analysis of the Sciatic Nerve Transcriptomes of Mice after 30-Day Spaceflight on Board the Bion-M1 Biosatellite	30 Days	Animals	Comparative bioinformatic analysis of sciatic nerve transcriptomes of C57BL/6J mice was carried out. Animals were divided into three groups: Flight, 30-day spaceflight; Recovery, 30-day spaceflight with subsequent 7-day readaptation; and Control. A significant pool of genes with an absolute difference in expression of more than 32 times compared to the control group was revealed in mice after the 30-day spaceflight (Flight and Recovery groups). Comparative analysis of the Flight and Recovery groups of murine transcriptomes did not reveal any significant differences in gene expression. In animals after spaceflight on board the biosatellite, using the KEGG database (Kyoto Encyclopedia of Genes and Genomes), we identified genes related to the state of metabolic and signaling pathways involved in actin cytoskeleton regulation, regulation of potential-dependent calcium, sodium, and potassium channels, and myelination of nerve fibers.
Spaceflight Study	BION-M1	SLID-111	Vestibular cerebellum of thick-toed geckos (Chondrodactylus turnery GRAY, 1864) and C57/BL6N mice after the long-term space flight on the biosatellite BION-M1	30 Days	Animals	The aim of this study was to estimate the effects of long-term space flights on neuronal and glial cells of the vestibular cerebellum of C57/BL6N mice and thick-toed geckos (Chondrodactylus turnery GRAY, 1864). The cerebella from 26 mice and 13 geckos were used in this study. Ten mice and five geckos were flown aboard the BION-M1 biosatellite. The other animals were used as controls. We used immunohistochemical techniques and classical histological method to reveal cell types in the vestibular cerebellum. Nonspecific pathomorphological changes in the Purkinje cells (such as chromatolysis, vacuolization and hyperchromatosis) were observed in the flight groups. However, these changes are reversible and were also found in some neurons in the control groups. In addition, as the vestibular cerebellum is an evolutionarily stable structure, thick-toed geckos may be a useful model for space flight studies on the vertebrate cerebellum.
Spaceflight Study	COSMOS 2229	SLID-112	Effects of spaceflight on ocular counterrolling and the spatial orientation of the vestibular system	12 Days	Animals	We recorded the horizontal (yaw), vertical (pitch), and torsional (roll) eye movements of two rhesus monkeys with scleral search coils before and after the COSMOS Biosatellite 2229 Flight. The aim was to determine effects of adaptation to microgravity on the vestibulo-ocular reflex (VOR). The animals flew for 11 days. The first postflight tests were 22 h and 55 h after landing, and testing extended for 11 days after reentry. There were four significant effects of spaceflight on functions related to spatial orientation: (1) Compensatory ocular counterrolling (OCR) was reduced by about 70% for static and dynamic head tilts with regard to gravity. The reduction in OCR persisted in the two animals throughout postflight testing. (2) The gain of the torsional component of the angular VOR (roll VOR) was decreased by 15% and 50% in the two animals over the same period. (3) An up-down asymmetry of nystagmus, present in the two monkeys before flight was reduced after exposure to microgravity. (4) The spatial orientation of velocity storage was shifted in the one monkey that could be tested soon after flight. Before flight, the yaw axis eigenvector of optokinetic afternystagmus was close to gravity when the animal was upright or tilted. After flight, the yaw orientation vector was shifted toward the body yaw axis. By 7 days after recovery, it had reverted to a gravitational orientation. We postulate that spaceflight causes changes in the vestibular system which reflect adaptation of spatial orientation from a gravitational to a body frame of reference. These changes are likely to play a role in the postural, locomotor, and gaze instability demonstrated on reentry after spaceflight.
Spaceflight Study	STS-76	SLID-113	Embryonic quail eye development in microgravity	8 Days	Animals	The US-Russian joint quail embryo project was designed to study the effects of microgravity on development of Japanese quail embryos incubated aboard Mir. For this part of the project, eyes from embryonic days 14 and 16 (E14 and E16) flight embryos were compared with eyes from several groups of ground-based control embryos. Measurements were recorded for eye weights; eye, corneal, and scleral ring diameters; and numbers of bones in scleral ossicle rings. Transparency of E16 corneas was documented, and immunohistochemical staining was performed to observe corneal innervation. In addition, corneal ultrastructure was observed at the electron microscopic level. Except for corneal diameter of E16 flight embryos, compared with that of one of the sets of controls, results reported here indicate that eye development occurred normally in microgravity. Fixation by cracking the shell and placing the egg in paraformaldehyde solution did not adequately preserve corneal nerves or cellular ultrastructure.
Spaceflight Study	STS-90	SLID-114	Weightlessness during spaceflight results in enhanced synapse formation in a fish brain vestibular nucleus	16 Days	Animals	Synapse counts were undertaken by conventional electron microscopy in primary vestibular integration centers, (i.e. nucleus descendens and nucleus magnocellularis of the brainstem area octavolateralis) and in the diencephalic visual nucleus corticalis of spaceflown neonate swordtail fish Xiphophorus helleri as well as in 1 g control siblings. Spaceflight (16 days microgravity, (μg), STS-90 Neurolab Mission) yielded an increase in synaptic contacts within the vestibular nucleus descendens indicating that lack of input resulted in compensation processes. No effect of μg, however, was observed in the visual nucleus corticalis and in the vestibular nucleus magnocellularis which is situated in the close vicinity of the nucleus descendens. In contrast to the latter, the nucleus magnocellularis does not receive exclusively vestibular input, but inputs from the lateral line as well, possibly providing sufficient input at microgravity.
Spaceflight Study	n.a.	SLID-115	Mechanism of vestibular adaptation of fish under microgravity	12 Days	Animals	In a space experiment, the adaptation of goldfish behavior during flight and readaptation after landing were investigated. Six goldfish (1 normal, 1 with otoliths removed on both sides, 4 with otoliths removed on one side) were flown in a fish package (F/P) of Aquatic Animal Experiment Unit (AAEU). The dorsal light responses (DLRs) of fish with otoliths removed were recorded after operation until launch and after landing. The behaviors of the fish were recorded with a video camera on Mission Elapsed Time (MET) Day-00, 02, 05, 08, 12. On MET Day-00, two fish with otoliths removed on one side showed flexion of body toward the operated side. These fish also showed rolling behavior toward the operated side. However, the body flexion disappeared on MET Day-05 or MET Day-08. No rolling behaviors were observed after that time. Five fish showed backward looping behaviors during the mission. Although the frequency of looping episodes decreased after MET Day-08, five fish still showed looping behavior on MET Day-12, that was the last day of video recording on orbit. In microgravity, visual system of fish did not seem to provide sufficient cues to prevent them from looping or rolling. After landing, no looping and rolling behavior was observed. However, the tilt angle of the DLR increased in the fish with otolith removed 5 month before launch but not in normal fish and those with otoliths removed 2 weeks before launch. These results suggest that the behavioral dysfunction and the adaptational process in space are dependent on vestibular inputs.
Spaceflight Study	STS-76	SLID-116	Calcium utilization by quail embryos during activities preceding space flight and during embryogenesis in microgravity aboard the orbital space station MIR	8 Days	Animals	A series of studies were conducted to determine the effect of activities preceding spaceflight and during space-flight on calcium utilization during quail embryonic development. In the pre-space trials, fertile quail eggs were subjected to pre-flight dynamics including forces of centrifugation, vibration, or a combination of vibration and centrifugation prior to incubation for 6 or 16 days. Quail eggs were also tested for survivability in a refrigerator stowage kit for eggs (RSKE) which was subsequently used to transport the eggs to space. Eggs in the RSKE were subjected to shuttle launch dynamics including G force and random vibration profiles. The space-flight trial involved 48 quail eggs launched on space shuttle Flight STS-76 which were subsequently incubated in a Slovakian incubator onboard space station, MIR. Two ground control trials, each with 48 eggs with and without exposure to shuttle launch dynamics were initiated 5 days post-launch. Eggshells from all study trials were retrieved and analyzed for calcium content. Results showed that neither pre-flight activities nor shuttle launch dynamics had an effect on calcium utilization by developing embryos. However, calcium utilization by developing embryos incubated in microgravity was impaired by 12.6% when compared to embryos incubated on earth under laboratory control environment. This impairment was believed to be due to unidentified factors of the microgravity environment.
Spaceflight Study	Bion-11 Biosatellite	SLID-117	The calcium endocrine system of adolescent rhesus monkeys and controls before and after spaceflight	14 Days	Animals	The calcium endocrine system of nonhuman primates can be influenced by chairing for safety and the weightless environment of spaceflight. The serum of two rhesus monkeys flown on the Bion 11 mission was assayed pre- and postflight for vitamin D metabolites, parathyroid hormone, calcitonin, parameters of calcium homeostasis, cortisol, and indexes of renal function. Results were compared with the same measures from five monkeys before and after chairing for a flight simulation study. Concentrations of 1,25-dihydroxyvitamin D were 72% lower after the flight than before, and more than after chairing on the ground (57%, P < 0.05). Decreases in parathyroid hormone did not reach significance. Calcitonin showed modest decreases postflight (P < 0.02). Overall, effects of spaceflight on the calcium endocrine system were similar to the effects of chairing on the ground, but were more pronounced. Reduced intestinal calcium absorption, losses in body weight, increases in cortisol, and higher postflight blood urea nitrogen were the changes in flight monkeys that distinguished them from the flight simulation study animals.
Spaceflight Study	Bion-11 Biosatellite	SLID-118	Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration	14 Days	Animals	Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.
Spaceflight Study	Andromède mission	SLID-119	Morphometric investigations of sensory vestibular structures in tadpoles (Xenopus laevis) after a spaceflight: implications for microgravity-induced alterations of the vestibuloocular reflex	9.5 Days	Animals	In lower vertebrates, gravity deprivation by orbital flights modifies the vestibuloocular reflex. Using the amphibian Xenopus laevis, the experiments should clarify to which extent macular structures of the labyrinth are responsible for these modifications. In particular, the shape of otoconia and number and size of sensory macular cells expressing CalBindin were considered. CalBindin is common in mature sensory cells including vestibular hair cells and is probably involved in otoconia formation. Two developmental stages were used for this study: stage 26/27 embryos, which were unable to perform the roll-induced vestibuloocular reflex (rVOR) at onset of microgravity, and stage 45 tadpoles, which had already developed the reflex. The main observations were that the developmental progress of the animals was not affected by microgravity; that in the young tadpole group with normal body shape the rVOR was not modified by microgravity, while in the older group with microgravity experience, the rVOR was augmented; and that significant effects on the shape of otoconia and on the number and size of CalBindin-expressing cells of the labyrinthine maculae cells were absent. In addition, behavioural data were never significantly correlated with morphological features of macular structures such as size and number of CalBindin-expressing cells. It is postulated that mechanisms of vestibular adaptation to microgravity during early development are probably based on mechanisms located in central structures of the vestibular system.
Spaceflight Study	Mir Space Station	SLID-120	Spaceflight-associated changes in immunoglobulin VH gene expression in the amphibian Pleurodeles waltl	148 Days	Animals	Understanding why the immune system is depressed during spaceflight is of obvious importance for future human deep-space missions, such as the foreseen missions to Mars. However, little is known about the effects of these flights on humoral immunity. We previously immunized adult Pleurodeles waltl (urodele amphibian) onboard the Mir space station and showed that heavy-chain variable (VH) domains of specific IgM antibodies are encoded by genes belonging to the VHII and VHVI families. We have now determined how these animals use their individual VHII and VHVI genes by screening IgM heavy-chain cDNA libraries and by quantifying IgM heavy-chain transcripts encoded by these genes. Results were compared with those obtained using control animals immunized on Earth under the same conditions as onboard Mir. Our experiments revealed an increase in the expression of IgM heavy-chain mRNAs encoded by the VHII and VHVI.C genes and a strong decrease in the expression of IgM heavy-chain mRNAs encoded by the VHVI.A and VHVI.B genes in spaceflight animals. Consequently, different heavy-chain mRNAs are expressed by spaceflight animals, demonstrating that this environment affects the humoral response. These observations may be due to a change in B-cell selection under spaceflight conditions.
Spaceflight Study	Mir Space Station	SLID-121	Decrease in antibody somatic hypermutation frequency under extreme, extended spaceflight conditions	148 Days	Animals	Somatic hypermutation（SHM） diversifies antibody binding sites by introducing point mutations in the variable domains of rearranged immunoglobulin genes. In this study, we analyzed somatic hypermutation in variable heavy-chain (VH) domains of specific IgM antibodies of the urodele amphibian Pleurodeles waltl, immunized either on Earth or onboard the Mir space station. To detect somatic hypermutation, we aligned the variable domains of IgM heavy-chain transcripts with the corresponding VH gene. We also quantified NF-κB and activation-induced cytidine deaminase transcripts. Results were compared with those obtained using control animals immunized on Earth. Our data show that, as in most species of ectotherms, somatic hypermutation in P. waltl exhibits a mutational bias toward G and C bases. Furthermore, we show for the first time that somatic hypermutation occurs in space following immunization but at a lower frequency. This decrease is not due to a decrease in food intake or of the B-cell receptor/antigen interaction or to the absence of the germinal center-associated nuclear protein. It likely results from the combination of several spaceflight-associated changes, such as the severe reduction in T-cell activation, important perturbations of the cytoskeleton, and changes in the distribution of lymphocyte subpopulations and adhesion molecule expression.
Spaceflight Study	International Space Station (ISS)	SLID-122	Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis	10 Days	Animals	Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.
Ground Study	n.a.	SLID-122	Gravity changes during animal development affect IgM heavy-chain transcription and probably lymphopoiesis	10 Days	Animals	Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.
Spaceflight Study	FOTON-M3 Satellite	SLID-123	Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight	12 Days	Animals	Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.
Spaceflight Study	TMA-06M	SLID-124	Microgravity promotes osteoclast activity in medaka fish reared at the international space station	27 Days	Animals	The bone mineral density (BMD) of astronauts decreases specifically in the weight-bearing sites during spaceflight. It seems that osteoclasts would be affected by a change in gravity; however, the molecular mechanism involved remains unclear. Here, we show that the mineral density of the pharyngeal bone and teeth region of TRAP-GFP/Osterix-DsRed double transgenic medaka fish was decreased and that osteoclasts were activated when the fish were reared for 56 days at the international space station. In addition, electron microscopy observation revealed a low degree of roundness of mitochondria in osteoclasts. In the whole transcriptome analysis, fkbp5 and ddit4 genes were strongly up-regulated in the flight group. The fish were filmed for abnormal behavior; and, interestingly, the medaka tended to become motionless in the late stage of exposure. These results reveal impaired physiological function with a change in mechanical force under microgravity, which impairment was accompanied by osteoclast activation.
Spaceflight Study	International Space Station (ISS)	SLID-125	Evaluation of rodent spaceflight in the NASA animal enclosure module for an extended operational period (up to 35 days)	35 Days	Animals	The National Aeronautics and Space Administration Animal Enclosure Module (AEM) was developed as a self-contained rodent habitat for shuttle flight missions that provides inhabitants with living space, food, water, ventilation, and lighting, and this study reports whether, after minimal hardware modification, the AEM could support an extended term up to 35 days for Sprague-Dawley rats and C57BL/6 female mice for use on the International Space Station. Success was evaluated based on comparison of AEM housed animals to that of vivarium housed and to normal biological ranges through various measures of animal health and well-being, including animal health evaluations, animal growth and body masses, organ masses, rodent food bar consumption, water consumption, and analysis of blood contents. The results of this study confirmed that the AEMs could support 12 adult female C57BL/6 mice for up to 35 days with self-contained RFB and water, and the AEMs could also support 5 adult male Sprague-Dawley rats for 35 days with external replenishment of diet and water. This study has demonstrated the capability and flexibility of the AEM to operate for up to 35 days with minor hardware modification. Therefore, with modifications, it is possible to utilize this hardware on the International Space Station or other operational platforms to extend the space life science research use of mice and rats.
Spaceflight Study	n.a.	SLID-126	Behavior of Stem-Like Cells, Precursors for Tissue Regeneration in Urodela, Under Conditions of Microgravity	n.a.	Animals	We summarize data from our experiments on stem-like cell-dependent regeneration in amphibians in microgravity. Considering its deleterious effect on many tissues, we asked whether microgravity is compatible with reparative processes, specifically activation and proliferation of source cells. Experiments were conducted using tailed amphibians, which combine profound regenerative capabilities with high robustness, allowing an in vivo study of lens, retina, limb, and tail regeneration in challenging settings of spaceflight. Microgravity promoted stem-like cell proliferation to a varying extent (up to 2-fold), and it seemed to speed up source cell dedifferentiation, as well as sequential differentiation in retina, lens, and limb, leading to formation of bigger and more developed regenerates than in 1g controls. It also promoted proliferation and hypertrophy of Müller glial cells, eliciting a response similar to reactive gliosis. A significant increase in stem-like cell proliferation was mostly beneficial for regeneration and only in rare cases caused moderate tissue growth abnormalities. It is important that microgravity yielded a lasting effect even if applied before operations. We hypothesize on the potential mechanisms of gravity-dependent changes in stem-like cell behavior, including fibroblast growth factor 2 signaling pathway and heat shock proteins, which were affected in our experimental settings. Taken together, our data indicate that microgravity does not disturb the natural regenerative potential of newt stem-like cells, and, depending on the system, even stimulates their dedifferentiation, proliferation, and differentiation. We discuss these data along with publications on mammalian stem cell behavior in vitro and invertebrate regeneration in vivo in microgravity. In vivo data are very scarce and require further research using contemporary methods of cell behavior analysis to elucidate mechanisms of stem cell response to altered gravity. They are relevant for both practical applications, such as managing human reparative responses in spaceflight, and fundamental understanding of stem cell biology.
Spaceflight Study	COSMOS 2044	SLID-127	Radiation and microgravity effects observed in the insect system Carausius morosus	14 Days	Animals	Among the biological problems that arise in long duration spaceflights, weightlessness and ionizing radiation appear to be the main risk factors. A precise differentiation between the effects of either energy deposition by heavy ions or microgravity alone and their combined action has succeeded for the first time with the experiment on Carausius morosus embryos flown in BIORACK during the D1 mission. It was clearly demonstrated that microgravity reduces the hatching rate and amplifies the effect of heavy ions with respect to the frequency of body anomalies. In the meantime, Carausius morosus eggs were exposed during two further spaceflights, Cosmos 1887 and 2044. The studies in these experiments were done with emphasis on the morphological differentiation during embryogenesis. The first results of the Cosmos 2044 flight of effects on hatching rate, growth kinetics, vitality and frequency of anomalies are presented and compared with those of the previous flights. These data indicate that in radiation protection an additional problem will be posed by a potential modification of radiobiological effects by microgravity.
Spaceflight Study	COSMOS 1129	SLID-128	Embryonic Development and Behaviour of Japanese Quail Exposed to Microgravity	12 Days	Animals	The effect of microgravity on the embryonic development of Japanese quail including hatching, the behaviour of newly hatched chickens and physiological functions of adult birds were studied. The feasibility of the entire embryonic development as well as hatching of quail in weightlessness was demonstrated. Priority data on sensory, motor and feeding behaviour of 1- to 4-day-old quail hatchlings as well as the behaviour of adult birds in microgravity were obtained. Under special conditions the quail were able to take food, but were generally unable to adapt their motor activity to microgravity at any time during the experiment. Their movements were uncoordinated and chaotic, particularly so in chicks. After a 7-day exposure to microgravity hormone levels (estradiol, triiodthyronine, corticosterone) and Ca in mature quail were determined. Changes in reproductive functions were manifested by the arrest of egg production, dystrophy of ovaries and testes and a decreased testosterone level. The first data on the effects of microgravity on the enteric microflora were also obtained.
Spaceflight Study	Mir Space Station	SLID-128	Embryonic Development and Behaviour of Japanese Quail Exposed to Microgravity	8 Days	Animals	The effect of microgravity on the embryonic development of Japanese quail including hatching, the behaviour of newly hatched chickens and physiological functions of adult birds were studied. The feasibility of the entire embryonic development as well as hatching of quail in weightlessness was demonstrated. Priority data on sensory, motor and feeding behaviour of 1- to 4-day-old quail hatchlings as well as the behaviour of adult birds in microgravity were obtained. Under special conditions the quail were able to take food, but were generally unable to adapt their motor activity to microgravity at any time during the experiment. Their movements were uncoordinated and chaotic, particularly so in chicks. After a 7-day exposure to microgravity hormone levels (estradiol, triiodthyronine, corticosterone) and Ca in mature quail were determined. Changes in reproductive functions were manifested by the arrest of egg production, dystrophy of ovaries and testes and a decreased testosterone level. The first data on the effects of microgravity on the enteric microflora were also obtained.
Spaceflight Study	Mir Space Station (Quant 2)	SLID-128	Embryonic Development and Behaviour of Japanese Quail Exposed to Microgravity	12 Days	Animals	The effect of microgravity on the embryonic development of Japanese quail including hatching, the behaviour of newly hatched chickens and physiological functions of adult birds were studied. The feasibility of the entire embryonic development as well as hatching of quail in weightlessness was demonstrated. Priority data on sensory, motor and feeding behaviour of 1- to 4-day-old quail hatchlings as well as the behaviour of adult birds in microgravity were obtained. Under special conditions the quail were able to take food, but were generally unable to adapt their motor activity to microgravity at any time during the experiment. Their movements were uncoordinated and chaotic, particularly so in chicks. After a 7-day exposure to microgravity hormone levels (estradiol, triiodthyronine, corticosterone) and Ca in mature quail were determined. Changes in reproductive functions were manifested by the arrest of egg production, dystrophy of ovaries and testes and a decreased testosterone level. The first data on the effects of microgravity on the enteric microflora were also obtained.
Spaceflight Study	Mir Space Station	SLID-129	The Quail Embryonic Development Under the Conditions of Weightlessness	21 Days	Animals	The experiment on board the orbital station “MIR” showed that normal development of quail embryos is possible and viable progeny can be obtained although at a considerably smaller hatching rate and a higher frequency of anomalies in the development of eyes, brain and beak. The morphological indices of embryos and hatchlings did not show any noticeable deviations in mass, and body size. An assumption was put forward concerning the possible secondary mechanism for development of these deviations and a decreased hatching rate. Numerous authors have successfully studied different phases of embryogenesis of amphibians, fish, insects, birds and mammals in the course of several spaceflights. The full cycle of embryogenesis in weightlessness has not been studied as yet except for experiments with Drosophylla carried out by G. P. Parfenov focused on genetics of gene rations of flies grown in space (Parfenov 1988). Unfortunately, these experiments have not been recognized in literature as the first embryological experiments using insects and covering the whole embryogenesis beginning with the moment of fertilization in weightlessness. The study of embryonic development of birds during space flight is especially important as this model enables investigation of the biological role of gravity as a general biology problem with its practical aspects, taking into consideration the fact that the quail is a potential component of a life-support system of man in long-time space flights. The first, though not full, realization of this idea took place in 1979 on board the Soviet satellite “Cosmos-1129”. The work was carried out together with specialists from CSFR. A 12-days-long experiment yielded results on quail egg incubation under the conditions of weightlessness and showed that normal development of about 33 % embryos during 2/3 of their embryogenesis is possible, which was determined by the period of existence of the biosputnik. It was possible to work out technology for an experiment embracing the whole embryogenesis and initial phases of post-embryonic development. The experiment was carried out in 1991 on board the orbital station “MIR”.
Spaceflight Study	International Space Station (ISS)	SLID-130	Ophthalmic changes and increased intracranial pressure associated with long duration spaceflight: An emerging understanding	6 Months	Vision and neuroanatomical changes	For many years, there have been anecdotal reports of vision changes by astronauts following short and long-duration spaceflight. Much of this was attributed to hyperopic shifts related to the age of the flying population. However, it has recently been recognized that vision changes are actually quite common in astronauts and are associated with a constellation of findings including elevated intracranial pressure, optic disc edema, globe flattening, optic nerve sheath thickening, hyperopic shifts and retinal changes. With advanced imaging modalities available on the ground along with the fidelity of in-flight diagnostic capabilities previously unavailable, information on this newly recognized syndrome is accumulating. As of this writing, 11 cases of visual impairment experienced by astronauts during missions on-board the International Space Station (ISS) have been documented and studied. Although the exact mechanisms of the vision changes are unknown, it is hypothesized that increased intracranial pressure (ICP) is a contributing factor. Microgravity is the dominant cause of many physiological changes during spaceflight and is thought to contribute significantly to the observed ophthalmic changes. However, several secondary factors that could contribute to increased ICP and vision changes in spaceflight have been proposed. Possible contributors include microgravity-induced cephalad fluid shift, venous obstruction due to microgravity-induced anatomical shifts, high levels of spacecraft cabin carbon dioxide, heavy resistive exercise, and high sodium diet. Individual susceptibility to visual impairment is not fully understood, though a demographic of affected astronauts is emerging. This paper describes the current understanding of this newly recognized syndrome, presents data from 11 individual cases, and discusses details of potential contributing factors. The occurrence of visual changes in long duration missions in microgravity is one of the most significant clinical issues to date for the human spaceflight community, and a comprehensive understanding of the issue at whole is critical to ensure safe space exploration in the future.
Spaceflight Study	International Space Station (ISS)	SLID-131	Circadian misalignment affects sleep and medication use before and during spaceflight	Average 155 ( ± 39) Days	Circadian misalignment is associated with sleep deficiency	Sleep deficiency and the use of sleep-promoting medication are prevalent during spaceflight. Operations frequently dictate work during the biological night and sleep during the biological day, which contribute to circadian misalignment. We investigated whether circadian misalignment was associated with adverse sleep outcomes before (preflight) and during spaceflight missions aboard the International Space Station (ISS). Actigraphy and photometry data for 21 astronauts were collected over 3,248 days of long-duration spaceflight on the ISS and 11 days prior to launch (n=231 days). Sleep logs, collected one out of every 3 weeks in flight and daily on Earth, were used to determine medication use and subjective ratings of sleep quality. Actigraphy and photometry data were processed using Circadian Performance Simulation Software to calculate the estimated endogenous circadian temperature minimum. Sleep episodes were classified as aligned or misaligned relative to the estimated endogenous circadian temperature minimum. Mixed-effects regression models accounting for repeated measures were computed by data collection interval (preflight, flight) and circadian alignment status. The estimated endogenous circadian temperature minimum occurred outside sleep episodes on 13% of sleep episodes during preflight and on 19% of sleep episodes during spaceflight. The mean sleep duration in low-Earth orbit on the ISS was 6.4±1.2 h during aligned and 5.4±1.4 h (P<0.01) during misaligned sleep episodes. During aligned sleep episodes, astronauts rated their sleep quality as significantly better than during misaligned sleep episodes (66.8±17.7 vs. 60.2±21.0, P<0.01). Sleep-promoting medication use was significantly higher during misaligned (24%) compared with aligned (11%) sleep episodes (P<0.01). Use of any medication was significantly higher on days when sleep episodes were misaligned (63%) compared with when sleep episodes were aligned (49%; P<0.01). Circadian misalignment is associated with sleep deficiency and increased medication use during spaceflight. These findings suggest that there is an immediate need to deploy and assess effective countermeasures to minimize circadian misalignment and consequent adverse sleep outcomes both before and during spaceflight.
Spaceflight Study	International Space Station (ISS)	SLID-132	Medication use by U.S. crewmembers on the International Space Station	Average, 159 ± 36 Days	Medication use	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long‐duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight‐related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule‐shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.—Wotring, V. E. Medication use by U.S. crewmembers on the International Space Station. FASEB J. 29, 4417‐4423 (2015). www.fasebj.org
Spaceflight Study	Spacecraft	SLID-133	Characterization of sleep-wake patterns in crew members under a short-duration spaceflight	15 Days	Sleep quality changes	Sleep loss is one of large challenges during space missions. A variety of sleep problems have been shown in space. We aimed to further characterize more representative and consecutive sleep-wake patterns and to investigate sleep-wake cycle on heart rate during short duration spaceflight mission. Here, we evaluated sleep quality using Pittsburgh Sleep Quality Index, analyzed sleep time, sleep efficiency, and periods of sleep-wake rhythm via the activity data from Actiwatch, and evaluated heart rate data via the Actiheart in three crew members before, during and after a 15-day spaceflight. The study showed that the sleep quality before and during the flight were lower than that after the space flight. Crew members slept less during spaceflight relative to on Earth. The mean sleep duration during spaceflight was 6.5 ± 1.3 h, but after flight, it was 7.2 ± 1.2 h, and the sleep efficiency was significantly higher during the flight than that on Earth. The periods of sleep-wake rhythm could keep at 23.92 h during flight mission. Moreover, the heart rate decreased during spaceflight and it was more obvious during wakeful state than during sleep. These data could help us better understand the effects of short-duration spaceflight on sleep pattern.
Spaceflight Study	International Space Station (ISS)	SLID-134	Effects of Spaceflight on Astronaut Brain Structure as Indicated on MRI	164.8 ± 18.9 Days	Narrowing of CSF spaces and the displacement of brain structures	BACKGROUND There is limited information regarding the effects of spaceflight on the anatomical configuration of the brain and on cerebrospinal fluid (CSF) spaces. METHODS We used magnetic resonance imaging (MRI) to compare images of 18 astronauts’ brains before and after missions of long duration, involving stays on the International Space Station, and of 16 astronauts’ brains before and after missions of short duration, involving participation in the Space Shuttle Program. Images were interpreted by readers who were unaware of the flight duration. We also generated paired preflight and postflight MRI cine clips derived from high-resolution, three-dimensional imaging of 12 astronauts after long-duration flights and from 6 astronauts after short-duration flights in order to assess the extent of narrowing of CSF spaces and the displacement of brain structures. We also compared preflight ventricular volumes with postflight ventricular volumes by means of an automated analysis of T1-weighted MRIs. The main prespecified analyses focused on the change in the volume of the central sulcus, the change in the volume of CSF spaces at the vertex, and vertical displacement of the brain. RESULTS Narrowing of the central sulcus occurred in 17 of 18 astronauts after long-duration flights (mean flight time, 164.8 days) and in 3 of 16 astronauts after short-duration flights (mean flight time, 13.6 days) (P<0.001). Cine clips from a subgroup of astronauts showed an upward shift of the brain after all long-duration flights (12 astronauts) but not after short-duration flights (6 astronauts) and narrowing of CSF spaces at the vertex after all long-duration flights (12 astronauts) and in 1 of 6 astronauts after short-duration flights. Three astronauts in the long-duration group had optic-disk edema, and all 3 had narrowing of the central sulcus. A cine clip was available for 1 of these 3 astronauts, and the cine clip showed upward shift of the brain. CONCLUSIONS Narrowing of the central sulcus, upward shift of the brain, and narrowing of CSF spaces at the vertex occurred frequently and predominantly in astronauts after long-duration flights. Further investigation, including repeated postflight imaging conducted after some time on Earth, is required to determine the duration and clinical significance of these changes. (Funded by the National Aeronautics and Space Administration.)
Spaceflight Study	Space Shuttle Program	SLID-134	Effects of Spaceflight on Astronaut Brain Structure as Indicated on MRI	13.6 ± 1.7 Days	Narrowing of CSF spaces and the displacement of brain structures	BACKGROUND There is limited information regarding the effects of spaceflight on the anatomical configuration of the brain and on cerebrospinal fluid (CSF) spaces. METHODS We used magnetic resonance imaging (MRI) to compare images of 18 astronauts’ brains before and after missions of long duration, involving stays on the International Space Station, and of 16 astronauts’ brains before and after missions of short duration, involving participation in the Space Shuttle Program. Images were interpreted by readers who were unaware of the flight duration. We also generated paired preflight and postflight MRI cine clips derived from high-resolution, three-dimensional imaging of 12 astronauts after long-duration flights and from 6 astronauts after short-duration flights in order to assess the extent of narrowing of CSF spaces and the displacement of brain structures. We also compared preflight ventricular volumes with postflight ventricular volumes by means of an automated analysis of T1-weighted MRIs. The main prespecified analyses focused on the change in the volume of the central sulcus, the change in the volume of CSF spaces at the vertex, and vertical displacement of the brain. RESULTS Narrowing of the central sulcus occurred in 17 of 18 astronauts after long-duration flights (mean flight time, 164.8 days) and in 3 of 16 astronauts after short-duration flights (mean flight time, 13.6 days) (P<0.001). Cine clips from a subgroup of astronauts showed an upward shift of the brain after all long-duration flights (12 astronauts) but not after short-duration flights (6 astronauts) and narrowing of CSF spaces at the vertex after all long-duration flights (12 astronauts) and in 1 of 6 astronauts after short-duration flights. Three astronauts in the long-duration group had optic-disk edema, and all 3 had narrowing of the central sulcus. A cine clip was available for 1 of these 3 astronauts, and the cine clip showed upward shift of the brain. CONCLUSIONS Narrowing of the central sulcus, upward shift of the brain, and narrowing of CSF spaces at the vertex occurred frequently and predominantly in astronauts after long-duration flights. Further investigation, including repeated postflight imaging conducted after some time on Earth, is required to determine the duration and clinical significance of these changes. (Funded by the National Aeronautics and Space Administration.)
Spaceflight Study	International Space Station (ISS)	SLID-135	Changes in human skeletal muscle architecture and function induced by extended spaceflight	> 180  Days	Skeletal muscle changes	The aim of this study was to quantitatively describe the relationships between joint angles and muscle architecture (lengths (Lf) and angles (Θf) of fascicles) of human triceps surae [medial (MG) and lateral (LG) gastrocnemius and soleus (SOL) muscles] in vivo for three men-cosmonaut after long-duration spaceflight. Sagittal sonographs of MG, LG, SOL were taken at ankle was positioned at 15° (dorsiflexion), 0° (neutral position), +15°, and +30° (plantarflexion), with the knee at 90° at rest and after a long-duration spaceflight. At each position, longitudinal ultrasonic images of the MG and LG and SOL were obtained while the cosmonauts was relaxed from which fascicle lengths and angles with respect to the aponeuroses were determined. After space flight plantarflexor force declined significantly (26%; p < 0.001). The internal architecture of the GM, and LG, and SOL muscle was significantly altered. In the passive condition, Lf changed from 45, 53, and 39 mm (knee, 0°, ankle, −15°) to 26, 33, and 28 mm (knee, 90° ankle, 30°) for MG, LG, and SOL, respectively. Different lengths and angles of fascicles, and their changes by contraction, might be related to differences in force-producing capabilities of the muscles and elastic characteristics of tendons and aponeuroses. The three heads of the triceps surae muscle substantially differ in architecture, which probably reflects their functional roles. Differences in fiber length and pennation angle that were observed among the muscles and could be associated with differences in force production and in elastic properties of musculo-tendinous complex and aponeuroses.
Spaceflight Study	International Space Station (ISS)	SLID-136	The effect of long-term exposure to microgravity on the perception of upright	Mean duration 168 Days	Perceptual changes	Going into space is a disorienting experience. Many studies have looked at sensory functioning in space but the multisensory basis of orientation has not been systematically investigated. Here, we assess how prolonged exposure to microgravity affects the relative weighting of visual, gravity, and idiotropic cues to perceived orientation. We separated visual, body, and gravity (when present) cues to perceived orientation before, during, and after long-term exposure to microgravity during the missions of seven astronauts on the International Space Station (mean duration 168 days) and measuring perceived vertical using the subjective visual vertical and the perceptual upright. The relative influence of each cue and the variance of their judgments were measured. Fourteen ground-based control participants performed comparable measurements over a similar period. The variance of astronauts’ subjective visual vertical judgments in the absence of visual cues was significantly larger immediately upon return to earth than before flight. Astronauts’ perceptual upright demonstrated a reduced reliance on visual cues upon arrival on orbit that re-appeared long after returning to earth. For earth-bound controls, the contributions of body, gravity, and vision remained constant throughout the year-long testing period. This is the first multisensory study of orientation behavior in space and the first demonstration of long-term perceptual changes that persist after returning to earth. Astronauts showed a plasticity in the weighting of perceptual cues to orientation that could form the basis for future countermeasures.
Spaceflight Study	n.a.	SLID-137	Spaceflight-induced cardiovascular changes and recovery during NASA's Functional Task Test	10 - 15 Days	Cardiovascular changes	Microgravity-induced physiologic changes could impair a crewmember's performance upon return to a gravity environment. The Functional Task Test aims to correlate these physiologic alterations with changes in performance during mission-critical tasks. In this study, we evaluated spaceflight-induced cardiovascular changes during 11 functional tasks in 7 Shuttle astronauts before spaceflight, on landing day, and 1, 6, and 30 days after landing. Mean heart rate was examined during each task and autonomic activity was approximated by heart rate variability during the Recovery from Fall/Stand Test, a 2-min prone rest followed by a 3-min stand. Heart rate was increased on landing day during all of the tasks, and remained elevated 6 days after landing during 6 of the 11 tasks. Parasympathetic modulation was diminished and sympathovagal balance was increased on landing day. Additionally, during the stand test 6 days after landing, parasympathetic modulation remained suppressed and heart rate remained elevated compared to preflight levels. Heart rate and autonomic activity were not different from preflight levels 30 days after landing. We detected changes in heart rate and autonomic activity during a 3-min stand and a variety of functional tasks, where cardiovascular deconditioning was still evident 6 days after returning from short-duration spaceflight. The delayed recovery times for heart rate and parasympathetic modulation indicate the necessity of assessing functional performance after long-duration spaceflight to ensure crew health and safety.
Spaceflight Study	International Space Station (ISS)	SLID-138	Long-term exposure to microgravity impairs vestibulo-cardiovascular reflex	156 ± 9 Days (range, 127 - 188 Days)	Vestibulo-cardiovascular reflex changes	The vestibular system is known to have an important role in controlling blood pressure upon posture transition (vestibulo-cardiovascular reflex, VCR). However, under a different gravitational environment, the sensitivity of the vestibular system may be altered. Thus, the VCR may become less sensitive after spaceflight because of orthostatic intolerance potentially induced by long-term exposure to microgravity. To test this hypothesis in humans, we investigated the ability of the VCR to maintain blood pressure upon head-up tilt before and after a 4–6 months stay on the International Space Station. To detect the functional state of the VCR, galvanic vestibular stimulation (GVS) was applied. As GVS transiently interrupts the vestibular-mediated pressor response, impaired VCR is detected when the head-up tilt-induced blood pressure response does not depend on GVS. During the first 20 s of head-up tilt, a transient blood pressure increase (11.9 ± 1.6 mmHg) was observed at pre-spaceflight but not at 1–4 days after return from spaceflight. The magnitude of VCR recovered to the pre-spaceflight levels within 2 months after return. These results indicate that long-term exposure to microgravity induces VCR impairment, which may be involved in a mechanism of spaceflight-induced orthostatic intolerance.
Spaceflight Study	International Space Station (ISS)	SLID-139	Study of the impact of long-duration space missions at the International Space Station on the astronaut microbiome	n.a.	Microbiome changes	Over the course of a mission to the International Space Station (ISS) crew members are exposed to a number of stressors that can potentially alter the composition of their microbiomes and may have a negative impact on astronauts' health. Here we investigated the impact of long-term space exploration on the microbiome of nine astronauts that spent six to twelve months in the ISS. We present evidence showing that the microbial communities of the gastrointestinal tract, skin, nose and tongue change during the space mission. The composition of the intestinal microbiota became more similar across astronauts in space, mostly due to a drop in the abundance of a few bacterial taxa, some of which were also correlated with changes in the cytokine profile of crewmembers. Alterations in the skin microbiome that might contribute to the high frequency of skin rashes/hypersensitivity episodes experienced by astronauts in space were also observed. The results from this study demonstrate that the composition of the astronauts' microbiome is altered during space travel. The impact of those changes on crew health warrants further investigation before humans embark on long-duration voyages into outer space.
Spaceflight Study	International Space Station (ISS)	SLID-140	Long-duration spaceflight adversely affects post-landing operator proficiency	142 - 200 Days (mean 170.8, SD 20.4)	Post-flight performance decrements	Performance of astronaut pilots during space shuttle landing was degraded after a few weeks of microgravity exposure, and longer-term exposure has the potential to impact operator proficiency during critical landing and post-landing operations for exploration-class missions. Full-motion simulations of operationally-relevant tasks were utilized to assess the impact of long-duration spaceflight on operator proficiency in a group of 8 astronauts assigned to the International Space Station, as well as a battery of cognitive/sensorimotor tests to determine the underlying cause of any post-flight performance decrements. A ground control group (N = 12) and a sleep restriction cohort (N = 9) were also tested to control for non-spaceflight factors such as lack of practice between pre- and post-flight testing and fatigue. On the day of return after 6 months aboard the space station, astronauts exhibited significant deficits in manual dexterity, dual-tasking and motion perception, and a striking degradation in the ability to operate a vehicle. These deficits were not primarily due to fatigue; performance on the same tasks was unaffected after a 30-h period of sleep restriction. Astronauts experienced a general post-flight malaise in motor function and motion perception, and a lack of cognitive reserve apparent only when faced with dual tasks, which had recovered to baseline by four days after landing.
Spaceflight Study	International Space Station (ISS)	SLID-141	Inflight leg cuff test does not identify the risk for orthostatic hypotension after long-duration spaceflight	167 ± 12 Days	n.a.	Landing day symptoms from orthostatic hypotension after prolonged spaceflight can be debilitating, but severity of these symptoms can be unpredictable and highly individual. We tested the hypothesis that an impaired baroreflex response to an inflight leg cuff test could predict orthostatic intolerance on return to Earth. Eight male astronauts (44 ± 7 years of age (mean ± SD); mean mission length: 167 ± 12 days) participated in a standardized supine-to-sit-to-stand test (5 min–30s–3 min) pre- and postflight, and a 3 min thigh cuff occlusion test pre- and inflight with continuous monitoring of heart rate and arterial blood pressure. The arterial baroreflex was not changed inflight as shown by similar reductions in mean arterial pressure (MAP) response to leg cuff deflation (preflight −19 ± 2 mmHg vs. inflight −18 ± 5 mmHg). With the sit/stand test, the nadir of MAP was lower postflight (−17 ± 9 mmHg) than preflight (−11 ± 6 mmHg, p < 0.05). A greater increase in heart rate (25 ± 7; 16 ± 3 bpm) and decrease in stroke volume (−24 ± 11; −6 ± 4 mL) occurred with sit/stand postflight than leg cuffs inflight (p < 0.001). Inflight testing was influenced by elevated cardiac output resulting in a smaller drop in total peripheral resistance. Two of eight subjects exhibited orthostatic hypotension during the postflight stand test; their responses were not predicted by the inflight leg cuff deflation test. These results suggest that the baroreflex response examined by inflight leg cuff deflation was not a reliable indicator of postflight stand responses.
Spaceflight Study	n.a.	SLID-142	Mental performance in extreme environments: results from a performance monitoring study during a 438-day spaceflight	n.a.	Mental performance	During their stay in a space habitat, astronauts are exposed to many different stressors that may entail detrimental effects on mood and performance. In order to monitor the effects of the space environment on different human information processing functions during an extraordinary long-term space mission, the cognitive, visuo-motor and time-sharing performance of one Russian cosmonaut was repeatedly assessed (29 times) during his 438-day stay in space. The performance tasks used were chosen from the AGARD-STRES battery and included grammatical reasoning, Sternberg memory-search, unstable tracking, and a dual-task consisting of unstable tracking with concurrent memory-search. In addition to performance assessment, several subjective ratings concerning mood and workload were collected. Comparisons of pre-flight, in-flight, postflight and two follow-up assessments 6 months after the mission revealed, (1) no impairments of basic cognitive functions during the flight, (2) clear impairments of mood, feelings of raised workload, and disturbances of tracking performance and time-sharing during the first 3 weeks in space and the first 2 weeks after return to Earth, (3) an impressive stability of mood and performance during the second to fourteenth month in space, where mood and performance had returned to preflight baseline level, and (4) no long-lasting performance deficits at follow-up assessments. From these results it is concluded that the first 3 weeks of long-term spaceflights and the first 2 weeks back on Earth represent critical periods where adverse effects on attentional processes are to be expected, induced by the demands to adjust to the extreme environmental changes. The stability of mood and performance observed after successful adaptation to the space environment indicates that mental efficiency and emotional state can be maintained on a level as high as on Earth even during extraordinary long-term space missions.
Spaceflight Study	n.a.	SLID-143	Impaired pressor response after spaceflight and bed rest: evidence for cardiovascular dysfunction	179 Days (n=2), 208 Days (n=1), 188 Days (n=1), and 389 Days (n=1)	Pressor response after spaceflight and bed rest	We hypothesized that impaired cardiovascular responses to isometric muscle action contribute to the cardiovascular deconditioning that occurs after space flight (SF) and head-down-tilt bed rest (HDT). Six subjects were studied before, during and after 120 days of –6° HDT, and four subjects were studied before, during (two subjects) and after 179–389 days of SF. Subjects performed a sustained handgrip (SHG) at a force equivalent to 30% of maximum contraction force for 2 min, and heart-rate (HR) and pressor (mean arterial pressure, ΔMAP) responses were recorded. At the same relative force, both ΔHR and ΔMAP were significantly reduced during the first days after HDT (–54%, P<0.05 and –43%, P<0.05). In two subjects studied within 24 h after their return from SF, ΔMAP was practically absent (–79%, P<0.05) whereas in four subjects studied 1–4 days after return from SF, ΔMAP was reduced by 35% (P<0.05). ΔHR was not significantly changed. Our finding of attenuated pressor responses to SHG after HDT and SF supports the notion of impairments at both the neurocirculatory control and effector organ levels.
Spaceflight Study	Shuttle Missions	SLID-144	Heart rate variability and short duration spaceflight: relationship to post-flight orthostatic intolerance	8 - 16 Days	Heart rate variability	Background Upon return from space many astronauts experience symptoms of orthostatic intolerance. Research has implicated altered autonomic cardiovascular regulation due to spaceflight with further evidence to suggest that there might be pre-flight autonomic indicators of post-flight orthostatic intolerance. We used heart rate variability (HRV) to determine whether autonomic regulation of the heart in astronauts who did or did not experience post-flight orthostatic intolerance was different pre-flight and/or was differentially affected by short duration (8 – 16 days) spaceflight. HRV data from ten-minute stand tests collected from the 29 astronauts 10 days pre-flight, on landing day and three days post-flight were analysed using coarse graining spectral analysis. From the total power (PTOT), the harmonic component was extracted and divided into high (PHI: >0.15 Hz) and low (PLO: = 0.15 Hz) frequency power regions. Given the distribution of autonomic nervous system activity with frequency at the sinus node, PHI/PTOT was used as an indicator of parasympathetic activity; PLO/PTOT as an indicator of sympathetic activity; and, PLO/PHI as an estimate of sympathovagal balance. Results Twenty-one astronauts were classified as finishers, and eight as non-finishers, based on their ability to remain standing for 10 minutes on landing day. Pre-flight, non-finishers had a higher supine PHI/PTOT than finishers. Supine PHI/PTOT was the same pre-flight and on landing day in the finishers; whereas, in the non-finishers it was reduced. The ratio PLO/PHI was lower in non-finishers compared to finishers and was unaffected by spaceflight. Pre-flight, both finishers and non-finishers had similar supine values of PLO/PTOT, which increased from supine to stand. Following spaceflight, only the finishers had an increase in PLO/PTOT from supine to stand. Conclusions Both finishers and non-finishers had an increase in sympathetic activity with stand on pre-flight, yet only finishers retained this response on landing day. Non-finishers also had lower sympathovagal balance and higher pre-flight supine parasympathetic activity than finishers. These results suggest pre-flight autonomic status and post-flight impairment in autonomic control of the heart may contribute to orthostatic intolerance. The mechanism by which higher pre-flight parasympathetic activity might contribute to post-flight orthostatic intolerance is not understood and requires further investigation.
Spaceflight Study	International Space Station (ISS)	SLID-145	B cell homeostasis is maintained during long-duration spaceflight	6 - 12 Months	B cell numbers and phenotypes changes	Long-duration spaceflights reportedly induce immune dysregulation, which is considered a risk to astronaut safety and mission success. Recent studies have examined the impact of spaceflight on markers of adaptive and innate immunity, but no study, to date, has comprehensively evaluated humoral immunity and serological markers of B cell function. The aim of this study was to characterize changes in B cell numbers and phenotypes, along with plasma Igs and polyclonal free light chains (FLCs)-near-"real-time" biomarkers of Ig synthesis-in response to an ~6-mo mission to the International Space Station (ISS). Whole-blood samples were collected before flight, during flight ("Early flight," "Mid-flight," and "Late flight"), immediately upon return, and during a recovery period (R + 18, R + 30/R + 33, and R + 60/R + 66) from 23 ISS crew members. B Cell counts and phenotypes were measured throughout the duration of the mission, along with total plasma Ig and FLC levels. There was no effect of spaceflight on the number and proportion of the different B cell subsets. There was no difference in kappa FLC between preflight samples and either in-flight or recovery samples ( P > 0.05), and only a marginal reduction was observed in lambda FLC levels upon return to Earth ( P < 0.05). Furthermore, IgG and IgM remained unchanged during and after spaceflight compared with preflight values ( P > 0.05). Of note, plasma IgA concentrations were elevated in-flight compared with baseline and recovery values ( P < 0.05). These results indicate that B cell homeostasis is maintained during long-duration spaceflight, advocating for potential in-flight vaccination as viable countermeasures against viral reactivation during exploration-class missions.
Spaceflight Study	International Space Station (ISS)	SLID-146	Brain ventricular volume changes induced by long-duration spaceflight	n.a.	Quantitative changes in cerebrospinal fluid (CSF) volume of the brain ventricular regions	Long-duration spaceflight induces detrimental changes in human physiology. Its residual effects and mechanisms remain unclear. We prospectively investigated the changes in cerebrospinal fluid (CSF) volume of the brain ventricular regions in space crew by means of a region of interest analysis on structural brain scans. Cosmonaut MRI data were investigated preflight (n = 11), postflight (n = 11), and at long-term follow-up 7 mo after landing (n = 7). Post hoc analyses revealed a significant difference between preflight and postflight values for all supratentorial ventricular structures, i.e., lateral ventricle (mean % change ± SE = 13.3 ± 1.9), third ventricle (mean % change ± SE = 10.4 ± 1.1), and the total ventricular volume (mean % change ± SE = 11.6 ± 1.5) (all P < 0.0001), with higher volumes at postflight. At follow-up, these structures did not quite reach baseline levels, with still residual increases in volume for the lateral ventricle (mean % change ± SE = 7.7 ± 1.6; P = 0.0009), the third ventricle (mean % change ± SE = 4.7 ± 1.3; P = 0.0063), and the total ventricular volume (mean % change ± SE = 6.4 ± 1.3; P = 0.0008). This spatiotemporal pattern of CSF compartment enlargement and recovery points to a reduced CSF resorption in microgravity as the underlying cause. Our results warrant more detailed and longer longitudinal follow-up. The clinical impact of our findings on the long-term cosmonauts’ health and their relation to ocular changes reported in space travelers requires further prospective studies.
Spaceflight Study	International Space Station (ISS)	SLID-147	Resistive exercise in astronauts on prolonged spaceflights provides partial protection against spaceflight-induced bone loss	154 ± 24 Days	Densitometry and bone biochemical data collection	Bone loss in astronauts during spaceflight may be a risk factor for osteoporosis, fractures and renal stone formation. We previously reported that the bisphosphonate alendronate, combined with exercise that included an Advanced Resistive Exercise Device (ARED), can prevent or attenuate group mean declines in areal bone mineral density (aBMD) measured soon after ~ 6-month spaceflights aboard the International Space Station (ISS). It is unclear however if the beneficial effects on postflight aBMD were due to individual or combined effects of alendronate and ARED. Hence, 10 additional ISS astronauts were recruited who used the ARED (ARED group) without drug administration using similar measurements in the previous study, i.e., densitometry, biochemical assays and analysis of finite element (FE) models. In addition densitometry data (DXA and QCT only) were compared to published data from crewmembers (n = 14–18) flown prior to in-flight access to the ARED (Pre-ARED). Group mean changes from preflight (± SD %) were used to evaluate effects of countermeasures as sequentially modified on the ISS (i.e., Pre-ARED vs. ARED; ARED vs. Bis+ARED). Spaceflight durations were not significantly different between groups. Postflight bone density measurements were significantly reduced from preflight in the Pre-ARED group. As previously reported, combined Bis+ARED prevented declines in all DXA and QCT hip densitometry and in estimates of FE hip strengths; increased the aBMD of lumbar spine; and prevented elevations in urinary markers for bone resorption during spaceflight. ARED without alendronate partially attenuated declines in bone mass but did not suppress biomarkers for bone resorption or prevent trabecular bone loss. Resistive exercise in the ARED group did not prevent declines in hip trabecular vBMD, but prevented reductions in cortical vBMD of the femoral neck, in FE estimate of hip strength for non-linear stance (NLS) and in aBMD of the femoral neck. We conclude that a bisphosphonate, when combined with resistive exercise, enhances the preservation of bone mass because of the added suppression of bone resorption in trabecular bone compartment not evident with ARED alone.
Spaceflight Study	International Space Station (ISS)	SLID-148	Exercise as a countermeasure for latent viral reactivation during long duration space flight	> 100 Days	Countermeasure for latent viral reactivation	Latent viral reactivation is a commonly reported manifestation of immune system dysregulation during spaceflight. As physical fitness and exercise training have been shown to benefit multiple arms of the immune system, we hypothesized that higher levels of preflight physical fitness and/or maintaining fitness during a mission would protect astronauts from latent viral reactivation. Standardized tests of maximal strength, muscular endurance, flexibility, and cardiorespiratory fitness (CRF) were performed in 22 international space station (ISS) crewmembers before and after a ~6‐month mission. Reactivation of cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and varicella zoster virus (VZV) was determined in crewmembers and ground‐based controls before, during, and after spaceflight. Crewmembers with higher CRF before spaceflight had a 29% reduced risk of latent viral reactivation compared to crew with lower CRF. Higher preflight upper body muscular endurance was associated with a 39% reduced risk of viral reactivation, a longer time to viral reactivation, and lower peak viral DNA concentrations, particularly for EBV and VZV. Latent viral reactivation rates were highest in crew with lower preflight CRF and higher levels of CRF deconditioning on return to Earth. We conclude that physical fitness may protect astronauts from latent viral reactivation during long duration spaceflight missions.
Spaceflight Study	Odissea mission; Cervantes Mission and Delta Mission	SLID-149	Non-linear heart rate control in orthostatic tolerant cosmonauts after short-duration spaceflight	10 Days	Non-linear heart rate control changes	Spaceflight causes changes in the cardiovascular system. These changes contribute to the occurrence of orthostatic intolerance after spaceflight. The study of heart rate variations (HRV) provides a non-invasive means to study the autonomic modulation of heart rate. Non-linear control mechanisms have been suggested to be involved in normal heart rate variations. Non-linear control would be helpful in maintaining healthy cardiovascular values. However, these methods have not yet been used in combination with data after spaceflight. In this study we examined the non-linear response of heart rate to standing before and after spaceflight. Methods: ECG was measured for at least 10 minutes in supine and standing position 30 days before launch; and at 1, 4, 9, and 25 days after return to earth. These measurements were performed in 5 cosmonauts who were in the ISS for 10 days. The Approximate entropy of HRV was calculated. Results: The non-linear stand response was slightly altered immediately after spaceflight but was restored after 25 days. Conclusion: Nonlinear control of heart rate was affected immediately after short duration spaceflight, but not to the extent that the astronauts experienced orthostatic intolerance.
Spaceflight Study	International Space Station (ISS)	SLID-150	Dysfunctional vestibular system causes a blood pressure drop in astronauts returning from space	n.a.	Deconditioned otolith system on blood pressure and cardiac autonomic control	It is a challenge for the human body to maintain stable blood pressure while standing. The body’s failure to do so can lead to dizziness or even fainting. For decades it has been postulated that the vestibular organ can prevent a drop in pressure during a position change – supposedly mediated by reflexes to the cardiovascular system. We show – for the first time – a significant correlation between decreased functionality of the vestibular otolith system and a decrease in the mean arterial pressure when a person stands up. Until now, no experiments on Earth could selectively suppress both otolith systems; astronauts returning from space are a unique group of subjects in this regard. Their otolith systems are being temporarily disturbed and at the same time they often suffer from blood pressure instability. In our study, we observed the functioning of both the otolith and the cardiovascular system of the astronauts before and after spaceflight. Our finding indicates that an intact otolith system plays an important role in preventing blood pressure instability during orthostatic challenges. Our finding not only has important implications for human space exploration; they may also improve the treatment of unstable blood pressure here on Earth.
Spaceflight Study	International Space Station (ISS)	SLID-151	Macro- and microstructural changes in cosmonauts' brains after long-duration spaceflight	Average of 171 Days	Alterations of white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) composition	Long-duration spaceflight causes widespread physiological changes, although its effect on brain structure remains poorly understood. In this work, we acquired diffusion magnetic resonance imaging to investigate alterations of white matter (WM), gray matter (GM), and cerebrospinal fluid (CSF) compositions in each voxel, before, shortly after, and 7 months after long-duration spaceflight. We found increased WM in the cerebellum after spaceflight, providing the first clear evidence of sensorimotor neuroplasticity. At the region of interest level, this increase persisted 7 months after return to Earth. We also observe a widespread redistribution of CSF, with concomitant changes in the voxel fractions of adjacent GM. We show that these GM changes are the result of morphological changes rather than net tissue loss, which remained unclear from previous studies. Our study provides evidence of spaceflight-induced neuroplasticity to adapt motor strategies in space and evidence of fluid shift–induced mechanical changes in the brain.
Spaceflight Study	International Space Station (ISS)	SLID-152	Understanding the effects of spaceflight on head–trunk coordination during walking and obstacle avoidance	159 ± 17 Days	Sensorimotor changes	Prolonged exposure to spaceflight conditions results in a battery of physiological changes, some of which contribute to sensorimotor and neurovestibular deficits. Upon return to Earth, functional performance changes are tested using the Functional Task Test (FTT), which includes an obstacle course to observe post-flight balance and postural stability, specifically during turning. The goal of this study was to quantify changes in movement strategies during turning events by observing the latency between head-and-trunk coordinated movements. It was hypothesized that subjects experiencing neurovestibular adaptations would exhibit head-to-trunk locking (‘en bloc’ movement) during turning, exhibited by a decrease in latency between head and trunk movement. FTT data samples were collected from 13 ISS astronauts and 26 male 70-day head down tilt bed rest subjects, including bed rest controls (10 BRC) and bed rest exercisers (16 BRE). Samples were analyzed three times pre-exposure, immediately post-exposure (0 or 1-day post) and 2–3 times during recovery from the unloading environment. Two 3D inertial measurements units (XSens MTx) were attached to subjects, one on the head and one on the upper back. This study focused primarily on the yaw movements about the subject׳s center of rotation. Time differences (latency) between head and trunk movement were averaged across a slalom obstacle portion, consisting of three turns (approximately three 60° turns). All participants were grouped as ‘decreaser’ or ‘increaser’, relating to their change in head-to-trunk movement latency between pre- and post-environmental adaptation measures. Spaceflight unloading (ISS) showed a bimodal response between the ‘increaser’ and ‘decreaser’ group, while both bed rest control (BRC) and bed rest exercise (BRE) populations showed increased preference towards a ‘decreaser’ categorization, displaying greater head–trunk locking. It is clear that changes in movement strategies are adopted during exposure to an unloading environment. These results further the understanding of vestibular–somatosensory convergence and support the use of bed rest as an exclusionary model to better understand sensorimotor changes in spaceflight.
Spaceflight Study	International Space Station (ISS)	SLID-153	A microbial survey of the International Space Station (ISS)	n.a.	Bacteria	Background: Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Results: Sterile swabs were used to sample 15 surfaces onboard the ISS. The sites sampled were designed to be analogous to samples collected for (1) the Wildlife of Our Homes project and (2) a study of cell phones and shoes that were concurrently being collected for another component of Project MERCCURI. Sequencing of the 16S rDNA genes amplified from DNA extracted from each swab was used to produce a census of the microbes present on each surface sampled. We compared the microbes found on the ISS swabs to those from both homes on Earth and data from the Human Microbiome Project. Conclusions: While significantly different from homes on Earth and the Human Microbiome Project samples analyzed here, the microbial community composition on the ISS was more similar to home surfaces than to the human microbiome samples. The ISS surfaces are species-rich with 1,036-4,294 operational taxonomic units (OTUs per sample). There was no discernible biogeography of microbes on the 15 ISS surfaces, although this may be a reflection of the small sample size we were able to obtain.
Spaceflight Study	International Space Station (ISS)	SLID-153	A microbial survey of the International Space Station (ISS)	n.a.	Bacteria	Background: Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Results: Sterile swabs were used to sample 15 surfaces onboard the ISS. The sites sampled were designed to be analogous to samples collected for (1) the Wildlife of Our Homes project and (2) a study of cell phones and shoes that were concurrently being collected for another component of Project MERCCURI. Sequencing of the 16S rDNA genes amplified from DNA extracted from each swab was used to produce a census of the microbes present on each surface sampled. We compared the microbes found on the ISS swabs to those from both homes on Earth and data from the Human Microbiome Project. Conclusions: While significantly different from homes on Earth and the Human Microbiome Project samples analyzed here, the microbial community composition on the ISS was more similar to home surfaces than to the human microbiome samples. The ISS surfaces are species-rich with 1,036-4,294 operational taxonomic units (OTUs per sample). There was no discernible biogeography of microbes on the 15 ISS surfaces, although this may be a reflection of the small sample size we were able to obtain.
Spaceflight Study	Cygnus Spacecraft	SLID-154	Spaceflight Modifies Escherichia coli Gene Expression in Response to Antibiotic Exposure and Reveals Role of Oxidative Stress Response	49 Hours	Bacteria	Bacteria grown in space experiments under microgravity conditions have been found to undergo unique physiological responses, ranging from modified cell morphology and growth dynamics to a putative increased tolerance to antibiotics. A common theory for this behavior is the loss of gravity-driven convection processes in the orbital environment, resulting in both reduction of extracellular nutrient availability and the accumulation of bacterial byproducts near the cell. To further characterize the responses, this study investigated the transcriptomic response of Escherichia coli to both microgravity and antibiotic concentration. E. coli was grown aboard International Space Station in the presence of increasing concentrations of the antibiotic gentamicin with identical ground controls conducted on Earth. Here we show that within 49 h of being cultured, E. coli adapted to grow at higher antibiotic concentrations in space compared to Earth, and demonstrated consistent changes in expression of 63 genes in response to an increase in drug concentration in both environments, including specific responses related to oxidative stress and starvation response. Additionally, we find 50 stress-response genes upregulated in response to the microgravity when compared directly to the equivalent concentration in the ground control. We conclude that the increased antibiotic tolerance in microgravity may be attributed not only to diminished transport processes, but also to a resultant antibiotic cross-resistance response conferred by an overlapping effect of stress response genes. Our data suggest that direct stresses of nutrient starvation and acid-shock conveyed by the microgravity environment can incidentally upregulate stress response pathways related to antibiotic stress and in doing so contribute to the increased antibiotic stress tolerance observed for bacteria in space experiments. These results provide insights into the ability of bacteria to adapt under extreme stress conditions and potential strategies to prevent antimicrobial-resistance in space and on Earth.
Spaceflight Study	International Space Station (ISS)	SLID-155	Succession and persistence of microbial communities and antimicrobial resistance genes associated with International Space Station environmental surfaces	n.a.	Fungi	Background The International Space Station (ISS) is an ideal test bed for studying the effects of microbial persistence and succession on a closed system during long space flight. Culture-based analyses, targeted gene-based amplicon sequencing (bacteriome, mycobiome, and resistome), and shotgun metagenomics approaches have previously been performed on ISS environmental sample sets using whole genome amplification (WGA). However, this is the first study reporting on the metagenomes sampled from ISS environmental surfaces without the use of WGA. Metagenome sequences generated from eight defined ISS environmental locations in three consecutive flights were analyzed to assess the succession and persistence of microbial communities, their antimicrobial resistance (AMR) profiles, and virulence properties. Metagenomic sequences were produced from the samples treated with propidium monoazide (PMA) to measure intact microorganisms. Results The intact microbial communities detected in Flight 1 and Flight 2 samples were significantly more similar to each other than to Flight 3 samples. Among 318 microbial species detected, 46 species constituting 18 genera were common in all flight samples. Risk group or biosafety level 2 microorganisms that persisted among all three flights were Acinetobacter baumannii, Haemophilus influenzae, Klebsiella pneumoniae, Salmonella enterica, Shigella sonnei, Staphylococcus aureus, Yersinia frederiksenii, and Aspergillus lentulus. Even though Rhodotorula and Pantoea dominated the ISS microbiome, Pantoea exhibited succession and persistence. K. pneumoniae persisted in one location (US Node 1) of all three flights and might have spread to six out of the eight locations sampled on Flight 3. The AMR signatures associated with β-lactam, cationic antimicrobial peptide, and vancomycin were detected. Prominent virulence factors were cobalt-zinc-cadmium resistance and multidrug-resistance efflux pumps. Conclusions There was an increase in AMR and virulence gene factors detected over the period sampled, and metagenome sequences of human pathogens persisted over time. Comparative analysis of the microbial compositions of ISS with Earth analogs revealed that the ISS environmental surfaces were different in microbial composition. Metagenomics coupled with PMA treatment would help future space missions to estimate problematic risk group microbial pathogens. Cataloging AMR/virulence characteristics, succession, accumulation, and persistence of microorganisms would facilitate the development of suitable countermeasures to reduce their presence in the closed built environment.
Spaceflight Study	International Space Station (ISS)	SLID-155	Succession and persistence of microbial communities and antimicrobial resistance genes associated with International Space Station environmental surfaces	n.a.	Bacteria	Background The International Space Station (ISS) is an ideal test bed for studying the effects of microbial persistence and succession on a closed system during long space flight. Culture-based analyses, targeted gene-based amplicon sequencing (bacteriome, mycobiome, and resistome), and shotgun metagenomics approaches have previously been performed on ISS environmental sample sets using whole genome amplification (WGA). However, this is the first study reporting on the metagenomes sampled from ISS environmental surfaces without the use of WGA. Metagenome sequences generated from eight defined ISS environmental locations in three consecutive flights were analyzed to assess the succession and persistence of microbial communities, their antimicrobial resistance (AMR) profiles, and virulence properties. Metagenomic sequences were produced from the samples treated with propidium monoazide (PMA) to measure intact microorganisms. Results The intact microbial communities detected in Flight 1 and Flight 2 samples were significantly more similar to each other than to Flight 3 samples. Among 318 microbial species detected, 46 species constituting 18 genera were common in all flight samples. Risk group or biosafety level 2 microorganisms that persisted among all three flights were Acinetobacter baumannii, Haemophilus influenzae, Klebsiella pneumoniae, Salmonella enterica, Shigella sonnei, Staphylococcus aureus, Yersinia frederiksenii, and Aspergillus lentulus. Even though Rhodotorula and Pantoea dominated the ISS microbiome, Pantoea exhibited succession and persistence. K. pneumoniae persisted in one location (US Node 1) of all three flights and might have spread to six out of the eight locations sampled on Flight 3. The AMR signatures associated with β-lactam, cationic antimicrobial peptide, and vancomycin were detected. Prominent virulence factors were cobalt-zinc-cadmium resistance and multidrug-resistance efflux pumps. Conclusions There was an increase in AMR and virulence gene factors detected over the period sampled, and metagenome sequences of human pathogens persisted over time. Comparative analysis of the microbial compositions of ISS with Earth analogs revealed that the ISS environmental surfaces were different in microbial composition. Metagenomics coupled with PMA treatment would help future space missions to estimate problematic risk group microbial pathogens. Cataloging AMR/virulence characteristics, succession, accumulation, and persistence of microorganisms would facilitate the development of suitable countermeasures to reduce their presence in the closed built environment.
Spaceflight Study	BRIC-21 Space Flight;BRIC-23 Space Flight	SLID-156	Comparison of Bacillus subtilis transcriptome profiles from two separate missions to the International Space Station	25 Hours	Bacteria	The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium Bacillus subtilis was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. A total of 91 genes were significantly differentially expressed in both experiments; 55 exhibiting higher transcript levels in flight samples and 36 showing higher transcript levels in ground control samples. Genes upregulated in flight samples notably included those involved in biofilm formation, biotin and arginine biosynthesis, siderophores, manganese transport, toxin production and resistance, and sporulation inhibition. Genes preferentially upregulated in ground control samples notably included those responding to oxygen limitation, e.g., fermentation, anaerobic respiration, subtilosin biosynthesis, and anaerobic regulatory genes. The results indicated differences in oxygen availability between flight and ground control samples, likely due to differences in cell sedimentation and the toroidal shape assumed by the liquid cultures in microgravity.
Spaceflight Study	BRIC-21 Space Flight;BRIC-23 Space Flight	SLID-156	Comparison of Bacillus subtilis transcriptome profiles from two separate missions to the International Space Station	25 Hours	Bacteria	The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium Bacillus subtilis was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. A total of 91 genes were significantly differentially expressed in both experiments; 55 exhibiting higher transcript levels in flight samples and 36 showing higher transcript levels in ground control samples. Genes upregulated in flight samples notably included those involved in biofilm formation, biotin and arginine biosynthesis, siderophores, manganese transport, toxin production and resistance, and sporulation inhibition. Genes preferentially upregulated in ground control samples notably included those responding to oxygen limitation, e.g., fermentation, anaerobic respiration, subtilosin biosynthesis, and anaerobic regulatory genes. The results indicated differences in oxygen availability between flight and ground control samples, likely due to differences in cell sedimentation and the toroidal shape assumed by the liquid cultures in microgravity.
Spaceflight Study	BRIC-21 Space Flight;BRIC-24 Space Flight	SLID-156	Comparison of Bacillus subtilis transcriptome profiles from two separate missions to the International Space Station	36 Hours	Bacteria	The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium Bacillus subtilis was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. A total of 91 genes were significantly differentially expressed in both experiments; 55 exhibiting higher transcript levels in flight samples and 36 showing higher transcript levels in ground control samples. Genes upregulated in flight samples notably included those involved in biofilm formation, biotin and arginine biosynthesis, siderophores, manganese transport, toxin production and resistance, and sporulation inhibition. Genes preferentially upregulated in ground control samples notably included those responding to oxygen limitation, e.g., fermentation, anaerobic respiration, subtilosin biosynthesis, and anaerobic regulatory genes. The results indicated differences in oxygen availability between flight and ground control samples, likely due to differences in cell sedimentation and the toroidal shape assumed by the liquid cultures in microgravity.
Spaceflight Study	BRIC-21 Space Flight;BRIC-24 Space Flight	SLID-156	Comparison of Bacillus subtilis transcriptome profiles from two separate missions to the International Space Station	36 Hours	Bacteria	The human spaceflight environment is notable for the unique factor of microgravity, which exerts numerous physiologic effects on macroscopic organisms, but how this environment may affect single-celled microbes is less clear. In an effort to understand how the microbial transcriptome responds to the unique environment of spaceflight, the model Gram-positive bacterium Bacillus subtilis was flown on two separate missions to the International Space Station in experiments dubbed BRIC-21 and BRIC-23. Cells were grown to late-exponential/early stationary phase, frozen, then returned to Earth for RNA-seq analysis in parallel with matched ground control samples. A total of 91 genes were significantly differentially expressed in both experiments; 55 exhibiting higher transcript levels in flight samples and 36 showing higher transcript levels in ground control samples. Genes upregulated in flight samples notably included those involved in biofilm formation, biotin and arginine biosynthesis, siderophores, manganese transport, toxin production and resistance, and sporulation inhibition. Genes preferentially upregulated in ground control samples notably included those responding to oxygen limitation, e.g., fermentation, anaerobic respiration, subtilosin biosynthesis, and anaerobic regulatory genes. The results indicated differences in oxygen availability between flight and ground control samples, likely due to differences in cell sedimentation and the toroidal shape assumed by the liquid cultures in microgravity.
Spaceflight Study	China’s Space Station (CSS)	SLID-157	Microbiomes of China's Space Station During Assembly, Integration, and Test Operations	n.a.	Fungi	Sufficient evidence indicates that orbiting space stations contain diverse microbial populations, which may threaten astronaut health and equipment reliability. Understanding the composition of microbial communities in space stations will facilitate further development of targeted biological safety prevention and maintenance practices. Therefore, this study systematically investigated the microbial community of China's Space Station (CSS). Air and surface samples from 46 sites on the CSS and Assembly Integration and Test (AIT) center were collected, from which 40 bacteria strains were isolated and identified. Most isolates were cold- and desiccation-resistant and adapted to oligotrophic conditions. Bacillus was the dominant bacterial genus detected by both cultivation-based and Illumina MiSeq amplicon sequencing methods. Microbial contamination on the CSS was correlated with encapsulation staff activities. Analysis by spread plate and qPCR revealed that the CSS surface contained 2.24 × 103-5.47 × 103 CFU/100 cm2 culturable bacteria and 9.32 × 105-5.64 × 106 16S rRNA gene copies/100cm2; BacLight™ analysis revealed that the viable/total bacterial cell ratio was 1.98-13.28%. This is the first study to provide important systematic insights into the microbiome of the CSS during assembly that describes the pre-launch microbial diversity of the space station. Our findings revealed the following. (1) Bacillus strains and staff activities should be considered major concerns for future biological safety. (2) Autotrophic and multi-resistant microbial communities were widespread in the AIT environment. Although harsh cleaning methods reduced the number of microorganisms, stress-resistant strains were not completely removed. (3) Sampling, storage and analytical methods for the space station were thoroughly optimized, and are expected to be applicable to low-biomass environments in general. Microbiology-related future works will follow up to comprehensively understand the changing characteristics of microbial communities in CSS.
Spaceflight Study	China’s Space Station (CSS)	SLID-157	Microbiomes of China's Space Station During Assembly, Integration, and Test Operations	n.a.	Bacteria	Sufficient evidence indicates that orbiting space stations contain diverse microbial populations, which may threaten astronaut health and equipment reliability. Understanding the composition of microbial communities in space stations will facilitate further development of targeted biological safety prevention and maintenance practices. Therefore, this study systematically investigated the microbial community of China's Space Station (CSS). Air and surface samples from 46 sites on the CSS and Assembly Integration and Test (AIT) center were collected, from which 40 bacteria strains were isolated and identified. Most isolates were cold- and desiccation-resistant and adapted to oligotrophic conditions. Bacillus was the dominant bacterial genus detected by both cultivation-based and Illumina MiSeq amplicon sequencing methods. Microbial contamination on the CSS was correlated with encapsulation staff activities. Analysis by spread plate and qPCR revealed that the CSS surface contained 2.24 × 103-5.47 × 103 CFU/100 cm2 culturable bacteria and 9.32 × 105-5.64 × 106 16S rRNA gene copies/100cm2; BacLight™ analysis revealed that the viable/total bacterial cell ratio was 1.98-13.28%. This is the first study to provide important systematic insights into the microbiome of the CSS during assembly that describes the pre-launch microbial diversity of the space station. Our findings revealed the following. (1) Bacillus strains and staff activities should be considered major concerns for future biological safety. (2) Autotrophic and multi-resistant microbial communities were widespread in the AIT environment. Although harsh cleaning methods reduced the number of microorganisms, stress-resistant strains were not completely removed. (3) Sampling, storage and analytical methods for the space station were thoroughly optimized, and are expected to be applicable to low-biomass environments in general. Microbiology-related future works will follow up to comprehensively understand the changing characteristics of microbial communities in CSS.
Spaceflight Study	Tiangong-2 Space Flight	SLID-158	Identification of potential tobramycin-resistant mutagenesis of Escherichia coli strains after spaceflight	64 Days	Bacteria	Aim: This study aimed to explore potential tobramycin-resistant mutagenesis of Escherichia coli strains after spaceflight. Materials & methods: A spaceflight-induced mutagenesis of multidrug resistant E. coli strain (T1_13) on the outer space for 64 days (ST5), and a ground laboratory with the same conditions (GT5) were conducted. Both whole-genome sequencing and RNA-sequencing were performed. Results: A total of 75 single nucleotide polymorphisms and 20 InDels were found to be associated with the resistance mechanism. Compared with T1_13, 1242 genes were differentially expressed in more than 20 of 38 tobramycin-resistant E. coli isolates while not in GT5. Function annotation of these single nucleotide polymorphisms/InDels related genes and differentially expressed genes was performed. Conclusion: This study provided clues for potential tobramycin-resistant spaceflight-induced mutagenesis of E. coli.
Spaceflight Study	Tiangong-2 Space Flight	SLID-158	Identification of potential tobramycin-resistant mutagenesis of Escherichia coli strains after spaceflight	64 Days	Bacteria	Aim: This study aimed to explore potential tobramycin-resistant mutagenesis of Escherichia coli strains after spaceflight. Materials & methods: A spaceflight-induced mutagenesis of multidrug resistant E. coli strain (T1_13) on the outer space for 64 days (ST5), and a ground laboratory with the same conditions (GT5) were conducted. Both whole-genome sequencing and RNA-sequencing were performed. Results: A total of 75 single nucleotide polymorphisms and 20 InDels were found to be associated with the resistance mechanism. Compared with T1_13, 1242 genes were differentially expressed in more than 20 of 38 tobramycin-resistant E. coli isolates while not in GT5. Function annotation of these single nucleotide polymorphisms/InDels related genes and differentially expressed genes was performed. Conclusion: This study provided clues for potential tobramycin-resistant spaceflight-induced mutagenesis of E. coli.
Spaceflight Study	Shenzhou 11 Space Flight	SLID-159	Increased growth rate and amikacin resistance of Salmonella enteritidis after one-month spaceflight on China's Shenzhou-11 spacecraft	31 Days	Bacteria	China launched the Tiangong-2 space laboratory in 2016 and will eventually build a basic space station by the early 2020s. These spaceflight missions require astronauts to stay on the space station for more than 6 months, and they inevitably carry microbes into the space environment. It is known that the space environment affects microbial behavior, including growth rate, biofilm formation, virulence, drug resistance, and metabolism. However, the mechanisms of these alternations have not been fully elucidated. Therefore, it is beneficial to monitor microorganisms for preventing infections among astronauts in a space environment. Salmonella enteritidis is a Gram-negative bacterial pathogen that commonly causes acute gastroenteritis in humans. In this study, to better understand the effects of the space environment on S. enteritidis, a S. enteritidis strain was taken into space by the Shenzhou-11 spacecraft from 17 October 2016 to 18 November 2016, and a ground simulation with similar temperature conditions was simultaneously performed as a control. It was found that the flight strain displayed an increased growth rate, enhanced amikacin resistance, and some metabolism alterations compared with the ground strain. Enrichment analysis of proteome revealed that the increased growth rate might be associated with differentially expressed proteins involved in transmembrane transport and energy production and conversion assembly. A combined transcriptome and proteome analysis showed that the amikacin resistance was due to the downregulation of the oppA gene and oligopeptide transporter protein OppA. In conclusion, this study is the first systematic analysis of the phenotypic, genomic, transcriptomic, and proteomic variations in S. enteritidis during spaceflight and will provide beneficial insights for future studies on space microbiology.
Spaceflight Study	Shenzhou 11 Space Flight	SLID-159	Increased growth rate and amikacin resistance of Salmonella enteritidis after one-month spaceflight on China's Shenzhou-11 spacecraft	31 Days	Bacteria	China launched the Tiangong-2 space laboratory in 2016 and will eventually build a basic space station by the early 2020s. These spaceflight missions require astronauts to stay on the space station for more than 6 months, and they inevitably carry microbes into the space environment. It is known that the space environment affects microbial behavior, including growth rate, biofilm formation, virulence, drug resistance, and metabolism. However, the mechanisms of these alternations have not been fully elucidated. Therefore, it is beneficial to monitor microorganisms for preventing infections among astronauts in a space environment. Salmonella enteritidis is a Gram-negative bacterial pathogen that commonly causes acute gastroenteritis in humans. In this study, to better understand the effects of the space environment on S. enteritidis, a S. enteritidis strain was taken into space by the Shenzhou-11 spacecraft from 17 October 2016 to 18 November 2016, and a ground simulation with similar temperature conditions was simultaneously performed as a control. It was found that the flight strain displayed an increased growth rate, enhanced amikacin resistance, and some metabolism alterations compared with the ground strain. Enrichment analysis of proteome revealed that the increased growth rate might be associated with differentially expressed proteins involved in transmembrane transport and energy production and conversion assembly. A combined transcriptome and proteome analysis showed that the amikacin resistance was due to the downregulation of the oppA gene and oligopeptide transporter protein OppA. In conclusion, this study is the first systematic analysis of the phenotypic, genomic, transcriptomic, and proteomic variations in S. enteritidis during spaceflight and will provide beneficial insights for future studies on space microbiology.
Spaceflight Study	International Space Station (ISS)	SLID-160	Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces	n.a.	Fungi	Background: The International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth. Results: The cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured. Conclusions: The results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries.
Spaceflight Study	International Space Station (ISS)	SLID-160	Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces	n.a.	Bacteria	Background: The International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth. Results: The cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured. Conclusions: The results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries.
Spaceflight Study	SJ-10 Satellite	SLID-161	Integrated proteomic and metabolomic analysis to study the effects of spaceflight on Candida albicans	12 Days	Fungi	Background Candida albicans is an opportunistic pathogenic yeast, which could become pathogenic in various stressful environmental factors including the spaceflight environment. In this study, we aim to explore the phenotypic changes and possible mechanisms of C. albicans after exposure to spaceflight conditions. Results The effect of C. albicans after carried on the “SJ-10” satellite for 12 days was evaluated by proliferation, morphology, environmental resistance and virulence experiment. The result showed that the proliferation rate, biofilm formation, antioxidant capacity, cytotoxicity and filamentous morphology of C. albicans were increased in the spaceflight group compared to the control group. Proteomics and metabolomics technologies were used to analyze the profiles of proteins and metabolites in C. albicans under spaceflight conditions. Proteomic analysis identified 548 up-regulated proteins involved in the ribosome, DNA replication, base excision repair and sulfur metabolism in the spaceflight group. Moreover, 332 down-regulated proteins related to metabolic processes were observed. The metabolomic analysis found five differentially expressed metabolites. The combined analysis of proteomic and metabolomic revealed the accumulation of cysteine and methionine in C. albicans after spaceflight. Conclusions Mechanisms that could explain the results in the phenotypic experiment of C. albicans were found through proteomic and metabolomic analysis. And our data provide an important basis for the assessment of the risk that C. albicans could cause under spaceflight environment.
Spaceflight Study	SJ-10 Satellite	SLID-161	Integrated proteomic and metabolomic analysis to study the effects of spaceflight on Candida albicans	12 Days	Fungi	Background Candida albicans is an opportunistic pathogenic yeast, which could become pathogenic in various stressful environmental factors including the spaceflight environment. In this study, we aim to explore the phenotypic changes and possible mechanisms of C. albicans after exposure to spaceflight conditions. Results The effect of C. albicans after carried on the “SJ-10” satellite for 12 days was evaluated by proliferation, morphology, environmental resistance and virulence experiment. The result showed that the proliferation rate, biofilm formation, antioxidant capacity, cytotoxicity and filamentous morphology of C. albicans were increased in the spaceflight group compared to the control group. Proteomics and metabolomics technologies were used to analyze the profiles of proteins and metabolites in C. albicans under spaceflight conditions. Proteomic analysis identified 548 up-regulated proteins involved in the ribosome, DNA replication, base excision repair and sulfur metabolism in the spaceflight group. Moreover, 332 down-regulated proteins related to metabolic processes were observed. The metabolomic analysis found five differentially expressed metabolites. The combined analysis of proteomic and metabolomic revealed the accumulation of cysteine and methionine in C. albicans after spaceflight. Conclusions Mechanisms that could explain the results in the phenotypic experiment of C. albicans were found through proteomic and metabolomic analysis. And our data provide an important basis for the assessment of the risk that C. albicans could cause under spaceflight environment.
Spaceflight Study	International Space Station (ISS)	SLID-162	Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station	n.a.	Bacteria	The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.
Spaceflight Study	International Space Station (ISS)	SLID-162	Genomic and phenotypic characterization of Burkholderia isolates from the potable water system of the International Space Station	n.a.	Bacteria	The opportunistic pathogens Burkholderia cepacia and Burkholderia contaminans, both genomovars of the Burkholderia cepacia complex (BCC), are frequently cultured from the potable water dispenser (PWD) of the International Space Station (ISS). Here, we sequenced the genomes and conducted phenotypic assays to characterize these Burkholderia isolates. All recovered isolates of the two species fall within monophyletic clades based on phylogenomic trees of conserved single-copy core genes. Within species, the ISS-derived isolates all demonstrate greater than 99% average nucleotide identity (with 95-99% of genomes aligning) and share around 90% of the identified gene clusters from a pangenomic analysis-suggesting that the two groups are each composed of highly similar genomic lineages and their members may have all stemmed from the same two founding populations. The differences that can be observed between the recovered isolates at the pangenomic level are primarily located within putative plasmids. Phenotypically, macrophage intracellularization and lysis occurred at generally similar rates between all ISS-derived isolates, as well as with their respective type-terrestrial strain references. All ISS-derived isolates exhibited antibiotic sensitivity similar to that of the terrestrial reference strains, and minimal differences between isolates were observed. With a few exceptions, biofilm formation rates were generally consistent across each species. And lastly, though isolation date does not necessarily provide any insight into how long a given isolate had been aboard the ISS, none of the assayed physiology correlated with either date of isolation or distances based on nucleotide variation. Overall, we find that while the populations of Burkholderia present in the ISS PWS each maintain virulence, they are likely are not more virulent than those that might be encountered on planet and remain susceptible to clinically used antibiotics.
Spaceflight Study	International Space Station (ISS)	SLID-163	Space Station conditions are selective but do not alter microbial characteristics relevant to human health	n.a.	Fungi	The International Space Station (ISS) is a unique habitat for humans and microorganisms. Here, we report the results of the ISS experiment EXTREMOPHILES, including the analysis of microbial communities from several areas aboard at three time points. We assess microbial diversity, distribution, functional capacity and resistance profile using a combination of cultivation-independent analyses (amplicon and shot-gun sequencing) and cultivation-dependent analyses (physiological and genetic characterization of microbial isolates, antibiotic resistance tests, co-incubation experiments). We show that the ISS microbial communities are highly similar to those present in ground-based confined indoor environments and are subject to fluctuations, although a core microbiome persists over time and locations. The genomic and physiological features selected by ISS conditions do not appear to be directly relevant to human health, although adaptations towards biofilm formation and surface interactions were observed. Our results do not raise direct reason for concern with respect to crew health, but indicate a potential threat towards material integrity in moist areas.
Spaceflight Study	International Space Station (ISS)	SLID-163	Space Station conditions are selective but do not alter microbial characteristics relevant to human health	n.a.	Bacteria	The International Space Station (ISS) is a unique habitat for humans and microorganisms. Here, we report the results of the ISS experiment EXTREMOPHILES, including the analysis of microbial communities from several areas aboard at three time points. We assess microbial diversity, distribution, functional capacity and resistance profile using a combination of cultivation-independent analyses (amplicon and shot-gun sequencing) and cultivation-dependent analyses (physiological and genetic characterization of microbial isolates, antibiotic resistance tests, co-incubation experiments). We show that the ISS microbial communities are highly similar to those present in ground-based confined indoor environments and are subject to fluctuations, although a core microbiome persists over time and locations. The genomic and physiological features selected by ISS conditions do not appear to be directly relevant to human health, although adaptations towards biofilm formation and surface interactions were observed. Our results do not raise direct reason for concern with respect to crew health, but indicate a potential threat towards material integrity in moist areas.
Spaceflight Study	International Space Station (ISS)	SLID-164	Expression of stress response genes in barley Hordeum vulgare in a spaceflight environment	26 Days	Grain	The transcriptome of barley Hordeum vulgare grown aboard the International Space Station was studied using microarray analysis. In the spaceflight environment, mRNA levels of over 500 genes were changed more than twofold; among them, genes of stress response proteins, in particular, heat shock proteins, pathogenesis-related proteins, and antioxidant proteins. Further analysis by real-time PCR confirmed enhanced transcription of reactive oxygen species scavenging genes. The superoxide dismutase (sod) mRNA level in the space environment was 6-fold higher than in earth conditions. The transcript levels of glutamyl transferase (gst), catalase (cat), and ascorbate peroxidase (apx) were increased in spaceflight 24, 18, and 3 times in comparison to ground control, respectively. For the first time, it has been shown that spaceflight environment can induce oxidative stress in plants.
Spaceflight Study	International Space Station (ISS)	SLID-165	Microgravity effects on thylakoid, single leaf, and whole canopy photosynthesis of dwarf wheat	21 - 24 Days	Grain	The concept of using higher plants to maintain a sustainable life support system for humans during long-duration space missions is dependent upon photosynthesis. The effects of extended exposure to microgravity on the development and functioning of photosynthesis at the leaf and stand levels were examined onboard the International Space Station (ISS). The PESTO (Photosynthesis Experiment Systems Testing and Operations) experiment was the first long-term replicated test to obtain direct measurements of canopy photosynthesis from space under well-controlled conditions. The PESTO experiment consisted of a series of 21-24 day growth cycles of Triticum aestivum L. cv. USU Apogee onboard ISS. Single leaf measurements showed no differences in photosynthetic activity at the moderate (up to 600 micromol m(-2) s(-1)) light levels, but reductions in whole chain electron transport, PSII, and PSI activities were measured under saturating light (>2,000 micromol m(-2) s(-1)) and CO(2) (4000 micromol mol(-1)) conditions in the microgravity-grown plants. Canopy level photosynthetic rates of plants developing in microgravity at approximately 280 micromol m(-2) s(-1) were not different from ground controls. The wheat canopy had apparently adapted to the microgravity environment since the CO(2) compensation (121 vs. 118 micromol mol(-1)) and PPF compensation (85 vs. 81 micromol m(-2) s(-1)) of the flight and ground treatments were similar. The reduction in whole chain electron transport (13%), PSII (13%), and PSI (16%) activities observed under saturating light conditions suggests that microgravity-induced responses at the canopy level may occur at higher PPF intensity.
Spaceflight Study	International Space Station (ISS), Increment IV	SLID-166	Microgravity effects on leaf morphology, cell structure, carbon metabolism and mRNA expression of dwarf wheat	n.a.	Grain	The use of higher plants as the basis for a biological life support system that regenerates the atmosphere, purifies water, and produces food has been proposed for long duration space missions. The objective of these experiments was to determine what effects microgravity (microg) had on chloroplast development, carbohydrate metabolism and gene expression in developing leaves of Triticum aestivum L. cv. USU Apogee. Gravity naive wheat plants were sampled from a series of seven 21-day experiments conducted during Increment IV of the International Space Station. These samples were fixed in either 3% glutaraldehyde or RNAlater or frozen at -25 degrees C for subsequent analysis. In addition, leaf samples were collected from 24- and 14-day-old plants during the mission that were returned to Earth for analysis. Plants grown under identical light, temperature, relative humidity, photoperiod, CO(2), and planting density were used as ground controls. At the morphological level, there was little difference in the development of cells of wheat under microg conditions. Leaves developed in mug have thinner cross-sectional area than the 1g grown plants. Ultrastructurally, the chloroplasts of microg grown plants were more ovoid than those developed at 1g, and the thylakoid membranes had a trend to greater packing density. No differences were observed in the starch, soluble sugar, or lignin content of the leaves grown in microg or 1g conditions. Furthermore, no differences in gene expression were detected leaf samples collected at microg from 24-day-old leaves, suggesting that the spaceflight environment had minimal impact on wheat metabolism.
Spaceflight Study	STS-110/8A;STS-111/UF-2;STS-112	SLID-166	Microgravity effects on leaf morphology, cell structure, carbon metabolism and mRNA expression of dwarf wheat	73 Days	Grain	The use of higher plants as the basis for a biological life support system that regenerates the atmosphere, purifies water, and produces food has been proposed for long duration space missions. The objective of these experiments was to determine what effects microgravity (microg) had on chloroplast development, carbohydrate metabolism and gene expression in developing leaves of Triticum aestivum L. cv. USU Apogee. Gravity naive wheat plants were sampled from a series of seven 21-day experiments conducted during Increment IV of the International Space Station. These samples were fixed in either 3% glutaraldehyde or RNAlater or frozen at -25 degrees C for subsequent analysis. In addition, leaf samples were collected from 24- and 14-day-old plants during the mission that were returned to Earth for analysis. Plants grown under identical light, temperature, relative humidity, photoperiod, CO(2), and planting density were used as ground controls. At the morphological level, there was little difference in the development of cells of wheat under microg conditions. Leaves developed in mug have thinner cross-sectional area than the 1g grown plants. Ultrastructurally, the chloroplasts of microg grown plants were more ovoid than those developed at 1g, and the thylakoid membranes had a trend to greater packing density. No differences were observed in the starch, soluble sugar, or lignin content of the leaves grown in microg or 1g conditions. Furthermore, no differences in gene expression were detected leaf samples collected at microg from 24-day-old leaves, suggesting that the spaceflight environment had minimal impact on wheat metabolism.
Spaceflight Study	Chinese Recoverable Satellite “Shen Zhou”	SLID-167	Quantification of four active ingredients and fingerprint analysis of Licorice (Glycyrrhiza uralensis Fisch.) after spaceflight by HPLC–DAD	18 Days	n.a.	The rhizomes growing from Licorice (Glycyrrhiza uralensis Fisch.) seeds carried by the Chinese recoverable satellite named “Shen Zhou” and from the parallel ground-based seeds were selected as the samples. Seeds were flown under the space condition for 18 days. Average radiation dose in the flight recovery module was 0.102 mGy/day and the distance from flight apogee to earth was 350 km and the gravity was 10−6 g. After returning to earth, the seeds carried by the satellite and the parallel ground control were cultivated to maturity under the same condition. In this paper, fingerprint was obtained and contents of the four main ingredients of the samples were investigated on a high performance liquid chromatography with diode-array detection. The differentiation between the 12 samples induced by cosmic factors was observed. Contents of the detected four components of space flight group were much higher than that of the control group. Areas of the 26 common peaks of the 12 samples were analyzed by principal component analysis and three components were extracted. The results demonstrated that extraterrestrial environment could affect the amount of its secondary metabolites. This report provided the scientific data for spaceflight breeding of a medicinal plant and indicated that extraterrestrial orbit is a possible way in accelerating the rate of breeding and select eximious germplasm resources.
Spaceflight Study	BRIC-AUX on STS-95	SLID-168	STS-95 space experiment for plant growth and development, and auxin polar transport	9 Days	Grain	The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.
Spaceflight Study	BRIC-AUX on STS-95	SLID-168	STS-95 space experiment for plant growth and development, and auxin polar transport	9 Days	Vegetable	The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.
Spaceflight Study	International Space Station (ISS)	SLID-169	Polar auxin transport is essential to maintain growth and development of etiolated pea and maize seedlings grown under 1 g conditions: Relevance to the international space station experiment	n.a.	Grain	We conducted "Auxin Transport" space experiments in 2016 and 2017 in the Japanese Experiment Module (JEM) on the International Space Station (ISS), with the principal objective being integrated analyses of the growth and development of etiolated pea (Pisum sativum L. cv Alaska) and maize (Zea mays L. cv Golden Cross Bantam) seedlings under true microgravity conditions in space relative to auxin dynamics. Etiolated pea seedlings grown under microgravity conditions in space for 3 days showed automorphogenesis. Epicotyls and roots bent ca. 45° and 20° toward the direction away from the cotyledons, respectively, whereas those grown under artificial 1 g conditions produced by a centrifuge in the Cell Biology Experimental Facility (CBEF) in space showed negative and positive gravitropic response in epicotyls and in roots, respectively. On the other hand, the coleoptiles of 4-day-old etiolated maize seedlings grew almost straight, but the mesocotyls curved and grew toward a random direction under microgravity conditions in space. In contrast, the coleoptiles and mesocotyls of etiolated maize seedlings grown under 1 g conditions on Earth were almost straight and grew upward or toward the direction against the gravity vector. The polar auxin transport activity in etiolated pea epicotyls and in maize shoots was significantly inhibited and enhanced, respectively, under microgravity conditions in space as compared with artificial 1 g conditions in space or 1 g conditions on Earth. An inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA) substantially affected the growth direction and polar auxin transport activity in etiolated pea seedlings grown under both artificial 1 g and microgravity conditions in space. These results strongly suggest that adequate polar auxin transport is essential for gravitropic response in plants. Possible mechanisms enhancing polar auxin transport in etiolated maize seedlings grown under microgravity conditions in space are also proposed.
Spaceflight Study	International Space Station (ISS)	SLID-169	Polar auxin transport is essential to maintain growth and development of etiolated pea and maize seedlings grown under 1 g conditions: Relevance to the international space station experiment	n.a.	Vegetable	We conducted "Auxin Transport" space experiments in 2016 and 2017 in the Japanese Experiment Module (JEM) on the International Space Station (ISS), with the principal objective being integrated analyses of the growth and development of etiolated pea (Pisum sativum L. cv Alaska) and maize (Zea mays L. cv Golden Cross Bantam) seedlings under true microgravity conditions in space relative to auxin dynamics. Etiolated pea seedlings grown under microgravity conditions in space for 3 days showed automorphogenesis. Epicotyls and roots bent ca. 45° and 20° toward the direction away from the cotyledons, respectively, whereas those grown under artificial 1 g conditions produced by a centrifuge in the Cell Biology Experimental Facility (CBEF) in space showed negative and positive gravitropic response in epicotyls and in roots, respectively. On the other hand, the coleoptiles of 4-day-old etiolated maize seedlings grew almost straight, but the mesocotyls curved and grew toward a random direction under microgravity conditions in space. In contrast, the coleoptiles and mesocotyls of etiolated maize seedlings grown under 1 g conditions on Earth were almost straight and grew upward or toward the direction against the gravity vector. The polar auxin transport activity in etiolated pea epicotyls and in maize shoots was significantly inhibited and enhanced, respectively, under microgravity conditions in space as compared with artificial 1 g conditions in space or 1 g conditions on Earth. An inhibitor of polar auxin transport, 2,3,5-triiodobenzoic acid (TIBA) substantially affected the growth direction and polar auxin transport activity in etiolated pea seedlings grown under both artificial 1 g and microgravity conditions in space. These results strongly suggest that adequate polar auxin transport is essential for gravitropic response in plants. Possible mechanisms enhancing polar auxin transport in etiolated maize seedlings grown under microgravity conditions in space are also proposed.
Spaceflight Study	Seed-Breeding Satellite SJ-8	SLID-170	A novel maize dwarf mutant generated by Ty1-copia LTR-retrotransposon insertion in Brachytic2 after spaceflight	15 Days	Grain	Plant height and ear height are two important agronomic traits in maize breeding. In this study, two dwarf mutants short internode length1 (sil1) and short internode length2 (sil2) were obtained from two of 398 spaceflighted seeds of inbred line 18-599. The decrease in longitudinal cell number and cell length led to the shortened internodes of sil1 and sil2. A Ty1-copia LTR-retrotransposon, termed ZmRE-1, inserted in the fifth exon of Brachytic2 (Br2) was identified in sil1 and sil2 at exactly the same site, which indicated the transposition of ZmRE-1 probably correlated with the spaceflight. This new dwarf mutant allele was named as br2-sil in this study. The insertion of ZmRE-1 not only led to the loss of normal transcript of Br2 allele, but also reduced the transcript expression of br2-sil allele. Chop-qPCR displayed that the promoter region DNA methylation level of br2-sil allele in sil1 was higher than that of Br2 allele in WT-sil1. We speculated that the increased methylation level might downregulate the br2-sil expression. There was no difference in the seed-setting rate between sil1 and WT-sil1. Meanwhile, br2-sil could reduce plant and ear height effectively in Br2/br2-sil genotype without negative effects on grain yield. Therefore, the application of br2-sil in breeding has the potential to improve the grain yield per unit area through increasing the planting density.
Spaceflight Study	International Space Station (ISS)	SLID-171	Genome-wide expression analysis of reactive oxygen species gene network in Mizuna plants grown in long-term spaceflight	27 Days	Vegetable	Background: Spaceflight environment have been shown to generate reactive oxygen species (ROS) and induce oxidative stress in plants, but little is known about the gene expression of the ROS gene network in plants grown in long-term spaceflight. The molecular response and adaptation to the spaceflight environment of Mizuna plants harvested after 27 days of cultivation onboard the International Space Station (ISS) were measured using genome-wide mRNA expression analysis (mRNA-Seq). Results: Total reads of transcripts from the Mizuna grown in the ISS as well as on the ground by mRNA-Seq showed 8,258 and 14,170 transcripts up-regulated and down-regulated, respectively, in the space-grown Mizuna 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 four hallmarks, and preferentially expressed genes associated with ROS-scavenging including thioredoxin, glutaredoxin, and alternative oxidase genes. In the transcription factors of the 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: This large-scale transcriptome analysis revealed that the spaceflight environment induced oxidative stress and the ROS gene network activation in the space-grown Mizuna. Among transcripts altered in expression by space conditions, some were common genes response to abiotic and biotic stress. Furthermore, certain genes were exclusively up-regulated in Mizuna grown on the ISS. Surprisingly, Mizuna grew in space normally, as well as on the ground, demonstrating that plants can acclimate to long-term exposure in the spaceflight environment by reprogramming the expression of the ROS gene network.
Spaceflight Study	SpaceX-10	SLID-172	Gravity-regulated localization of PsPIN1 is important for polar auxin transport in etiolated pea seedlings: Relevance to the International Space Station experiment	n.a.	Vegetable	To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment "Auxin Transport" (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (μg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under μg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under μg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under μg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the μg experiment conducted in space.
Spaceflight Study	SpaceX-8	SLID-172	Gravity-regulated localization of PsPIN1 is important for polar auxin transport in etiolated pea seedlings: Relevance to the International Space Station experiment	n.a.	Vegetable	To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment "Auxin Transport" (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (μg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under μg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under μg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under μg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the μg experiment conducted in space.
Spaceflight Study	International Space Station (ISS), Russian Segment (RS)	SLID-173	Spaceflight effects on consecutive generations of peas grown onboard the Russian segment of the International Space Station	n.a.	Vegetable	In the period from March 2003 to April 2005 we fulfilled five experimental cultivations of genetically marked dwarf pea species in greenhouse Lada installed in the Russian segment (RS) of the International Space Station (ISS). The purpose of this series of experiments was to make morphologic and genetic analysis of pea plants grown in successive generations.According to our results, pea growth and development over the full cycle of ontogenesis (from seed to seed) taking place in space greenhouse Lada were not different as compared with the ground control plants. In addition, four successive pea crops gathered in space flight did not loose their reproductive functions and formed viable seeds. Genetic analysis of the plants grown from the “space” and “ground” seeds produced by the first to fourth successive crops was performed using the methods of chromosomal aberrations count and Random Amplified Polymorphic DNA (molecular method).No genetic polymorphism was found either in the experimental or control crops. This can serve as a sound argument for the supposition that the genetic apparatus of plants is not impacted by exposure of several successive generations to the conditions of space flight.
Spaceflight Study	International Space Station (ISS)	SLID-174	Microbiological and Nutritional Analysis of Lettuce Crops Grown on the International Space Station	33 Days	Vegetable	The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv 'Outredgeous,' plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24-72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 - 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isolate identification and community characterization indicated variation in the diversity of genera between leaf and root tissue with diversity being higher in root tissue, and included differences in the dominant genera. The only difference between ground and flight experiments was seen in the third experiment, VEG-03A, with significant differences in the genera from leaf tissue. Flight and ground tissue showed differences in Fe, K, Na, P, S, and Zn content and total phenolic levels, but no differences in anthocyanin and ORAC levels. This study indicated that leafy vegetable crops can produce safe, edible, fresh food to supplement to the astronauts' diet, and provide baseline data for continual operation of the Veggie plant growth units on ISS.
Spaceflight Study	International Space Station (ISS)	SLID-174	Microbiological and Nutritional Analysis of Lettuce Crops Grown on the International Space Station	56 Days	Vegetable	The ability to grow safe, fresh food to supplement packaged foods of astronauts in space has been an important goal for NASA. Food crops grown in space experience different environmental conditions than plants grown on Earth (e.g., reduced gravity, elevated radiation levels). To study the effects of space conditions, red romaine lettuce, Lactuca sativa cv 'Outredgeous,' plants were grown in Veggie plant growth chambers on the International Space Station (ISS) and compared with ground-grown plants. Multiple plantings were grown on ISS and harvested using either a single, final harvest, or sequential harvests in which several mature leaves were removed from the plants at weekly intervals. Ground controls were grown simultaneously with a 24-72 h delay using ISS environmental data. Food safety of the plants was determined by heterotrophic plate counts for bacteria and fungi, as well as isolate identification using samples taken from the leaves and roots. Molecular characterization was conducted using Next Generation Sequencing (NGS) to provide taxonomic composition and phylogenetic structure of the community. Leaves were also analyzed for elemental composition, as well as levels of phenolics, anthocyanins, and Oxygen Radical Absorbance Capacity (ORAC). Comparison of flight and ground tissues showed some differences in total counts for bacteria and yeast/molds (2.14 - 4.86 log10 CFU/g), while screening for select human pathogens yielded negative results. Bacterial and fungal isolate identification and community characterization indicated variation in the diversity of genera between leaf and root tissue with diversity being higher in root tissue, and included differences in the dominant genera. The only difference between ground and flight experiments was seen in the third experiment, VEG-03A, with significant differences in the genera from leaf tissue. Flight and ground tissue showed differences in Fe, K, Na, P, S, and Zn content and total phenolic levels, but no differences in anthocyanin and ORAC levels. This study indicated that leafy vegetable crops can produce safe, edible, fresh food to supplement to the astronauts' diet, and provide baseline data for continual operation of the Veggie plant growth units on ISS.
Spaceflight Study	Space Shuttle STS-95	SLID-175	Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism	n.a.	Seedling	During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.
Spaceflight Study	Space Shuttle STS-95	SLID-175	Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism	n.a.	Seedling	During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.
Spaceflight Study	STS-95 Space Transport System	SLID-176	Growth and cell wall changes in rice roots during spaceflight	136 Hours	Grain	We analyzed the changes in growth and cell wall properties of roots of rice (Oryza sativa L. cv. Koshihikari) grown for 68.5, 91.5, and 136 h during the Space Shuttle STS-95 mission. In space, most of rice roots elongated in a direction forming a constant mean angle of about 55 degrees with the perpendicular base line away from the caryopsis in the early phase of growth, but later the roots grew in various directions, including away from the agar medium. In space, elongation growth of roots was stimulated. On the other hand, some of elasticity moduli and viscosity coefficients were higher in roots grown in space than on the ground, suggesting that the cell wall of space-grown roots has a lower capacity to expand than the controls. The levels of both cellulose and the matrix polysaccharides per unit length of roots decreased greatly, whereas the ratio of the high molecular mass polysaccharides in the hemicellulose fraction increased in space-grown roots. The prominent thinning of the cell wall could overwhelm the disadvantageous changes in the cell wall mechanical properties, leading to the stimulation of elongation growth in rice roots in space. Thus, growth and the cell wall properties of rice roots were strongly modified under microgravity conditions during spaceflight.
Spaceflight Study	Chinese 20th Recoverable Satellite	SLID-177	Analysis of cytogenetic damage in rice seeds induced by energetic heavy ions on-ground and after spaceflight	18 Days	Grain	To analyze the biological effects of the space environment, we flew nine lines of rice seeds on the Chinese 20(th) recoverable satellite for a duration of 18 days. The same lines of seeds were also irradiated to low doses (2.0 mGy) of Carbon, or Neon or Iron ions (with different LET value of 13.3 keV/μm, 31 keV/μm and 500 keV/μm respectively) at National Institute of Radiological Sciences in Chiba, Japan. The total number of mitotic cells and chromosomal aberrations were analyzed. The mitotic index (MI) and the frequency of chromosomal aberration were evaluated in order to compare the cytogenetic damages from spaceflight and from exposure to similar doses of charged particle on the ground. The results of the present study show that the space environment and heavy energy ion can alter cell growth, and induce various chromosome aberrations including micronuclei, chromosomal bridges, fragments and laggards. With all the lines combined, the frequency of chromosome aberrations and MI in seeds flown in space are the highest. The effectiveness of cytogenetic damage from spaceflight (SP) and the heavy ion irradiations is SP > Fe > Ne > C.
Spaceflight Study	Recoverable Satellite JB-1	SLID-178	Diversity and Stability Study on Rice Mutants Induced in Space Environment	15 Days	Grain	To further study the characteristics of changes on the molecular level of rice mutants induced in space environment, we analyzed proteins in leaves and seeds of four rice mutants (two high-tillering and two low-tillering) in the 8(th) and 9(th) generations after a 15-day spaceflight, and compared with their ground controls by two-dimentional polyacrylamide gel electrophoresis and reverse phase liquid chromatography (RPLC). In addition, the albumin, globulin, prolamine, glutelin, and amylose of the mutant seeds were analyzed by RPLC and ultra-violet spectrometry. The results showed that the low-abundance proteins of leaves in the peak tillering stage are more likely to be induced compared with their corresponding controls. The albumin, globulin, and prolamine of the mutant seeds revealed changes when compared with their controls, and the characteristics of changes in different mutants were stably inherited in the 8(th) and 9(th) generations, suggesting that they can be used as bio markers to identity the mutants induced by spaceflight. Moreover, two proteins (SSP9111 and SSP6302) were found to be expressed with high intensity (two-fold change) in different mutants, which were both correlated with photosystem according to mass spectrometry and database searching.
Spaceflight Study	Chinese 20th Recoverable Satellite	SLID-179	Comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice (Oryza sativa)	18 Days	Grain	Rice seeds, after space flight and low dose heavy ion radiation treatment were cultured on ground. Leaves of the mature plants were obtained for examination of genomic/epigenomic mutations by using amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) method, respectively. The mutation sites were identified by fragment recovery and sequencing. The heritability of the mutations was detected in the next generation. Results showed that both space flight and low dose heavy ion radiation can induce significant alterations on rice genome and epigenome (P<0.05). For both genetic and epigenetic assays, while there was no significant difference in mutation rates and their ability to be inherited to the next generation, the site of mutations differed between the space flight and radiation treated groups. More than 50% of the mutation sites were shared by two radiation treated groups, radiated with different LET value and dose, while only about 20% of the mutation sites were shared by space flight group and radiation treated group. Moreover, in space flight group, we found that DNA methylation changes were more prone to occur on CNG sequence than CG sequence. Sequencing results proved that both space flight and heavy ion radiation induced mutations were widely spread on rice genome including coding region and repeated region. Our study described and compared the characters of space flight and low dose heavy ion radiation induced genomic/epigenomic mutations. Our data revealed the mechanisms of application of space environment for mutagenesis and crop breeding. Furthermore, this work implicated that the nature of mutations induced under space flight conditions may involve factors beyond ion radiation.
Spaceflight Study	International Space Station (ISS)	SLID-180	Gene expression of rice seeds surviving 13- and 20-month exposure to space environment	13 Months or 20 Months	Grain	Rice seeds were exposed outside of the international space station to assess the risk of space environment exposure on gene expression associated with seed germination. The germination percentages of the space-stored and ground-stored seeds exposed for 13 months were 48 and 96% respectively. Those for 20 months were 7 and 76%, respectively. Germination was defined 3 days after imbibition, except for the space-stored seeds exposed for 20 months, which germinated 5 days after imbibition. Subsequent RNA-seq analyses of the dry seeds, germinated seeds, and roots and shoots of seedlings revealed that the mutation rates of mRNA sequences were not significantly different between space-stored and ground-stored samples exposed for 13 months and 20 months. In all, 4 and 16 transcripts of glycolysis-related genes were increased in the germinated seeds after 13-month and 20-month exposure, respectively. Also, 2 and 39 transcripts of long-lived mRNA required for germination were decreased more than 2-fold in the dry seeds after 13-month and 20-month exposure, respectively. These results suggest that damage to long-lived mRNA in seeds by a space environment delays and reduces germination.
Spaceflight Study	SJ-10 Returning Satellite	SLID-181	Proteomic analysis in different development stages on SP0 generation of rice seeds after space flight	12.5 Days	Grain	The space biological effects of plants will drive the development of aerospace science and breeding science. The aim of this study is to reveal changes in the proteome of contemporary plants at different growth and development stages after space flight of rice seeds. We carried the rice seeds (DN416) through the SJ-10 returning satellite and returned to the ground for planting to the three-leaf stage (TLP) and tillering stage (TS) after a 12.5-day orbital flight. We found that the space flight caused the rice germination rate, the TLP plant height, and the number of tillers in the TS decreased by 11.64%, 9.75%, and 9.80%, respectively. In addition, the treatment group ROS and MDA level increased in the TLP and TS. The abundance patterns of proteins in these leaves identified 214 proteins in the TLP and 286 in the TS leaves that were markedly changed. Moreover, our study identified D14 proteins that control plant height and tiller. Our results show that the space environment may affect the downstream signaling mechanism by regulating the level of ROS in the body to achieve a response to the space environment. Meanwhile, the space environment may affect the plant height and tiller of rice by altering the expression of D14 protein and hormone-regulated proteins. Our results reveal changes in the proteome of different growth stages of rice plants, and also reveal the molecular mechanism of space environment regulation of rice plant height and tiller, which provides a new direction for further understanding of space biological effects and space mutation breeding.
Spaceflight Study	Recoverable Satellite JB-1	SLID-182	Space environment induced mutations prefer to occur at polymorphic sites of rice genomes	15 Days	Grain	To explore the genomic characteristics of rice mutants induced by space environment, space-induced mutants 971-5, 972-4, and R955, which acquired new traits after space flight such as increased yield, reduced resistance to rice blast, and semi-dwarfism compared with their on-ground controls, 971ck, 972ck, and Bing95-503, respectively, together with other 8 japonica and 3 indica rice varieties, 17 in total, were analyzed by amplified fragment length polymorphism (AFLP) method. We chose 16 AFLP primer-pairs which generated a total of 1251 sites, of which 745 (59.6%) were polymorphic over all the genotypes. With the 16 pairs of primer combinations, 54 space-induced mutation sites were observed in 971-5, 86 in 972-4, and 5 in R955 compared to their controls, and the mutation rates were 4.3%, 6.9% and 0.4%, respectively. Interestingly, 75.9%, 84.9% and 100% of the mutation sites identified in 971-5, 972-4, and R955 occurred in polymorphic sites. This result suggests that the space environment preferentially induced mutations at polymorphic sites in rice genomes and might share a common mechanism with other types of mutagens. It also implies that polymorphic sites in genomes are potential “hotspots” for mutations induced by the space environment.
Spaceflight Study	JB-1 Recoverable Satellite	SLID-183	Transcriptomic analyses of space-induced rice mutants with enhanced susceptibility to rice blast	15 Days	Grain	Mutagenic factors of the space environment influence organisms in different aspects. To elucidate the transcriptomic effects of space flight, a space flight-induced rice mutant, 972-4, and its on-ground control, 972ck, were inoculated with rice blast pathogens. Compared to the control, the mutant exhibited reduced resistance to the rice blast pathogen CH45. Microarray technique was employed to analyze affected genes and revealed that 481 genes were expressed at higher levels in the mutant strain and 188 genes were expressed at higher levels in the control strain under normal growth conditions, indicating that transcriptomic changes of rice seeds are induced by the space environment. After inoculation with the rice blast pathogen CH45, however, 2680 genes were differentially expressed in 972ck and 1863 genes were differentially expressed in 972-4. In addition, disease evaluation indicated that the control strain 972ck is more resistant to the rice blast pathogen CH45 than mutant strain 972-4. In addition, genes in both strains that were co-regulated after blast inoculation account for only 36.8% and 53.3% of the genes expressed in 972ck and 972-4, respectively. A large percentage of blast-regulated genes were not consistently expressed in 972-4 and 972ck, and the mutant and control strains exhibit different gene expression patterns after blast inoculation. Interestingly, 84 genes constitutively expressed higher in 972ck were up-regulated by blast inoculation, and 105 genes that were expressed at constitutively higher levels in 972-4 were down-regulated by blast inoculation. Of the differentially expressed, 7 encoded genes associated with pathogen resistance. Taken together, our results suggest that gene expression patterns are different between a space flight-induced rice mutant and its on-ground control, and the differential expression of resistance genes may be a potential mechanism that modulates the resistance of 972-4 to rice blast. Our results also suggest that the rice plants are suitable plant models for further research of the effects of the space environment on gene expression and function.
Spaceflight Study	Chinese Recoverable Satellite, JB-1	SLID-184	Proteomic analysis of high yield rice variety mutated from spaceflight	15 Days	Grain	Seeds of pure rice varieties were flown on Chinese recoverable satellite, JB-1, for a 15-day flight in 1996. Many mutant rice varieties with various phenotypes were generated after continuous selection and breeding. Among the mutants, a variety 971-5 showed a significant increase in grain yield compared to its control (971ck). In this study, proteomic analysis of both mutant variety 971-5 and control variety 971ck were carried out to investigate the changes of protein expression level in their leaves at three different growth stages (early and middle stage of tillering, and booting stage). Results showed that (1) almost all differentially expressed proteins were down-regulated in 971-5 with only one exception, (2) the percentages of differentially expressed proteins were 3.1%, 2.1% and 3.1% at the three stages, respectively, and (3) one protein showed a significant alteration in its molecular weight (MW). These data demonstrated that the space environment can alter the expression level of rice proteins both quantitatively and qualitatively.
Spaceflight Study	Shenzhou 3 Space Flight	SLID-185	Characteristics of phenotype and genetic mutations in rice after spaceflight	6 Days	Grain	To investigate the mechanism of spaceflight induced mutations, seeds of 11 pure rice varieties carried by Shenzhou-3 spaceship of China in 2002 for six-day flight were planted and investigated. Results showed that mutations could be induced in the first generation (M1). Five tall mutants were found in DongnongV7 variety, and the average height of the mutants was 31% taller than that of the control. Other traits such as the panicle length were also remarkably different from the control. In the second generation (M2), various changes of traits were observed in all 11 varieties, including the height, heading date, leaf color, leaf shape, flag leaf angle, awns, panicle length, panicle type, rice shape (length–width ratio), and maturity. The mutation rate for the changes of the plant height and of the rice color (purple) varied from 0.05% to 0.52% among ten varieties except Xixuan-1. Changes of the height, fresh weight, dry weight, and culm width of the five DongnongV7 tall mutants were observed in the progeny individually. By using the AFLP (amplified fragment length polymorphism) method, 21 pairs of primers were employed and the mutated loci rate of the genome in 10 M2 mutants from 10 varieties was found between 1.7% and 6.2%. In the third generation (M3), many traits, such as the awn length, main panicle exertion date and plant height, were still segregated widely and diversely. In addition, the leaf color and awn color varied in the progenies of purple rice mutants. Our study suggested that spaceflight induced mutations were dependent on different rice varieties.
Spaceflight Study	Shenzhou 4 Space Flight	SLID-186	A Comparative Study on Mutagenic Effects of Space Flight and Irradiation of γ-rays on Rice	6.75 Days	Grain	The experiment was conducted to study the mutagenic effects of space environment on seedling growth in M1 generation and plant height and heading date in M2 generation in rice. Two types of lines of growth promotion (GP) and growth suppression (GS) were selected from the recombinant inbred lines (RILs) derived from a cross between a sensitive japonica Lemont and a nonsensitive indica Teqing to compare the mutation frequency and mutagenic efficiency for plant height and heading date in the M2 generation. Space environment resulted in 34.9% higher seedling height (SH) in the GP group than in the control, and 39.1% lower in the GS group than in the control, and there was no difference in seed fertility (SF) between the two groups. In M2, mutants of plant height and heading date can be induced by space treatment in both the two groups with lower mutation frequency and mutagenic efficiency in the GP group than in the GS group. There were no significant differences in the physiological damages in M1 between the two groups after γ-rays irradiation treatment. Mutation frequency and mutagenic efficiency of heading date in M2 were higher in the GS group than in the GP group, and the opposite was true for mutagenic efficiency of plant height although the mutation frequency varied between the two groups. The mutation frequencies of plant height and heading date induced by space environment were obviously lower than those by γ-rays irradiation, but the mutagenic efficiency was the opposite for most of the traits. For the GP and GS of seedlings induced by space environment, the GS had higher mutation frequency and mutagenic efficiency for plant height and heading date than the GP in M2.
Spaceflight Study	FOTON-M2 Satellite	SLID-187	Xylem development and cell wall changes of soybean seedlings grown in space	5 Days	Grain	Background and aims: Plants growing in altered gravity conditions encounter changes in vascular development and cell wall deposition. The aim of this study was to investigate xylem anatomy and arrangement of cellulose microfibrils in vessel walls of different organs of soybean seedlings grown in Space. Methods: Seeds germinated and seedlings grew for 5 d in Space during the Foton-M2 mission. The environmental conditions, other than gravity, of the ground control repeated those experienced in orbit. The seedlings developed in space were compared with those of the control test on the basis of numerous anatomical and ultrastructural parameters such as number of veins, size and shape of vessel lumens, thickness of cell walls and deposition of cellulose microfibrils. Key results: Observations made with light, fluorescence and transmission electron microscopy, together with the quantification of the structural features through digital image analysis, showed that the alterations due to microgravity do not occur at the same level in the various organs of soybean seedlings. The modifications induced by microgravity or by the indirect effect of space-flight conditions, became conspicuous only in developing vessels at the ultrastructural level. The results suggested that the orientation of microfibrils and their assembly in developing vessels are perturbed by microgravity at the beginning of wall deposition, while they are still able to orient and arrange in thicker and ordered structures at later stages of secondary wall deposition. Conclusions: The process of proper cell-wall building, although not prevented, is perturbed in Space at the early stage of development. This would explain the almost unaltered anatomy of mature structures, accompanied by a slower growth observed in seedlings grown in Space than on Earth.
Spaceflight Study	STS-87	SLID-188	Growth patterns for etiolated soybeans germinated under spaceflight conditions	15 Days	Grain	In the GENEX (GENe EXpression) spaceflight experiment (flown on STS-87), six surface sterilized soybean seeds (Glycine max cv McCall) were inserted into each of 32 autoclaved plastic seed growth pouches containing an inner germination paper sleeve (for a total of 192 seeds). The pouches were stowed within a mid-deck locker until Mission Flight Day 10, at which time an astronaut added water to initiate the process of seed germination on-orbit and subsequently transferred them to four light-tight aluminum canisters called BRIC-60s (Biological Research In Canisters). We report here on the morphological characteristics of: (1) the recovered flight plants (N = 177), (2) the corresponding ground control population (N = 183), plus (3) additional controls grown on the ground under clinostat conditions (N = 93). No significant morphological differences were found between the flight, ground control and clinorotated treatments for either the cotyledons or hypocotyls. There were, however, significantly longer primary roots produced in the flight population relative to the ground control population, which in turn had significantly longer primary roots than the clinorotated population. This same pattern was observed relative to the production of lateral roots (flight > control > clinorotated). Taken together with previous literature reports, we believe that there is now sufficient evidence to conclude that plants grown under conditions of microgravity will generally exhibit enhanced root production relative to their ground control counterparts. Some causes underlying this phenomenon are speculated on.
Spaceflight Study	Russian Space Station Mir	SLID-189	Comparative floral development of Mir-grown and ethylene-treated, earth-grown Super Dwarf wheat	123 Days	Grain	To study plant growth in microgravity, we grew Super Dwarf wheat (Triticum aestivum L.) in the Svet growth chamber onboard the orbiting Russian space station, Mir, and in identical ground control units at the Institute of BioMedical Problems in Moscow, Russia. Seedling emergence was 56% and 73% in the two root-module compartments on Mir and 75% and 90% on earth. Growth was vigorous (produced ca. 1 kg dry mass), and individual plants produced 5 to 8 tillers on Mir compared with 3 to 5 on earth-grown controls. Upon harvest in space and return to earth, however, all inflorescences of the flight-grown plants were sterile. To ascertain if Super Dwarf wheat responded to the 1.1 to 1.7 micromoles mol-1 atmospheric levels of ethylene measured on the Mir prior to and during flowering, plants on earth were exposed to 0, 1, 3, 10, and 20 micromoles mol-1 of ethylene gas and 1200 micromoles mol-1 CO2 from 7 d after emergence to maturity. As in our Mir wheat, plant height, awn length, and the flag leaf were significantly shorter in the ethylene-exposed plants than in controls; inflorescences also exhibited 100% sterility. Scanning-electron-microscopic (SEM) examination of florets from Mir-grown and ethylene-treated, earth-grown plants showed that development ceased prior to anthesis, and the anthers did not dehisce. Laser scanning confocal microscopic (LSCM) examination of pollen grains from Mir and ethylene-treated plants on earth exhibited zero, one, and occasionally two, but rarely three nuclei; pollen produced in the absence of ethylene was always trinucleate, the normal condition. The scarcity of trinucleate pollen, abrupt cessation of floret development prior to anthesis, and excess tillering in wheat plants on Mir and in ethylene-containing atmospheres on earth build a strong case for the ethylene on Mir as the agent for the induced male sterility and other symptoms, rather than microgravity.
Spaceflight Study	Shenzhou 8 Spacecraft	SLID-190	Effects of spaceflight and simulated microgravity on cell sub-microstructure and antioxidant enzyme activity in tomato	17 Days	Fruit	Controlled ecological life support systems provide food, air, water, and other basic living resources for crew members on long-duration spaceflight missions. Plants are an important basic requirement of these systems and their biological characteristics in space have very high research value. Based on experiments of spaceflight in Shenzhou 8 spacecraft and simulating microgravity effects on three-dimensional (3-D) clinostat, the biological characteristics of tomato’s leaf cell sub-microstructure and antioxidant enzyme activities were studied and compared in this work. Results showed that leaf cell sub-microstructure of the tomato samples experiencing spaceflight had more changes than that of the samples processed by simulated microgravity effects, and both peroxidase (POD) and superoxide dismutase (SOD) activities increase obviously in both the environments.
Spaceflight Study	International Space Station (ISS)	SLID-191	Physical, chemical and biological characteristics of space flown tomato ( Lycopersicum esculentum ) seeds	3 Months	Fruit	Several research showed that space flown treated seeds had a different characteristic with that of ground treated seed, which eventually produced a different characteristic of growth and productivity. Research was conducted to study the physical, chemical and biological properties, such as the rate of germination and the growth of tomato (Lycopersicum esculentum) space flown seeds compared with that of control one. Observations of physical properties using a SEM showed that there were pores on the surface of some tomato space flown seeds. Observations using a stereo and inverted microscope showed that the coat layer of space flown seeds was thinner than control seeds. The total mineral content in the control seeds (22.88%) was averagely higher than space flown seeds (18.66%), but the average carbohydrate content in control seed was lower (15.2 ± 2.79%) than the space flown seeds (9.02 ± 1.87%). The level of auxin (IAA) of control seeds (142 ± 6.88 ppm) was averagely lower than the space flown seeds (414 ± 78.84 ppm), whereas the level of cytokinins (zeatin) for the control seeds (381 ± 68.86 ppm) was higher than the space flown seeds (68 ± 9.53 ppm), and the level of gibberellin (GA3) for the control seeds (335 ± 10.7 ppm) was higher than the space flown seeds (184 ± 7.4 ppm). The results of this study showed that the physical and chemical properties of tomato space flown seeds were generally different compare with that to control seeds, so that it might also be resulted in different germination and growth characteristic. The germination test showed that space flown seeds had lower germination rate compare to control. The growth pattern indicated that planted space flown seeds generally grew better than control. However, those data were more homogenous in control seeds compare to that in space flown tomato seeds.
Spaceflight Study	Recoverable Chinese Satellite (FSW-2-3)	SLID-192	The effects of spaceflight on soluble protein, isoperoxidase, and genomic DNA in ural licorice (Glycyrrhiza uralensis fisch.)	15 Days	n.a.	We investigated the effects of weightlessness, and ionizing radiation plus weightlessness, on changes in the levels of soluble protein, isoperoxidase, and genomic DNA, respectively, in a medicinal plant-ural licorice (Glycyrrhiza uralensis)-after a 15-d spaceflight in a recoverable satellite. Both the weightlessness samples (Ws) and the ionizing radiation plus weightlessness samples (IR/Ws) showed increases in soluble protein content or peroxidase activity, compared with the ground control (Gc). Moreover, the increased isoperoxidase activity for the IR/Ws group was significantly greater than for the Ws, compared with the controls. Likewise, distinctive RAPD profiles were generated among the Ws, the IR/Ws, and the Gc. The Ws and IR/Ws yielded 66 and 78 polymorphic RAPD fragments, respectively, based on bulk template DNA, along with 19 selected primers. Therefore, weightlessness alone can trigger genomic alterations, to some extent, and may even result in modulation of gene expression, whereas ionizing radiation would probably enhance the effect of weightlessness.
Spaceflight Study	n.a.	SLID-193	Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta	n.a.	Grain	Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs) and proteins (DEPs), respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.
Spaceflight Study	Shijian-8 Satellite	SLID-194	RNAseq analysis reveals pathways and candidate genes associated with salinity tolerance in a spaceflight-induced wheat mutant	15 Days	Grain	Salinity stress has become an increasing threat to food security worldwide and elucidation of the mechanism for salinity tolerance is of great significance. Induced mutation, especially spaceflight mutagenesis, is one important method for crop breeding. In this study, we show that a spaceflight-induced wheat mutant, named salinity tolerance 1 (st1), is a salinity-tolerant line. We report the characteristics of transcriptomic sequence variation induced by spaceflight, and show that mutations in genes associated with sodium ion transport may directly contribute to salinity tolerance in st1. Furthermore, GO and KEGG enrichment analysis of differentially expressed genes (DEGs) between salinity-treated st1 and wild type suggested that the homeostasis of oxidation-reduction process is important for salt tolerance in st1. Through KEGG pathway analysis, "Butanoate metabolism" was identified as a new pathway for salinity responses. Additionally, key genes for salinity tolerance, such as genes encoding arginine decarboxylase, polyamine oxidase, hormones-related, were not only salt-induced in st1 but also showed higher expression in salt-treated st1 compared with salt-treated WT, indicating that these genes may play important roles in salinity tolerance in st1. This study presents valuable genetic resources for studies on transcriptome variation caused by induced mutation and the identification of salt tolerance genes in crops.
Spaceflight Study	Shijian-8 Recoverable Satellite	SLID-195	Characterization of a Novel Chlorophyll-Deficient Mutant Mt6172 in Wheat	15 Days	Grain	Identification of new chlorophyll-deficient mutants will provide materials for studying signaling components and pathways between plastid and nucleus. A novel chlorophyll-deficient mutant, named Mt6172, was obtained by spaceflight environment induction. Genetic analysis showed that its inheritance was controlled by nuclear and cytoplamic genes. Leaf color of its self-fertilized progenies was albino, narrow-white striped, or green. Only a few cells with abnormal chloroplasts were observed in albino plants and white section of narrow-white striped plants. These chloroplasts had obvious flaws in inner structure, and granum lamellae was extremely disordered. The narrow-white striped plants were characterized with green-and-narrow-white striped leaves, and the width of stripes between different plants was even, their plant height, number of productive tillers, and 1000-grain weight were lower than those of the wild type. The narrow-white striped plants and the wild type had significant difference in the value of potential activity of photosystem II at all tested stages. At elongation stage, which was impacted the most seriously, effective quantum yield significantly decreased, whereas the energy for photoprotection and photodamage significantly increased. Under different photosynthetic active radiation conditions, changes of electron transport rate, photochemical dissipation, and effective quantum yield were different, electron transport rate was more impacted than other parameters. Therefore, the leaf morphology and inheritance of mutant Mt6172 was different from the other reported mutants in wheat, and it was a novel mutant of chlorophyll deficiency.
Spaceflight Study	n.a.	SLID-196	A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings	n.a.	Vegetable	Seedlings of Cucurbitaceae plants form a protuberance, termed peg, on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction.
Spaceflight Study	International Space Station (ISS), (launched on STS-132 space shuttle Atlantis, returned on space shuttle Discovery (STS-133) )	SLID-197	Gravitropism interferes with hydrotropism via counteracting auxin dynamics in cucumber roots: clinorotation and spaceflight experiments	5 Months	Vegetable	Roots of land plants show gravitropism and hydrotropism in response to gravity and moisture gradients, respectively, for controlling their growth orientation. Gravitropism interferes with hydrotropism, although the mechanistic aspects are poorly understood. Here, we differentiated hydrotropism from gravitropism in cucumber roots by conducting clinorotation and spaceflight experiments. We also compared mechanisms regulating hydrotropism and auxin-regulated gravitropism. Clinorotated or microgravity (μG)-grown cucumber seedling roots hydrotropically bent toward wet substrate in the presence of moisture gradients, but they grew straight in the direction of normal gravitational force at the Earth's surface (1G) on the ground or centrifuge-generated 1G in space. The roots appeared to become hydrotropically more sensitive to moisture gradients under μG conditions in space. Auxin transport inhibitors significantly reduced the hydrotropic response of clinorotated seedling roots. The auxin efflux protein CsPIN5 was differentially expressed in roots of both clinorotated and μG-grown seedlings; with higher expression in the high-humidity (concave) side than the low-humidity (convex) side of hydrotropically responding roots. Our results suggest that roots become hydrotropically sensitive in μG, and CsPIN5-mediated auxin transport has an important role in inducing root hydrotropism. Thus, hydrotropic and gravitropic responses in cucumber roots may compete via differential auxin dynamics established in response to moisture gradients and gravity.
Spaceflight Study	International Space Station (ISS), (launched on STS-133, returned on STS-135)	SLID-198	The gravity-induced re-localization of auxin efflux carrier CsPIN1 in cucumber seedlings: spaceflight experiments for immunohistochemical microscopy	n.a.	Vegetable	Reorientation of cucumber seedlings induces re-localization of CsPIN1 auxin efflux carriers in endodermal cells of the transition zone between hypocotyl and roots. This study examined whether the re-localization of CsPIN1 was due to the graviresponse. Immunohistochemical analysis indicated that, when cucumber seedlings were grown entirely under microgravity conditions in space, CsPIN1 in endodermal cells was mainly localized to the cell side parallel to the minor axis of the elliptic cross-section of the transition zone. However, when cucumber seeds were germinated in microgravity for 24 h and then exposed to 1g centrifugation in a direction crosswise to the seedling axis for 2 h in space, CsPIN1 was re-localized to the bottom of endodermal cells of the transition zone. These results reveal that the localization of CsPIN1 in endodermal cells changes in response to gravity. Furthermore, our results suggest that the endodermal cell layer becomes a canal by which auxin is laterally transported from the upper to the lower flank in response to gravity. The graviresponse-regulated re-localization of CsPIN1 could be responsible for the decrease in auxin level, and thus for the suppression of peg formation, on the upper side of the transition zone in horizontally placed seedlings of cucumber.
Spaceflight Study	International Space Station (ISS) Expedition-14	SLID-199	Molecular Cytogenetics of Pisum sativum L. Grown under Spaceflight-Related Stress	78 Days	Vegetable	The ontogenesis and reproduction of plants cultivated aboard a spacecraft occur inside the unique closed ecological system wherein plants are subjected to serious abiotic stresses. For the first time, a comparative molecular cytogenetic analysis of Pisum sativum L. (Fabaceae) grown on board the RS ISS during the Expedition-14 and Expedition-16 and also plants of their succeeding (F1 and F2) generations cultivated on Earth was performed in order to reveal possible structural chromosome changes in the pea genome. The karyotypes of these plants were studied by multicolour fluorescence in situ hybridization (FISH) with five different repeated DNA sequences (45S rDNA, 5S rDNA, PisTR-B/1, microsatellite motifs (AG)12, and (GAA)9) as probes. A chromosome aberration was revealed in one F1 plant. Significant changes in distribution of the examined repeated DNAs in karyotypes of the "space grown" pea plants as well as in F1 and F2 plants cultivated on Earth were not observed if compared with control plants. Additional oligo-(GAA)9 sites were detected on chromosomes 6 and 7 in karyotypes of F1 and F2 plants. The detected changes might be related to intraspecific genomic polymorphism or plant cell adaptive responses to spaceflight-related stress factors. Our findings suggest that, despite gradual total trace contamination of the atmosphere on board the ISS associated with the extension of the space station operating life, exposure to the space environment did not induce serious chromosome reorganizations in genomes of the "space grown" pea plants and generations of these plants cultivated on Earth.
Spaceflight Study	International Space Station (ISS) Expedition-16	SLID-199	Molecular Cytogenetics of Pisum sativum L. Grown under Spaceflight-Related Stress	79 Days	Vegetable	The ontogenesis and reproduction of plants cultivated aboard a spacecraft occur inside the unique closed ecological system wherein plants are subjected to serious abiotic stresses. For the first time, a comparative molecular cytogenetic analysis of Pisum sativum L. (Fabaceae) grown on board the RS ISS during the Expedition-14 and Expedition-16 and also plants of their succeeding (F1 and F2) generations cultivated on Earth was performed in order to reveal possible structural chromosome changes in the pea genome. The karyotypes of these plants were studied by multicolour fluorescence in situ hybridization (FISH) with five different repeated DNA sequences (45S rDNA, 5S rDNA, PisTR-B/1, microsatellite motifs (AG)12, and (GAA)9) as probes. A chromosome aberration was revealed in one F1 plant. Significant changes in distribution of the examined repeated DNAs in karyotypes of the "space grown" pea plants as well as in F1 and F2 plants cultivated on Earth were not observed if compared with control plants. Additional oligo-(GAA)9 sites were detected on chromosomes 6 and 7 in karyotypes of F1 and F2 plants. The detected changes might be related to intraspecific genomic polymorphism or plant cell adaptive responses to spaceflight-related stress factors. Our findings suggest that, despite gradual total trace contamination of the atmosphere on board the ISS associated with the extension of the space station operating life, exposure to the space environment did not induce serious chromosome reorganizations in genomes of the "space grown" pea plants and generations of these plants cultivated on Earth.
Spaceflight Study	Long-March-2 Spaceship	SLID-200	Spaceflight induces both transient and heritable alterations in DNA methylation and gene expression in rice (Oryza sativa L.)	18 Days	Grain	Spaceflight represents a complex environmental condition in which several interacting factors such as cosmic radiation, microgravity and space magnetic fields are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic as well as external perturbations, it is conceivable that epigenetic markers like DNA methylation may undergo alterations in response to spaceflight. We report here that extensive alteration in both DNA methylation and gene expression occurred in rice plants subjected to a spaceflight, as revealed by a set of characterized sequences including 6 transposable elements (TEs) and 11 cellular genes. We found that several features characterize the alterations: (1) All detected alterations are hypermethylation events; (2) whereas alteration in both CG and CNG methylation occurred in the TEs, only alteration in CNG methylation occurred in the cellular genes; (3) alteration in expression includes both up- and down-regulations, which did not show a general correlation with alteration in methylation; (4) altered methylation patterns in both TEs and cellular genes are heritable to progenies at variable frequencies; however, stochastic reversion to wild-type patterns and further de novo changes in progenies are also apparent; and (5) the altered expression states in both TEs and cellular genes are also heritable to selfed progenies but with markedly lower transmission frequencies than altered DNA methylation states. Furthermore, we found that a set of genes encoding for the various putative DNA methyltransferases, 5-methylcytosine DNA glycosylases, the SWI/SNF chromatin remodeller (DDM1) and siRNA-related proteins are extremely sensitive to perturbation by spaceflight, which might be an underlying cause for the altered methylation patterns in the space-flown plants. We discuss implications of spaceflight-induced epigenetic variations with regard to health safety issues of spaceship crews and potentiality of spaceflight as a means for mutagenesis in crop breeding.
Spaceflight Study	Spaceship Long-March-2	SLID-201	The spaceflight environment can induce transpositional activation of multiple endogenous transposable elements in a genotype-dependent manner in rice	18 Days	Grain	Spaceflight represents a unique environmental condition whereby dysregulated gene expression and genomic instability can be provoked. However, detailed molecular characterization of the nature of genetic changes induced by spaceflight is yet to be documented in a higher eukaryote. Transposable elements (TEs) are ubiquitous and have played a significant role in genome evolution. Mounting evidence indicates that TEs constitute the genomic fraction that is susceptible and responsive to environmental perturbations, and hence, most likely manifesting genetic instabilities in times of stress. A predominant means for TEs to cause genetic instability is via their transpositional activation. Here we show that spaceflight has induced transposition of several endogenous TEs in rice, which belong to distinct classes including the miniature inverted terminal repeat TEs (MITEs) and long-terminal repeat (LTR) retrotransposons. Of three rice lines studied, transposition of TEs were detected in the plants germinated from space-flown dry seeds of two lines (RZ1 and RZ35), which are genetically homogeneous and stabilized recombinant inbred lines (RILs) derived from a pure-line rice cultivar, Matsumae. In contrast, the TEs remained immobile in plants derived from space-flown seeds of Matsumae itself, indicating a genotype-dependent manner of TE transposition under the spaceflight environment. Further examination showed that at least in some cases transposition of TEs was associated with cytosine demethylation within the elements. Moreover, the spaceflight-induced TE activity was heritable to organismal progenies. Thus, our results implicate that the spaceflight environment represents a potent mutagenic environment that can cause genetic instabilities by eliciting transposition of otherwise totally quiescent endogenous TEs in a higher eukaryote.
Spaceflight Study	Long-March-2 Spaceship	SLID-202	Spaceflight-induced genetic and epigenetic changes in the rice (Oryza sativa L.) genome are independent of each other	18 Days	Grain	An array of studies have reported that the spaceflight environment is mutagenic and may induce phenotypic and genetic changes in diverse organisms. We reported recently that in at least some plant species (e.g., rice) the spaceflight environment can be particularly potent in generating heritable epigenetic changes in the form of altered cytosine methylation patterns and activation of transposable elements. To further study the issue of spaceflight-induced genomic instability, and in particular to test whether the incurred genetic and epigenetic changes are connected or independent of each other, we performed the present study. We subjected seeds of the standard laboratory rice (Oryza sativa L.) cultivar Nipponbare to a spaceflight in the spaceship Long March 2 for 18 days. We then investigated the genetic and DNA methylation stabilities of 11 randomly selected plants germinated from the spaceflown seeds by using two kinds of DNA markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP). For AFLP, by using 15 primer combinations, we assessed 460 genomic loci and found that the frequencies of genetic changes across the 11 plants ranged from 0.7% to 6.7% with an average frequency of 3.5%. For MSAP, by using 14 primer combinations, we assessed 467 loci and detected the occurrence of four major types of cytosine methylation alterations at the CCGG sites, namely CG or CNG hypomethylation and CG or CNG hypermethylation. Collectively, the frequencies of the two kinds of hypermethylation, CG (1.95%) and CNG (1.44%), are about two times higher than those of the two kinds of hypomethylation, CG (0.76%) and CNG (0.80%), though different plants showed variable frequencies for each type of alteration. Further analysis suggested that both the genetic and cytosine methylation changes manifested apparent mutational bias towards specific genomic regions, but the two kinds of instabilities are independent of each other based on correlation analysis.
Spaceflight Study	International Space Station (ISS)	SLID-203	Rocket Science: The Effect of Spaceflight on Germination Physiology, Ageing, and Transcriptome of Eruca sativa Seeds	6 Months	Vegetable	In the 'Rocket Science' project, storage of Eruca sativa (salad rocket) seeds for six months on board the International Space Station resulted in delayed seedling establishment. Here we investigated the physiological and molecular mechanisms underpinning the spaceflight effects on dry seeds. We found that 'Space' seed germination vigor was reduced, and ageing sensitivity increased, but the spaceflight did not compromise seed viability and the development of normal seedlings. Comparative analysis of the transcriptomes (using RNAseq) in dry seeds and upon controlled artificial ageing treatment (CAAT) revealed differentially expressed genes (DEGs) associated with spaceflight and ageing. DEG categories enriched by spaceflight and CAAT included transcription and translation with reduced transcript abundances for 40S and 60S ribosomal subunit genes. Among the 'spaceflight-up' DEGs were heat shock proteins (HSPs), DNAJ-related chaperones, a heat shock factor (HSFA7a-like), and components of several DNA repair pathways (e.g., ATM, DNA ligase 1). The 'response to radiation' category was especially enriched in 'spaceflight-up' DEGs including HSPs, catalases, and the transcription factor HY5. The major finding from the physiological and transcriptome analysis is that spaceflight causes vigor loss and partial ageing during air-dry seed storage, for which space environmental factors and consequences for seed storage during spaceflights are discussed.
Spaceflight Study	Shenzhou 4 Space Flight	SLID-204	Morphological variation of mutant sunflowers (Helianthus annuus) induced by space flight and their genetic background detection by SSR primers	n.a.	Oil crop	After sunflower seeds were exposed to space conditions, various mutant plants were screened from the descendent plants. The morphological characters of plants changed in flower color from golden to yellow, light yellow, or even to yellowish green. The ligulate petals of the unisexual floret broadened, or became thin, while the short tubular petals of bisexual floret elongated to some extent, or even turned into semi-ligulate petals or ligulate petals, making the phenotype of the whole inflorescence like a chrysanthemum. The shape and thickness of leaves varied in some of these plants. Absolute sterile plants in mutant plants were found to possess neither normal bisexual florets nor unisexual florets, but the "pseudo-floret" only consisted of pieces of shield-like bracts on protuberant floral disc. Thirty-five pairs of simple sequence of repeat primers were used to detect the genetic variation of the plants, and the results showed that only a variation was tested in the mutant plants from 4 primers. The different PCR products obtained were extracted for sequencing and alignment analysis, and the aligned results showed that the DNA sequence changed by deletion, insertion and replacement that occurred at some sites. The results proved the high mutagenic efficacy of space flight, and ways of DNA transformation due to space conditions.
Spaceflight Study	Russian Space Station Mir	SLID-205	Analysis of the Spaceflight Effects on Growth and Development of Super Dwarf Wheat Grown on the Space Station Mir	123 Days	Grain	The hypothesis being tested is that Super Dwarf wheat, Triticum aestivum L., plants in the Svet Greenhouse onboard the Russian Space Station Mir will complete a life cycle in spaceflight, providing that the environmental conditions necessary for adequate growth on Earth are supplied. Twenty six seeds of wheat were planted in each of 2 rows of 2 root compartments for a total of 104 seeds in Svet. Germination rate at 7 d was 56 and 73% on Mir and 75 and 90% in ground-based controls. Plants were grown throughout the whole cycle of ontogenesis (123 d) with samples gathered at different times to validate the morphological and reproductive stages of the plants. Young plants showed vigorous early seedling growth, with large biomass production, including the formation of 280 floral spikes. Upon return to Earth, comparative analyses showed that the number of tillers and flowers per spikelet were 63.2% and 40% greater, respectively, in Mir-grown plants than in the controls. By contrast, the stem length (52.4%), spike mass (49.2%) and length (23.1%), awn length (75.7%), number of spikelets per spike (42.8%) and number of seeds per spike (100% sterile) from Mir-grown plants were substantially less than the controls. Distribution of moisture and roots throughout the substrate was very good. All florets on Mir-grown spikes ceased development at the same stage of ontogeny. Lack of caryopses formation was attributed to male sterility occurring at different stages of staminal development. Anthers failed to dehisce and pollen grains were smaller and shriveled compared to the controls, suggesting a chronic stress had occurred in the Svet growth chamber. Recent ground-based studies indicated that ethylene, which was measured at 0.3 to 1.8 mg kg-1 in the Mir, almost certainly could have induced male sterility in the wheat plants grown on the Mir.
Spaceflight Study	Long Duration Exposure Facility (LDEF) Satellite	SLID-206	Effects of Exposure in Space on Tomato Seeds: Photosynthesis, Biomass, and Water Relations of Well-Watered and Drought-Stressed Plants	6 Years	Fruit	Tomato seeds exposed to space conditions for nearly six years on board the Long Duration Exposure Facility (LDEF) satellite were subsequently germinated and the resultant seedlings grown on earth under controlled conditions for analysis. Photosynthesis, biomass, and water relations were compared between mature plants grown from earth-based control seeds and space-exposed seeds under both well-watered and drought-stressed conditions. No consistent significant differences in photosynthesis and water relations were observed in the two sets of plants at any level of drought stress. Fruit production, however, though limited and variable, was significantly greater in plants grown from space-exposed seeds than in plants grown from earth-based seeds. Overall, exposure of seeds to space had only minor effects on the physiology and growth of plants grown from such seed.
Spaceflight Study	China Recovered Satellite	SLID-207	Effect of spaceflight duration of subcellular morphologies and defense enzyme activities in earth-grown tomato seedlings propagated from space-flown seeds	27 Days	Fruit	Subcellular changes and enzymes activities were compared between Earth-grown plants from the original tomato seeds, 6-year long-term flown in the Mir Station and 27-day short-term flown in the satellite. In some first generation Mir-flown plants, the lamellae’s structure of some chloroplasts became curved and loose, and some mitochondrial outer membranes were broken. In some satellite-flown plants, the number of mitochondria increased, the lamellae’s structure of some chloroplasts became curved and loose, and some mitochondrial cristae disappeared. The number of starch grains per chloroplast in first generation, second generation, third generation Mir-flown plants and satellite-flown plants increased significantly compared with the ground control, but the difference between the number of starch grains per chloroplast in Mir-flown plants (include three generations) and satellite-flown plants was not significant. The number of chloroplasts per leaf cell in Mir-flown plants and satellite-flown plants increased significantly compared with the ground control. The activities of three defense enzymes supperoxide dismutase (SOD), peroxidase dismutase (POD) and catalase (CAT) in the satellite-flown plants increased significantly as compared with those of the ground controls and the Mir-flown plants, but the content of malondialdehyde (MDA) decreased significantly. Coefficients of variation of the activities of SOD, POD, CAT and the content of MDA in the satellite-flown plants were lest, and those in the Mir-flown plants were maximum. Coefficient of variation of the activities of SOD, POD, and CAT in the first, second and third generation Mir-flown plants were more than that in the ground control. The activities of defense enzyme in some Mir-flown plants increased significantly.
Spaceflight Study	Mir Space Station	SLID-208	Effects of a spaceflight environment on heritable changes in wheat gene expression	167 Days	Grain	Once it was established that the spaceflight environment was not a drastic impediment to plant growth, a remaining space biology question was whether long-term spaceflight exposure could cause changes in subsequent generations, even if they were returned to a normal Earth environment. In this study, we used a genomic approach to address this question. We tested whether changes in gene expression patterns occur in wheat plants that are several generations removed from growth in space, compared to wheat plants with no spaceflight exposure in their lineage. Wheat flown on Mir for 167 days in 1991 formed viable seeds back on Earth. These seeds were grown on the ground for three additional generations. Gene expression of fourth-generation Mir flight leaves was compared to that of the control leaves by using custom-made wheat microarrays. The data were evaluated using analysis of variance, and transcript abundance of each gene was contrasted among samples with t-tests. After corrections were made for multiple tests, none of the wheat genes represented on the microarrays showed a statistically significant difference in expression between wheat that has spaceflight exposure in their lineage and plants with no spaceflight exposure. This suggests that exposure to the spaceflight environment in low Earth orbit space stations does not cause significant, heritable changes in gene expression patterns in plants.
Spaceflight Study	A Biocosmos Satellite	SLID-209	Effects of space flight on Xenopus laevis larval development	11.5 Days	Animals	Fifty-three fertilized Xenopus laevis embryos at early tail bud stage were launched into orbit aboard a Biocosmos satellite and remained in microgravity for 11.5 days. During this period, the embryos hatched and continued to develop as free-living larvae. Forty-eight individuals survived the mission. Upon recovery these tadpoles had smaller heads/bodies and proportionately longer tails than ground controls. Almost all the flight animals had caudal lordosis and consequently swam in backward somersaults. Compared to ground-based controls, their notochords were significantly larger in cross-sectional area and were deformed. Caudal muscle fibers were less dense and involuted in a fashion indicative of degeneration. In contrast, cranial muscles associated with buccal pumping did not differ between the flight and control animals. Upon landing, the flight larvae were found to be negatively buoyant and lay on the bottom when they were not swimming. They had significantly smaller lungs than controls, suggesting that they had failed to inflate their lungs in microgravity. Additionally, the branchial baskets, gill filters and thymuses all showed signs of retarded development or degeneration. The caudal deformity that we observed in the flight X. laevis has been independently observed in three other space flight experiments where embryos were launched then hatched in space. In contrast, Xenopus larvae from another orbital experiment that were raised from fertilization through hatching in space did not exhibit any caudal abnormalities. These divergent results suggest that either features of the launch itself (i.e., high acceleration and vibration) or an abrupt decrease in gravity during the tail bud stage detrimentally affects musculoskeletal development in anurans.
Spaceflight Study	STS-55	SLID-210	Altered gravitational forces affect the development of the static vestibuloocular reflex in fish (Oreochromis mossambicus)	9 - 10 Days	Animals	Young fish (Oreochromis mossambicus) were exposed to microgravity (micro g) for 9 to 10 days during space missions STS-55 and STS-84, or to hypergravity (hg) for 9 days. Young animals (stages 11-12), which had not yet developed the roll-induced static vestibuloocular reflex (rVOR) at micro g- and hg-onset, and older ones (stages 14-16), which had already developed the rVOR, were used. For several weeks afterwards, the rVOR was recorded after termination of mug and hg. Here are the main results: (1) In the stage 11-12 fish, the rVOR gain (response angle/roll angle) measured for roll angles 15 degrees, 30 degrees, and 45 degrees was not affected by microgravity if animals were rolled from the horizontal to the inclined posture, but was increased significantly if animals were rolled in the opposite manner. The rVOR amplitude (maximal eye movement during a complete 360 degrees roll) of micro g animals increased significantly by 25% compared to 1g controls during the first postflight week, but decreased to the control level during the second postflight week. Microgravity had no effect in stage 14-16 fish on either rVOR gain or amplitude. (2) After 3g exposure, both rVOR gain and amplitude were significantly reduced for both stage 11-12 and stage 15 fish. One g readaptation was completed during the second post-3g week. Hypergravity at 2 or 2.5 g had no effect. (3) Hypergravity at all three levels tested (2g, 2.5g, and 3g) accelerated the morphological development as assessed by external morphological markers. Exposure to micro g- or 3g-periods during an early developmental period modifies the physiological properties of the neuronal network underlying the static rVOR; in susceptible developmental stages, these modifications include sensitization by microgravity and desensitization by hypergravity.
Spaceflight Study	STS-84	SLID-210	Altered gravitational forces affect the development of the static vestibuloocular reflex in fish (Oreochromis mossambicus)	9 Days	Animals	Young fish (Oreochromis mossambicus) were exposed to microgravity (micro g) for 9 to 10 days during space missions STS-55 and STS-84, or to hypergravity (hg) for 9 days. Young animals (stages 11-12), which had not yet developed the roll-induced static vestibuloocular reflex (rVOR) at micro g- and hg-onset, and older ones (stages 14-16), which had already developed the rVOR, were used. For several weeks afterwards, the rVOR was recorded after termination of mug and hg. Here are the main results: (1) In the stage 11-12 fish, the rVOR gain (response angle/roll angle) measured for roll angles 15 degrees, 30 degrees, and 45 degrees was not affected by microgravity if animals were rolled from the horizontal to the inclined posture, but was increased significantly if animals were rolled in the opposite manner. The rVOR amplitude (maximal eye movement during a complete 360 degrees roll) of micro g animals increased significantly by 25% compared to 1g controls during the first postflight week, but decreased to the control level during the second postflight week. Microgravity had no effect in stage 14-16 fish on either rVOR gain or amplitude. (2) After 3g exposure, both rVOR gain and amplitude were significantly reduced for both stage 11-12 and stage 15 fish. One g readaptation was completed during the second post-3g week. Hypergravity at 2 or 2.5 g had no effect. (3) Hypergravity at all three levels tested (2g, 2.5g, and 3g) accelerated the morphological development as assessed by external morphological markers. Exposure to micro g- or 3g-periods during an early developmental period modifies the physiological properties of the neuronal network underlying the static rVOR; in susceptible developmental stages, these modifications include sensitization by microgravity and desensitization by hypergravity.
Spaceflight Study	Mir Space Station (Cassiopée mission)	SLID-211	Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl	16 Days	Animals	The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.
Spaceflight Study	Mir Space Station (Pégase mission)	SLID-211	Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl	21 Days	Animals	The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.
Spaceflight Study	TEXUS-17 Flight	SLID-212	Fertilization of frog eggs on a sounding rocket in space	21 Days	Animals	During the TEXUS-17 flight (April/May 1988) eggs of a higher organism, the anuran amphibian Xenopus laevis, have for the first time been successfully fertilized under microgravity on a Sounding Rocket. This result also implies that Life Sciences Experiments of Short Duration can be carried out on Sounding Rockets. The latter can therefore function as additional carriers for such experiments. Histological sections of the experimental material demonstrated the penetration of sperm into eggs, while SEM analysis revealed the differentiation of characteristic egg surface structures. Our TEXUS-17 experiment convincingly shows that the modified automatic experiment container, originally designed for experimental BR 52NL on the D1-mission, now functions flawlessly. Eight containers were flown in an airtight, well-isolated box (TEM 06-15), and a similar set was activated on Earth, two hours later. The analysis of the biological material is in progress.
Spaceflight Study	German Spacelab Mission D1	SLID-213	Embryogenesis and organogenesis of Carausius morosus under spaceflight conditions	7 Days	Animals	The influence of cosmic radiation and/or microgravity on insect development was studied during the 7 day German Spacelab Mission D1. Eggs of Carausius morosus of five stages differing in sensitivity to radiation and in capacity to regeneration were allowed to continue their development in the BIORACK 22 degrees C incubator, either at microgravity conditions or on the 1 g reference centrifuge. Using the Biostack concept--eggs in monolayers were sandwiched between visual track detectors--and the 1 g reference centrifuge, we were able to separate radiation effects from microgravity effects and also from combined effects of these two factors in space. After retrieval, hatching rates, growth kinetics and anomaly frequencies were determined in the different test samples. The early stages of development turned out to be highly sensitive to single hits of cosmic ray particles as well as to the temporary exposure to microgravity during their development. In some cases, the combined action of radiation and microgravity even amplified the effects exerted by the single parameters of space. Hits by single HZE particles caused early effects, such as body anomalies, as well as late effects, such as retarded growth after hatching. Microgravity exposure lead to a reduced hatching rate. A synergistic action of HZE particle hits and microgravity was established in the unexpectedly high frequency of anomal larvae. However, it cannot be excluded, that cosmic background radiation or low LET HZE particles are also causally involved in damage observed in the microgravity samples.
Spaceflight Study	Russian 10th Biosatellite	SLID-214	Space flight experiment on chinese silkworm on board the russian 10th biosatellite	12 Days	Animals	Space flight experiments on Chinese silkworm (Bombyx mori L.) were conducted on board the Russian 10th Biosatellite for 12 days. The samples included silkworm eggs, larvae, cocoons, pupae and moths. The processes of spinning, cocooning, mating, oviposition, larval hatching, pupation and moth emergence all completed well in space. The following effects of space flight on silkworm development were observed: The times of hatching and oviposition in the flight group were 2 to 3 days earlier than in the control group; the hatching rate of diapause eggs during space flight seemed higher than that of the control group; the life span of 2 of the 7 varieties flown was shortened; genetical variations appeared in 3 varieties. The results showed that the embryonic stage was probably the period most sensitive to the space flight environment.
Spaceflight Study	STS-107	SLID-215	Development and swimming behavior of Medaka fry in a spaceflight aboard the Space Shuttle Columbia (STS-107)	16 Days	Animals	A space experiment aimed at closely observing the development and swimming activity of medaka fry under microgravity was carried out as a part of the STARS Program, a space shuttle mission, in STS-107 in January 2003. Four eggs laid on earth in an artificially controlled environment were put in a container with a functionally closed ecological system and launched on the Space Shuttle Columbia. Each egg was held in place by a strip of Velcro in the container to be individually monitored by closeup CCD cameras. In the control experiment, four eggs prepared using the same experimental set-up remained on the ground. There was no appreciable difference in the time course of development between space- and ground-based embryos. In the ground experiment, embryos were observed to rotate in place enclosed with the egg membrane, whereas those in the flight unit did not rotate. One of the four eggs hatched on the 8th day after being launched into space. All four eggs hatched in the ground unit. The fry hatched in space was mostly motionless, but with occasional control of its posture with respect to references in the experimental chamber. The fry hatched on ground were observed to move actively, controlling their posture with respect to the gravity vector. These findings suggest that the absence of gravity affects the initiation process of motility of embryos and hatched fry.
Spaceflight Study	FOTON-M3 Satellite	SLID-216	Tardigrades survive exposure to space in low Earth orbit	12 Days	Animals	Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.
Spaceflight Study	Russian Soyuz Spacecraft	SLID-217	Biochemical and Molecular Biological Analyses of space-flown nematodes in Japan, the First International Caenorhabditis elegans Experiment (ICE-First)	10 Days	Animals	The first International Caenorhabditis elegans Experiment (ICE-First) was carried out using a Russian Soyuz spacecraft from April 19-30, 2004. This experiment was a part of the program of the DELTA (Dutch Expedition for Life science Technology and Atmospheric research) mission, and the space agencies that participate in the International Space Station (ISS) program formed international research teams. A Japanese research team that conducted by Japan aerospace Exploration Agency (JAXA) investigated the following aspects of the organism: (1) whether meiotic chromosomal dynamics and apoptosis in the germ cells were normal under microgravity conditions, (2) the effect of the space flight on muscle cell development, and (3) the effect of the space flight on protein aggregation. In this article, we summarize the results of these biochemical and molecular biological analyses.
Spaceflight Study	FOTON-M3 Satellite	SLID-218	Tardigrade Resistance to Space Effects: first results of experiments on the LIFE-TARSE mission on FOTON-M3 (September 2007)	12 Days	Animals	The Tardigrade Resistance to Space Effects (TARSE) project, part of the mission LIFE on FOTON-M3, analyzed the effects of the space environment on desiccated and active tardigrades. Four experiments were conducted in which the eutardigrade Macrobiotus richtersi was used as a model species. Desiccated (in leaf litter or on paper) and hydrated tardigrades (fed or starved) were flown on FOTON-M3 for 12 days in September 2007, which, for the first time, allowed for a comparison of the effects of the space environment on desiccated and on active animals. In this paper, we report the experimental design of the TARSE project and data on tardigrade survival. In addition, data on survival, genomic DNA integrity, Hsp70 and Hsp90 expressions, antioxidant enzyme contents and activities, and life history traits were compared between hydrated starved tardigrades flown in space and those maintained on Earth as a control. Microgravity and radiation had no effect on survival or DNA integrity of active tardigrades. Hsp expressions between the animals in space and the control animals on Earth were similar. Spaceflight induced an increase of glutathione content and its related enzymatic activities. Catalase and superoxide dismutase decreased with spaceflight, and thiobarbituric acid reactive substances did not change. During the flight mission, tardigrades molted, and females laid eggs. Several eggs hatched, and the newborns exhibited normal morphology and behavior.
Spaceflight Study	n.a.	SLID-219	Innate immune responses of Drosophila melanogaster are altered by spaceflight	12 Days and 18.5 Hours	Animals	Alterations and impairment of immune responses in humans present a health risk for space exploration missions. The molecular mechanisms underpinning innate immune defense can be confounded by the complexity of the acquired immune system of humans. Drosophila (fruit fly) innate immunity is simpler, and shares many similarities with human innate immunity at the level of molecular and genetic pathways. The goals of this study were to elucidate fundamental immune processes in Drosophila affected by spaceflight and to measure host-pathogen responses post-flight. Five containers, each containing ten female and five male fruit flies, were housed and bred on the space shuttle (average orbit altitude of 330.35 km) for 12 days and 18.5 hours. A new generation of flies was reared in microgravity. In larvae, the immune system was examined by analyzing plasmatocyte number and activity in culture. In adults, the induced immune responses were analyzed by bacterial clearance and quantitative real-time polymerase chain reaction (qPCR) of selected genes following infection with E. coli. The RNA levels of relevant immune pathway genes were determined in both larvae and adults by microarray analysis. The ability of larval plasmatocytes to phagocytose E. coli in culture was attenuated following spaceflight, and in parallel, the expression of genes involved in cell maturation was downregulated. In addition, the level of constitutive expression of pattern recognition receptors and opsonins that specifically recognize bacteria, and of lysozymes, antimicrobial peptide (AMP) pathway and immune stress genes, hallmarks of humoral immunity, were also reduced in larvae. In adults, the efficiency of bacterial clearance measured in vivo following a systemic infection with E. coli post-flight, remained robust. We show that spaceflight altered both cellular and humoral immune responses in Drosophila and that the disruption occurs at multiple interacting pathways.
Spaceflight Study	Shenzhou 9 Space Flight	SLID-220	Effect of spaceflight on the circadian rhythm, lifespan and gene expression of Drosophila melanogaster	13 Days	Animals	Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.
Spaceflight Study	Shenzhou 9 Space Flight	SLID-220	Effect of spaceflight on the circadian rhythm, lifespan and gene expression of Drosophila melanogaster	13 Days	Animals	Space travelers are reported to experience circadian rhythm disruption during spaceflight. However, how the space environment affects circadian rhythm is yet to be determined. The major focus of this study was to investigate the effect of spaceflight on the Drosophila circadian clock at both the behavioral and molecular level. We used China’s Shenzhou-9 spaceship to carry Drosophila. After 13 days of spaceflight, behavior tests showed that the flies maintained normal locomotor activity rhythm and sleep pattern. The expression level and rhythm of major clock genes were also unaffected. However, expression profiling showed differentially regulated output genes of the circadian clock system between space flown and control flies, suggesting that spaceflight affected the circadian output pathway. We also investigated other physiological effects of spaceflight such as lipid metabolism and lifespan, and searched genes significantly affected by spaceflight using microarray analysis. These results provide new information on the effects of spaceflight on circadian rhythm, lipid metabolism and lifespan. Furthermore, we showed that studying the effect of spaceflight on gene expression using samples collected at different Zeitgeber time could obtain different results, suggesting the importance of appropriate sampling procedures in studies on the effects of spaceflight.
Spaceflight Study	FOTON-M4 Satellite	SLID-221	The Development Of Drosophila Melanogaster under Different Duration Space Flight and Subsequent Adaptation to Earth Gravity	44.5 Days	Animals	In prospective human exploration of outer space, the need to preserve a species over several generations under changed gravity conditions may arise. This paper demonstrates our results in the creation of the third generation of fruit fly Drosophila melanogaster (third-stage larvae) during the 44.5-day space flight (Foton-M4 satellite (2014, Russia)), then the fourth generation on Earth and the fifth generation again in conditions of the 12-day space flight (2014, in the Russian Segment of the ISS). The species preserves fertility despite a number of changes in the level of expression and content of cytoskeletal proteins, which are the key components of the cleavage spindle and the contractile ring of cells. The results of transcriptome screening and space analysis of cytoskeletal proteins show that the exposure to weightless conditions leads to the increased transcription of metabolic genes, cuticle components and the decreased transcription of genes involved in morphogenesis, cell differentiation, cytoskeletal organization and genes associated with the plasma membrane. “Subsequent” exposure to the microgravity for 12 days resulted in an even more significant increase/decrease in the transcription of the same genes. On the contrary, the transition from the microgravity conditions to the gravity of Earth leads to the increased transcription of genes whose products are involved in the morphogenesis, cytoskeletal organization, motility of cells and transcription regulation, and to the decreased transcription of cuticle genes and proteolytic processes.
Spaceflight Study	FOTON-M4 Satellite	SLID-221	The Development Of Drosophila Melanogaster under Different Duration Space Flight and Subsequent Adaptation to Earth Gravity	44.5 Days	Animals	In prospective human exploration of outer space, the need to preserve a species over several generations under changed gravity conditions may arise. This paper demonstrates our results in the creation of the third generation of fruit fly Drosophila melanogaster (third-stage larvae) during the 44.5-day space flight (Foton-M4 satellite (2014, Russia)), then the fourth generation on Earth and the fifth generation again in conditions of the 12-day space flight (2014, in the Russian Segment of the ISS). The species preserves fertility despite a number of changes in the level of expression and content of cytoskeletal proteins, which are the key components of the cleavage spindle and the contractile ring of cells. The results of transcriptome screening and space analysis of cytoskeletal proteins show that the exposure to weightless conditions leads to the increased transcription of metabolic genes, cuticle components and the decreased transcription of genes involved in morphogenesis, cell differentiation, cytoskeletal organization and genes associated with the plasma membrane. “Subsequent” exposure to the microgravity for 12 days resulted in an even more significant increase/decrease in the transcription of the same genes. On the contrary, the transition from the microgravity conditions to the gravity of Earth leads to the increased transcription of genes whose products are involved in the morphogenesis, cytoskeletal organization, motility of cells and transcription regulation, and to the decreased transcription of cuticle genes and proteolytic processes.
Spaceflight Study	BION-M1	SLID-222	Individual features of play behavior in thick-toed geckos in weightlessness and normal gravity conditions	30 Days	Animals	The object play behavior in thick-toed geckos (Chondrodactylus turneri GRAY 1864) was studied during a 30-day orbital experiment onboard the Bion-M1 biosatellite. The play object for five geckos was a marking collar that one of the geckos in the flight group removed immediately before the launch. The play behavior started when either the gecko observed the approaching floating collar or when the gecko independently approached the stationary collar, followed by manipulation of the collar and subsequent observation of its moving away. While playing with the collar, the individuality of geckos' behavior was manifested in the frequency and number of play episodes, the nature of manipulations, and the duration of interest in play during the flight. We found that thick-toed geckos could play not only with an unknown object (marking collar) but also with familiar molting skins. In weightlessness, the play behavior of geckos with molting skin fragments was similar to the play behavior with the collar and also varied between individuals. It was established that geckos maintained a similar individual level of play activity with different objects (collar and molting skins). It was found that geckos also played with fragments of molting skin under normal gravity conditions. In contrast to weightlessness, play behavior at normal gravity was rare and limited to short durations of object manipulation.
Spaceflight Study	SpaceX-19	SLID-223	Targeting myostatin/activin A protects against skeletal muscle and bone loss during spaceflight	33 Days	Animals	Among the physiological consequences of extended spaceflight are loss of skeletal muscle and bone mass. One signaling pathway that plays an important role in maintaining muscle and bone homeostasis is that regulated by the secreted signaling proteins, myostatin (MSTN) and activin A. Here, we used both genetic and pharmacological approaches to investigate the effect of targeting MSTN/activin A signaling in mice that were sent to the International Space Station. Wild type mice lost significant muscle and bone mass during the 33 d spent in microgravity. Muscle weights of Mstn -/- mice, which are about twice those of wild type mice, were largely maintained during spaceflight. Systemic inhibition of MSTN/activin A signaling using a soluble form of the activin type IIB receptor (ACVR2B), which can bind each of these ligands, led to dramatic increases in both muscle and bone mass, with effects being comparable in ground and flight mice. Exposure to microgravity and treatment with the soluble receptor each led to alterations in numerous signaling pathways, which were reflected in changes in levels of key signaling components in the blood as well as their RNA expression levels in muscle and bone. These findings have implications for therapeutic strategies to combat the concomitant muscle and bone loss occurring in people afflicted with disuse atrophy on Earth as well as in astronauts in space, especially during prolonged missions.
Spaceflight Study	BION-M1	SLID-224	Object play in thick-toed geckos during a space experiment	30 Days	Animals	Play behavior was observed in thick-toed geckos (Chondrodactylus turneri GRAY 1864) during a 30-day orbital experiment on the unmanned spacecraft “BION-M” No. 1. The geckos wore ornamented colored collars which made it possible to track the behavior of individual animals on video recordings. The object of the play behavior was a collar that one of the geckos had managed to remove in the pre-launch period and which floated weightless in the animal holding unit under microgravity. Four of the five geckos participated in play episodes, which were defined as one-time interactions with the collar, as well in a fuller form of play that included approaching the unmoving collar or observing its approach, manipulations with the collar and further tracking the collar. Manipulations with the collar could take the form of complicated play, such as pressing the snout against the edge of the collar rim, multiple episodes of pushing the collar with the snout, inserting the head into the collar, holding the collar by pressing the head to the container floor and tilting the head with the collar on the snout. There were individual variations in play pattern. Explanations for the rarity of play behavior in reptiles under normal conditions and the geckos’ playfulness in microgravity are discussed. Appropriate video is available at http://www.momo-p.com/showdetail-e.php?movieid=momo150224ct01a.
Spaceflight Study	FOTON-M3 Satellite	SLID-225	Fish Inner Ear Otolith Growth Under Real Microgravity (Spaceflight) and Clinorotation	12 Days	Animals	Using late larval stages of cichlid fish (Oreochromis mossambicus) we have shown earlier that the biomineralization of otoliths is adjusted towards gravity by means of a neurally guided feedback loop. Centrifuge experiments, e.g., revealed that increased gravity slows down otolith growth. Microgravity thus should yield an opposite effect, i.e., larger than normal otoliths. Consequently, late larval cichlids (stage 14, vestibular system operational) were subjected to real microgravity during the 12 days FOTON-M3 spaceflight mission (OMEGAHAB-hardware). Controls were kept at 1g on ground within an identical hardware. Animals of another batch were subsequently clinorotated within a submersed fast-rotating clinostat with one axis of rotation (2d-clinostat), a device regarded to simulate microgravity. Temperature and light conditions were provided in analogy to the spaceflight experiment. Controls were maintained at 1g within the same aquarium. After all experiments, animals had reached late stage 21 (fish can swim freely). Maintenance under real microgravity during spaceflight resulted in significantly larger than normal otoliths (both lapilli and sagittae, involved in sensing gravity and the hearing process, respectively). This result is fully in line with an earlier spaceflight study in the course of which otoliths from late-staged swordtails Xiphophorus helleri were analyzed. Clinorotation resulted in larger than 1g sagittae. However, no effect on lapilli was obtained. Possibly, an effect was present but too light to be measurable. Overall, spaceflight obviously induces an adaptation of otolith growth, whereas clinorotation does not fully mimic conditions of microgravity regarding late larval cichlids.
Ground Study	n.a.	SLID-225	Fish Inner Ear Otolith Growth Under Real Microgravity (Spaceflight) and Clinorotation	12 Days	Animals	Using late larval stages of cichlid fish (Oreochromis mossambicus) we have shown earlier that the biomineralization of otoliths is adjusted towards gravity by means of a neurally guided feedback loop. Centrifuge experiments, e.g., revealed that increased gravity slows down otolith growth. Microgravity thus should yield an opposite effect, i.e., larger than normal otoliths. Consequently, late larval cichlids (stage 14, vestibular system operational) were subjected to real microgravity during the 12 days FOTON-M3 spaceflight mission (OMEGAHAB-hardware). Controls were kept at 1g on ground within an identical hardware. Animals of another batch were subsequently clinorotated within a submersed fast-rotating clinostat with one axis of rotation (2d-clinostat), a device regarded to simulate microgravity. Temperature and light conditions were provided in analogy to the spaceflight experiment. Controls were maintained at 1g within the same aquarium. After all experiments, animals had reached late stage 21 (fish can swim freely). Maintenance under real microgravity during spaceflight resulted in significantly larger than normal otoliths (both lapilli and sagittae, involved in sensing gravity and the hearing process, respectively). This result is fully in line with an earlier spaceflight study in the course of which otoliths from late-staged swordtails Xiphophorus helleri were analyzed. Clinorotation resulted in larger than 1g sagittae. However, no effect on lapilli was obtained. Possibly, an effect was present but too light to be measurable. Overall, spaceflight obviously induces an adaptation of otolith growth, whereas clinorotation does not fully mimic conditions of microgravity regarding late larval cichlids.
Spaceflight Study	Soyuz Rocket (TMA-4)	SLID-226	Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans	10 Days	Animals	We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.
Spaceflight Study	Soyuz Rocket (TMA-4)	SLID-226	Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans	10 Days	Animals	We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.
Spaceflight Study	SJ-10 Recoverable Satellite	SLID-227	Development of mouse preimplantation embryos in space	n.a.	Animals	The development of life beyond planet Earth is a long-standing quest of the human race, but whether normal mammalian embryonic development can occur in space is still unclear. Here, we show unequivocally that preimplantation mouse embryos can develop in space, but the rate of blastocyst formation and blastocyst quality are compromised. Additionally, the cells in the embryo contain severe DNA damage, while the genome of the blastocysts developed in space is globally hypomethylated with a unique set of differentially methylated regions. The developmental defects, DNA damage and epigenetic abnormalities can be largely mimicked by the treatment with ground-based low-dose radiation. However, the exposure to simulated microgravity alone does not cause major disruptions of embryonic development, indicating that radiation is the main cause for the developmental defects. This work advances the understanding of embryonic development in space and reveals long-term extreme low-dose radiation as a hazardous factor for mammalian reproduction.
Spaceflight Study	FOTON-M3 Satellite	SLID-228	Extreme stress tolerance in tardigrades: surviving space conditions in low earth orbit	12 Days	Animals	Most terrestrial tardigrade species possess the ability to enter a reversible ametabolic state termed anhydrobiosis in response to desiccation. In the anhydrobiotic state, tardigrades display an incredible capacity to tolerate extreme environmental stress, not necessarily encountered in their natural habitat. In this study, we determine the effect of different extreme stresses on initial survival, long-term survival and fecundity of selected species of limno-terrestrial tardigrades. The primary focus was to assess the effect of cosmic radiation. This was achieved through the RoTaRad (Rotifers, Tardigrades and Radiation) project on the BIOPAN 6 mission, funded by Agenzia Spaziale Italiana under the European Space Agency. To test their tolerance of space environment, tardigrades were sent into low earth orbit, and exposed to cosmic radiation and a microgravity environment. Experiments on Whatman-3 filters show an effect of cosmic radiation on the survival of the eutardigrade Richtersius coronifer just after returning to Earth; however, after 2 years of desiccation on Whatman-3 filters, none of the tardigrades previously exposed to cosmic radiation could be revived. In a microcosmos experiment, the tardigrades R. coronifer, Ramazzottius oberhauseri and Echiniscus testudo were desiccated on a moss substrate together with rotifers and nematodes. Very low survival rates were observed in this experiment, likely due to the applied desiccation protocol. Embryos of the tardigrade Milnesium tardigradum were also exposed to cosmic radiation; they all hatched in the laboratory after the flight. In addition, experiments testing extreme cold and vacuum tolerance in R. coronifer show that tardigrades in anhydrobiosis are unaffected by these conditions.
Spaceflight Study	Apollo Missions	SLID-229	Spider Web-Building in Outer Space: Evaluation of Records from the Skylab Spider Experiment	n.a.	Animals	Two spiders built orb-webs in Skylab under zero gravity only four days after release from transpor tation vials. The first webs resembled pre-flight controls in size and regularity, but the unusual distribution of radial angles and thinness of thread together with a probably low number of turning points in the spiral indicated a deviation from earth webs which can be attributed to the absence of gravity as a cue. Other web changes like a gradual decrease in regularity, diminished web size, and the spiders' decrease in body weight are identified as being likely consequences of starvation, Skylab stress and unknown circumstances not directly connected with zero gravity.
Spaceflight Study	n.a.	SLID-230	Nematode Muscles Project in Spaceflight Experiment	4 Days	Animals	Our previous spaceflight experiment CERISE showed that gene and protein expression levels of muscular components, cytoskeleton, and mitochondrial enzymes are altered in space flown wild-type C. elegans. To confirm and clarify whether the C. elegans muscle fibers and mitochondrial network are physically altered in response to microgravity, this Nematode Muscles project was designed with wild-type and several mutant lines with GFP expression. This investigation also studied whether microgravity could affect the insulin/IGF-1 (Insulin-like growth factor -1) and/or TGF-β signaling by imaging DAF-16::GFP fusion protein. Wild-type and several mutants were grown in a culture bag kept under microgravity or 1G centrifuge conditions on board ISS for 4 days starting from L1 larva. All samples were fixed on board and recovered, to be analyzed on the earth. The worms did not grow well in the μG culture bag probably due to unexpected air bubbles. Therefore, DAF-16 activation observed in larval worms in μG and not in 1G may be attributed to starvation instead of μG response. In 1G samples, we could successfully find normal mitochondrial network. We also found that chemical fixation using CFA is an effective method for preservation of GFP containing C. elegans in space environment.
Ground Study	n.a.	SLID-231	Influence of long-term altered gravity on the swimming performance of developing cichlid fish: Including results from the 2nd German Spacelab Mission D-2	10 Days	Animals	This study presents qualitative and quantitative data concerning gravity-dependent changes in the swimming behaviour of developing cichlid fish larvae (Oreochromis mossambicus) after a 9 resp. 10 days exposure to increased acceleration (centrifuge experiments), to reduced gravity (fast-rotating clinostat), changed accelerations (parabolic aircraft flights) and to near weightlessness (2nd German Spacelab Mission D-2). Changes of gravity initially cause disturbances of the swimming performance of the fish larvae. With prolonged stay in orbit a step by step normalisation of the swimming behaviour took place in the fish. After return to 1g earth conditions no somersaulting or looping could be detected concerning the fish, but still slow and disorientated movements as compared to controls occurred. The fish larvae adapted to earth gravity within 3-5 days. Fish seem to be in a distinct early developmental stages extreme sensitive and adaptable to altered gravity; However, elder fish either do not react or show compensatory behaviour e.g. escape reactions.
Spaceflight Study	STS-55	SLID-231	Influence of long-term altered gravity on the swimming performance of developing cichlid fish: Including results from the 2nd German Spacelab Mission D-2	10 Days	Animals	This study presents qualitative and quantitative data concerning gravity-dependent changes in the swimming behaviour of developing cichlid fish larvae (Oreochromis mossambicus) after a 9 resp. 10 days exposure to increased acceleration (centrifuge experiments), to reduced gravity (fast-rotating clinostat), changed accelerations (parabolic aircraft flights) and to near weightlessness (2nd German Spacelab Mission D-2). Changes of gravity initially cause disturbances of the swimming performance of the fish larvae. With prolonged stay in orbit a step by step normalisation of the swimming behaviour took place in the fish. After return to 1g earth conditions no somersaulting or looping could be detected concerning the fish, but still slow and disorientated movements as compared to controls occurred. The fish larvae adapted to earth gravity within 3-5 days. Fish seem to be in a distinct early developmental stages extreme sensitive and adaptable to altered gravity; However, elder fish either do not react or show compensatory behaviour e.g. escape reactions.
Spaceflight Study	FOTON-M3 Satellite	SLID-232	Effect of 12-Day Spaceflight on Brain of Thick-Toed Geckos	12 Days	Animals	In the frames of Russian-American joint space experiment onboard Foton-M3 satellite there was undertaken a study of spaceflight influence on brain of the thick-toed gecko (Pachydactylus turneri Gray, 1864). Serial brain sections were stained according to Nissl and also the immunohistochemical method with antibodies to NGF-receptor (p75NGFR), CD95 (also known as Fas and APO-1), glial fibrillary acidic protein (GFAP) and transferrin-receptor (CD71). Detailed examination of the sections of rhombencephalon revealed cytological changes in the neuron bodies of vestibular nuclei inside the flight group. Immunohistochemicaly we found the increase density of CD95 and p75NGFR and decrease of GFAP expression in medial cortex and epithalamus in flight group compared both control.
Spaceflight Study	International Space Station (ISS)	SLID-233	Effect of potassium citrate therapy on the risk of renal stone formation during spaceflight	93 - 215 Days	n.a.	Purpose: Exposure to microgravity affects human physiology and results in changes in urinary chemical composition during and after spaceflight, favoring an increased risk of renal stones. We assessed the efficacy of potassium citrate to decrease the stone risk during and after spaceflight. Materials and Methods: The study was done in 30 long duration spaceflight crew members to the space stations Mir and International Space Station. Before, during and after spaceflight 24-hour urine samples were collected to assess the renal stone risk. Potassium citrate (20 mEq) was ingested daily by International Space Station crew members in a double-blind, placebo controlled study. Mir crew members performed the identical protocol but did not ingest medication. Results: Potassium citrate treated crew members had decreased urinary calcium excretion and maintained the calcium oxalate supersaturation risk at preflight levels compared to that in controls. Increased urinary pH in the treatment group decreased the risk of uric acid stones. Conclusions: Results from this investigation suggest that supplementation with potassium citrate may decrease the risk of renal stone formation during and immediately after spaceflight.
Spaceflight Study	NASA-Mir Spaceflights	SLID-233	Effect of potassium citrate therapy on the risk of renal stone formation during spaceflight	129 - 208 Days	n.a.	Purpose: Exposure to microgravity affects human physiology and results in changes in urinary chemical composition during and after spaceflight, favoring an increased risk of renal stones. We assessed the efficacy of potassium citrate to decrease the stone risk during and after spaceflight. Materials and Methods: The study was done in 30 long duration spaceflight crew members to the space stations Mir and International Space Station. Before, during and after spaceflight 24-hour urine samples were collected to assess the renal stone risk. Potassium citrate (20 mEq) was ingested daily by International Space Station crew members in a double-blind, placebo controlled study. Mir crew members performed the identical protocol but did not ingest medication. Results: Potassium citrate treated crew members had decreased urinary calcium excretion and maintained the calcium oxalate supersaturation risk at preflight levels compared to that in controls. Increased urinary pH in the treatment group decreased the risk of uric acid stones. Conclusions: Results from this investigation suggest that supplementation with potassium citrate may decrease the risk of renal stone formation during and immediately after spaceflight.
Spaceflight Study	Mir Space Station	SLID-234	The State of the Digestive Organs during Long Spaceflight	132 - 438 Days	Glycemic profile, the biochemical composition of capillary blood, and the ultrasonic pattern of the internal organs and the blood vessels of the abdominal cavity	The state of the digestive organs was comprehensively studied in 12 cosmonauts before, during, and after Mir missions 132-438 days long. The study consisted of glucose–milk loading during which the glycemic profile, the biochemical composition of capillary blood, and the ultrasonic pattern of the internal organs and the blood vessels of the abdominal cavity were recorded. As compared to the preflight data, an increase in the size of the parenchymatous organs, a decrease in their echogenicity, and the thickening of the walls of the hollow organs were observed during spaceflight, which was indicative of their being excessively plethoric. Therewith, an increase in the stomach fluid, intestinal dilatation, and an increased gallbladder tone were determined in most cases on an empty stomach, which suggested increased secretory activity. Flattened glycemic curves and decreased pancreas and gallbladder reactivities, as well as delayed gastric evacuation, were revealed after a glucose-mil load. It should be pointed out that the severity of the changes described was not directly related to the duration of exposure to weightlessness within the limit of six months. The changes revealed were reversible and, in most cases, completely disappeared two weeks after completion of the missions.
Spaceflight Study	n.a.	SLID-235	Stability of chromosome aberrations in the blood lymphocytes of astronauts measured after space flight by FISH chromosome painting	n.a.	Chromosome aberrations	Follow-up measurements of chromosome aberrations in the blood lymphocytes of astronauts were performed by FISH chromosome painting at various intervals from 5 months to more than 5 years after space flight and compared to preflight baseline measurements. For five of the six astronauts studied, the analysis of individual time courses for translocations revealed a temporal decline of yields with half-lives ranging from 10 to 58 months. The yield of exchanges remained unchanged for the sixth astronaut during an observation period of 5 months after flight. These results may indicate complications with the use of stable aberrations for retrospective dose reconstruction, and the differences in the decay time may reflect individual variability in risk from space radiation exposure.
Spaceflight Study	International Space Station (ISS)	SLID-236	Biochemical Markers of Bone Tissue Metabolism in Cosmonauts after a Prolonged Spaceflight	129 - 196 Days	Bone tissue metabolism	Parameters of calcium homeostasis and its hormonal regulation, including biochemical markers of bone metabolism, were measured in the blood serum of Russian cosmonauts after prolonged flights on the International Space Station during the period from 2000 to 2003. The duration of the spaceflights was 129–196 days. Flight factors had an impact on calcium and bone tissue metabolism after a flight. Increased levels of osteogenesis and resorption markers were detected in the blood of the cosmonauts in the early rehabilitation period after a spaceflight. The prevalence of resorption over the formation of new bone tissue was observed in the early rehabilitation period, when the hormonal system maintaining calcium homeostasis was activated.
Spaceflight Study	Space Shuttle; ISS	SLID-237	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	n.a.	Sleep deficiency and hypnotic drug use in space	Background: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. Methods: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. Findings: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. Interpretation: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Spaceflight Study	Space Shuttle; ISS	SLID-237	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	n.a.	Sleep deficiency and hypnotic drug use in space	Background: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. Methods: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. Findings: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. Interpretation: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Spaceflight Study	n.a.	SLID-238	Brain structural plasticity with spaceflight	n.a.	Brain structural changes	Humans undergo extensive sensorimotor adaptation during spaceflight due to altered vestibular inputs and body unloading. No studies have yet evaluated the effects of spaceflight on human brain structure despite the fact that recently reported optic nerve structural changes are hypothesized to occur due to increased intracranial pressure occurring with microgravity. This is the first report on human brain structural changes with spaceflight. We evaluated retrospective longitudinal T2-weighted MRI scans and balance data from 27 astronauts (thirteen ~2-week shuttle crew members and fourteen ~6-month International Space Station crew members) to determine spaceflight effects on brain structure, and whether any pre to postflight brain changes are associated with balance changes. Data were obtained from the NASA Lifetime Surveillance of Astronaut Health. Brain scans were segmented into gray matter maps and normalized into MNI space using a stepwise approach through subject specific templates. Non-parametric permutation testing was used to analyze pre to postflight volumetric gray matter changes. We found extensive volumetric gray matter decreases, including large areas covering the temporal and frontal poles and around the orbits. This effect was larger in International Space Station versus shuttle crew members in some regions. There were bilateral focal gray matter increases within the medial primary somatosensory and motor cortex; i.e., the cerebral areas where the lower limbs are represented. These intriguing findings are observed in a retrospective data set; future prospective studies should probe the underlying mechanisms and behavioral consequences.
Spaceflight Study	STS-135	SLID-239	Gene-metabolite profile integration to understand the cause of spaceflight induced immunodeficiency	6 Months	Metabolite profile of spaceflown human cells	Spaceflight presents a spectrum of stresses very different from those associated with terrestrial conditions. Our previous study (BMC Genom. 15: 659, 2014) integrated the expressions of mRNAs, microRNAs, and proteins and results indicated that microgravity induces an immunosuppressive state that can facilitate opportunistic pathogenic attack. However, the existing data are not sufficient for elucidating the molecular drivers of the given immunosuppressed state. To meet this knowledge gap, we focused on the metabolite profile of spaceflown human cells. Independent studies have attributed cellular energy deficiency as a major cause of compromised immunity of the host, and metabolites that are closely associated with energy production could be a robust signature of atypical energy fluctuation. Our protocol involved inoculation of human endothelial cells in cell culture modules in spaceflight and on the ground concurrently. Ten days later, the cells in space and on the ground were exposed to lipopolysaccharide (LPS), a ubiquitous membrane endotoxin of Gram-negative bacteria. Nucleic acids, proteins, and metabolites were collected 4 and 8 h post-LPS exposure. Untargeted profiling of metabolites was followed by targeted identification of amino acids and knowledge integration with gene expression profiles. Consistent with the past reports associating microgravity with increased energy expenditure, we identified several markers linked to energy deficiency, including various amino acids such as tryptophan, creatinine, dopamine, and glycine, and cofactors such as lactate and pyruvate. The present study revealed a molecular architecture linking energy metabolism and immunodeficiency in microgravity. The energy-deficient condition potentially cascaded into dysregulation of protein metabolism and impairment of host immunity. This project is limited by a small sample size. Although a strict statistical screening was carefully implemented, the present results further emphasize the need for additional studies with larger sample sizes. Validating this hypothesis using an in vivo model is essential to extend the knowledge towards identifying markers of diagnostic and therapeutic value.
Spaceflight Study	International Space Station (ISS)	SLID-240	High intensity training during spaceflight: results from the NASA Sprint Study	n.a.	The effectiveness of a a high intensity/lower volume exercise prescription for ISS crewmembers	Historically, International Space Station (ISS) exercise countermeasures have not fully protected astronauts’ musculoskeletal and cardiorespiratory fitness. Although these losses have been reduced on more recent missions, decreasing the time required to perform in-flight exercise would permit reallocation of that time to other tasks. To evaluate the effectiveness of a new training prescription, ISS crewmembers performed either the high intensity/lower volume integrated Sprint resistance (3 d wk−1) and aerobic (interval and continuous workouts, each 3 d wk−1 in alternating fashion) exercise program (n = 9: 8M/1F, 48 ± 7 y, 178 ± 5 cm, 77.7 ± 12.0 kg) or the standard ISS countermeasure consisting of daily resistance and aerobic exercise (n = 17: 14M/3F, 46 ± 6 y, 176 ± 6 cm, 80.6 ± 10.5 kg) during long-duration spaceflight. Bone mineral density (dual energy X-ray absorptiometry (DXA)), muscle strength (isokinetic dynamometry), muscle function (cone agility test), and cardiorespiratory fitness (VO2peak) were assessed pre- and postflight. Mixed-effects modeling was used to analyze dependent measures with alpha set at P < 0.05. After spaceflight, femoral neck bone mineral density (−1.7%), knee extensor peak torque (−5.8%), cone agility test time (+7.4%), and VO2peak (−6.1%) were decreased in both groups (simple main effects of time, all P < 0.05) with a few group × time interaction effects detected for which Sprint experienced either attenuated or no loss compared to control. Although physiologic outcomes were not appreciably different between the two exercise programs, to conserve time and optimally prepare crewmembers for the performance of physically demanding mission tasks, high intensity/lower volume training should be an indispensable component of spaceflight exercise countermeasure prescriptions.
Spaceflight Study	International Space Station (ISS)	SLID-241	Quantitative magnetic resonance image assessment of the optic nerve and surrounding sheath after spaceflight	n.a.	Optic nerve (ON) and ON sheath (ONS) cross-sectional areas and optic nerve deviation changes	A subset of long-duration spaceflight astronauts have experienced ophthalmic abnormalities, collectively termed spaceflight-associated neuro-ocular syndrome (SANS). Little is understood about the pathophysiology of SANS; however, microgravity-induced alterations in intracranial pressure (ICP) due to headward fluid shifts is the primary hypothesized contributor. In particular, potential changes in optic nerve (ON) tortuosity and ON sheath (ONS) distension may indicate altered cerebrospinal fluid dynamics during weightlessness. The present longitudinal study aims to provide a quantitative analysis of ON and ONS cross-sectional areas, and ON deviation, an indication of tortuosity, before and after spaceflight. Ten astronauts undergoing ~6-month missions on the International Space Station (ISS) underwent high-resolution magnetic resonance imaging (MRI) preflight and at five recovery time points extending to 1 year after return from the ISS. The mean changes in ON deviation, ON cross-sectional area, and ONS cross-sectional area immediately post flight were −0.14 mm (95% CI: −0.36 to 0.08, Bonferroni-adjusted P = 1.00), 0.13 mm2 (95% CI −0.66 to 0.91, Bonferroni-adjusted P = 1.00), and −0.22 mm2 (95% CI: −1.78 to 1.34, Bonferroni-adjusted P = 1.00), respectively, and remained consistent during the recovery period. Terrestrially, ONS distension is associated with increased ICP; therefore, these results suggest that, on average, ICP was not pathologically elevated immediately after spaceflight. However, a subject diagnosed with optic disc edema (Frisen Grade 1, right eye) displayed increased ONS area post flight, although this increase is relatively small compared to clinical populations with increased ICP. Advanced quantitative MRI-based assessment of the ON and ONS could help our understanding of SANS and the role of ICP.
Spaceflight Study	n.a.	SLID-242	Vestibulo-oculomotor testing during the course of a spaceflight mission	n.a.	Vestibulo-oculomotor response	The experimental concept and findings from a recent manned orbital spaceflight are presented. In a single-case, longitudinal study, vestibulo-oculomotor function was examined by caloric testing and active head oscillations. The results from preflight, inflight, and postflight measurements of the human vestibulo-ocular reflex, together with those of ongoing terrestrial studies, should enable separation of the canalicular and otolithic contributions to ocular torsion. This analysis enables an accurate evaluation of the adaptation of the otolithic system to the inflight microgravity and, after landing, to the 1- force environment. Video-oculography was employed throughout for the comprehensive measurement of eye and head movements. Caloric testing involved air insufflation at 15° C over 90 s, followed by an observation interval of 2 min. During inflight testing this was continued with a 30-s free-floating interval. Active head oscillations were performed at four discrete frequencies (0.12, 0.32, 0.80, 2.0 Hz) and over a frequency sweep between 0.1 and 2.0 Hz. These head oscillations were performed in yaw, pitch, and roll and for three visual conditions (head-fixed target, space-fixed target, no target). The concomitant stimulation of the semicircular canals and otolithic receptors during these oscillations should yield different oculomotor responses under 1-g and 0-g adaptations. Both the short-form caloric test and the active head movement test were performed on 4 of the 5 available mission days. The results of the caloric tests yield a caloric nystagmus intensity (slow-phase velocity) of approximately 60% of that measured before flight and indicate an adaptation in response over the 10-day period after landing. The preliminary results from the head movement tests about the roll axis indicate an adaptive response in this aspect of the vestibulo-ocular reflex during prolonged microgravity. Some changes in sensomotoric control were also apparent during the inflight and postflight phases.
Spaceflight Study	n.a.	SLID-243	Vestibular plasticity following orbital spaceflight: recovery from postflight postural instability	4 - 10 Days	Sensori-motor postural control	Results of previous studies suggested that the vestibular mediated postural instability observed in astronauts upon return to earth from orbital spaceflight may be exacerbated by an increased weighting of visual inputs for spatial orientation and control of movement. This study was performed to better understand the roles of visual and somatosensory contributions to recovery of normal sensori-motor postural control in returning astronauts. Preflight and postflight. 23 astronaut volunteers were presented randomly with three trials of six sensory organization test (SOT) conditions in the EquiTest system test battery. Sagittal plane center-of-gravity (COG) excursions computed from ground reaction forces were significantly higher on landing day than preflight for those test conditions presenting sway-referenced visual and/or somatosensory orientation cues. The ratio of summed peak-to-peak COG sway amplitudes on the two sway-referenced vision tests (SOTs 3 + 6) compared to the two eyes closed tests (SOTs 2 + 5) was increased on landing day, indicating an increased reliance on visual orientation cues for postural control. The ratio of peak-to-peak COG excursions on sway-referenced surfaces (SOTs 4, 5 & 6) to an earth fixed support surfaces (SOTs 1, 2 & 3) increased even more after landing suggesting primary reliance on somatosensory orientation cues for recovery of postflight postural stability. Readaptation to sway-referenced support surfaces took longer than readaptation to sway-referenced vision. The increased reliance on visual and somatosensory inputs disappeared in all astronauts 4-8 days following return to earth.
Spaceflight Study	n.a.	SLID-244	Impairments of manual tracking performance during spaceflight are associated with specific effects of microgravity on visuomotor transformations	n.a.	Manual tracking performance	In contrast to performance in cognitive tasks, tracking performance tends to deteriorate fairly consistently during spaceflight. We address the question whether this decrement results from specific effects of microgravity on motor control or from non-specific effects of the various other stressors present. In a case study we generalize the findings obtained with aiming movements, performed by the same cosmonaut with the same effectors as used for an unstable tracking task, to obtain hypotheses for specific changes of parameters of a simple model used to analyse tracking performance. Consistent with these hypotheses, we observed a reduction of limb stiffness in-flight, but a reduction of the tracking gain post-flight. The cross-task consistency of the observed changes does strongly suggest that the tracking impairment is at least partly caused by specific effects of microgravity on motor control, in particular by a mis-calibration of muscular forces which likely results from an underestimation of masses due to weightlessness.
Spaceflight Study	Shenzhou 9 Space Flight; Shenzhou 10 Space Flight	SLID-245	Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions	13 and 15 Days，respectively	Cell populations changes	Spaceflight leads to health risks including bone demineralization, skeletal muscle atrophy, cardiovascular dysfunction, and disorders of almost all physiologic systems. However, the impacts of microgravity on blood lineage cells and hematopoietic stem cells (HSCs) in vivo are largely unknown. In this study, we analyzed peripheral blood samples from 6 astronauts who had participated in spaceflight missions and found significant changes in several cell populations at different time points. These dynamic alterations of lineage cells and the role of HSCs were further studied in a mouse model, using hindlimb unloading (HU) to simulate microgravity. Large reductions in the frequency of NK cells, B cells, and erythrocyte precursors in the bone marrow of the HU mice were observed, together with an increased frequency of T cells, neutrophils, and HSCs. T cell levels recovered faster than those of B cells and erythrocyte precursors, whereas the recovery rates of NK cells and granulocytes were slow. In addition, competitive reconstitution experiments demonstrated the impaired function of HSCs, although these changes were reversible. Deep sequencing showed changes in the expression of regulatory molecules important for the differentiation of HSCs. This study provides the first determination of altered HSC function under simulated microgravity in vivo. The impairment of HSC function and differentiation provides an explanation for the immune disorders that occur under simulated microgravity. Thus, our findings demonstrated that spaceflight and simulated microgravity disrupt the homeostasis of immune system and cause dynamic alterations on both HSCs and lineage cells.
Spaceflight Study	International Space Station (ISS)	SLID-246	Persistent Globe Flattening in Astronauts following Long-Duration Spaceflight	6 Months	Scleral remodelling	Posterior globe flattening has been well-documented in astronauts both during and after long-duration space flight (LDSF) and has been observed as early as 10 days into a mission on the International Space Station. Globe flattening (GF) is thought to be caused by the disc centred anterior forces created by elevated volume and/or pressure within the optic nerve sheath (ONS). This might be the result of increased intracranial pressure, increased intraorbital ONS pressure from compartmentalisation or a combination of these mechanisms. We report posterior GF in three astronauts that has persisted for 7 years or more following their return from LDSFs suggesting that permanent scleral remodelling may have occurred.
Spaceflight Study	International Space Station (ISS)	SLID-247	Resilient microorganisms in dust samples of the International Space Station-survival of the adaptation specialists	n.a.	Bacteria	Background: The International Space Station (ISS) represents a unique biotope for the human crew but also for introduced microorganisms. Microbes experience selective pressures such as microgravity, desiccation, poor nutrient-availability due to cleaning, and an increased radiation level. We hypothesized that the microbial community inside the ISS is modified by adapting to these stresses. For this reason, we analyzed 8-12 years old dust samples from Russian ISS modules with major focus on the long-time surviving portion of the microbial community. We consequently assessed the cultivable microbiota of these samples in order to analyze their extremotolerant potential against desiccation, heat-shock, and clinically relevant antibiotics. In addition, we studied the bacterial and archaeal communities from the stored Russian dust samples via molecular methods (next-generation sequencing, NGS) and compared our new data with previously derived information from the US American ISS dust microbiome. Results: We cultivated and identified in total 85 bacterial, non-pathogenic isolates (17 different species) and 1 fungal isolate from the 8-12 year old dust samples collected in the Russian segment of the ISS. Most of these isolates exhibited robust resistance against heat-shock and clinically relevant antibiotics. Microbial 16S rRNA gene and archaeal 16S rRNA gene targeting Next Generation Sequencing showed signatures of human-associated microorganisms (Corynebacterium, Staphylococcus, Coprococcus etc.), but also specifically adapted extremotolerant microorganisms. Besides bacteria, the detection of archaeal signatures in higher abundance was striking. Conclusions: Our findings reveal (i) the occurrence of living, hardy microorganisms in archived Russian ISS dust samples, (ii) a profound resistance capacity of ISS microorganisms against environmental stresses, and (iii) the presence of archaeal signatures on board. In addition, we found indications that the microbial community in the Russian segment dust samples was different to recently reported US American ISS microbiota.
Spaceflight Study	International Space Station (ISS)	SLID-247	Resilient microorganisms in dust samples of the International Space Station-survival of the adaptation specialists	n.a.	Fungi	Background: The International Space Station (ISS) represents a unique biotope for the human crew but also for introduced microorganisms. Microbes experience selective pressures such as microgravity, desiccation, poor nutrient-availability due to cleaning, and an increased radiation level. We hypothesized that the microbial community inside the ISS is modified by adapting to these stresses. For this reason, we analyzed 8-12 years old dust samples from Russian ISS modules with major focus on the long-time surviving portion of the microbial community. We consequently assessed the cultivable microbiota of these samples in order to analyze their extremotolerant potential against desiccation, heat-shock, and clinically relevant antibiotics. In addition, we studied the bacterial and archaeal communities from the stored Russian dust samples via molecular methods (next-generation sequencing, NGS) and compared our new data with previously derived information from the US American ISS dust microbiome. Results: We cultivated and identified in total 85 bacterial, non-pathogenic isolates (17 different species) and 1 fungal isolate from the 8-12 year old dust samples collected in the Russian segment of the ISS. Most of these isolates exhibited robust resistance against heat-shock and clinically relevant antibiotics. Microbial 16S rRNA gene and archaeal 16S rRNA gene targeting Next Generation Sequencing showed signatures of human-associated microorganisms (Corynebacterium, Staphylococcus, Coprococcus etc.), but also specifically adapted extremotolerant microorganisms. Besides bacteria, the detection of archaeal signatures in higher abundance was striking. Conclusions: Our findings reveal (i) the occurrence of living, hardy microorganisms in archived Russian ISS dust samples, (ii) a profound resistance capacity of ISS microorganisms against environmental stresses, and (iii) the presence of archaeal signatures on board. In addition, we found indications that the microbial community in the Russian segment dust samples was different to recently reported US American ISS microbiota.
Spaceflight Study	Shijian-8 Seed Breeding Satellite	SLID-248	Effect of space flight factors on alfalfa seeds	15 Days	Seed	To explore the effect of space flight factors on the early development of alfalfa seedling, dry seeds were placed onboard a satellite for a 15-day flight. After retrieval, the ultra structure of seed coat and the chemical content of seed were tested, followed by tests for germinate ability, seedling growth, and mitotic and chromosome aberrations. Results showed that space flight factors have both positive and negative effects on alfalfa seeds. Positive effects include: (1) A 6.2% increase in germinate potential and(2) an 80% decrease in the number of hard seed in flight seeds. Meanwhile, negative effects included a decrease of 3.0 and 33.2% in the index of germination and vigor of flight seeds, respectively, which may be partly due to the inhibition of cell mitotic (26% less than ground control) and root growth (29.0% less than ground control) after the space flight. Moreover, the DNA and Ca2+ content of alfalfa seeds increased after the space flight, while the reserve energy content of alfalfa seeds, such as saccharine and fatty acid, decreased after the space flight. Conclusively, space flight factors accelerate the germination process of alfalfa seeds but restrain the root from growing due to chromosomal damage and abnormal mitosis induced by cosmic radiation.
Spaceflight Study	Russian Space Station Mir (STS-84 )	SLID-249	Gravitropism of hypocotyls of wild-type and starch-deficient Arabidopsis seedlings in spaceflight studies	n.a.	Seedling	The major purpose of this spaceflight project was to investigate the starch-statolith hypothesis for gravity perception, and a secondary goal was to study plant growth and development under spaceflight conditions. This research was based on our ground studies of gravity perception in the wild type and three starch-deficient (one starchless and two reduced starch) mutants of Arabidopsis thaliana (L.) Heynh. Dark-grown seedlings that developed in microgravity were given one of several (30 min, 60 min, or 90 min) 1-g stimuli by an on-board centrifuge, and additional controls for seedling development also were performed. These latter control experiments included a morphological study of plants that developed in space in microgravity (F microg), in space on a centrifuge (F 1g), on the ground (G 1g), and on a rotating clinostat on the ground. Since elevated levels of ethylene were reported in the spacecraft atmosphere, additional controls for morphology and gravitropism with added ethylene also were performed. While exogenous ethylene reduced the absolute magnitude of the response in all four strains of Arabidopsis, this gas did not appear to change the relative graviresponsiveness among the strains. The relative response of hypocotyls of microgravity-grown seedlings to the stimuli provided by the in-flight centrifuge was: wild type > starch-deficient mutants. Although the protoplast pressure model for gravity perception cannot be excluded, these results are consistent with a statolith-based model for perception in plants.
Spaceflight Study	Space Shuttle Mission STS-131	SLID-250	An endogenous growth pattern of roots is revealed in seedlings grown in microgravity	15 Days	Seedling	In plants, sensitive and selective mechanisms have evolved to perceive and respond to light and gravity. We investigated the effects of microgravity on the growth and development of Arabidopsis thaliana (ecotype Landsberg) in a spaceflight experiment. These studies were performed with the Biological Research in Canisters (BRIC) hardware system in the middeck region of the space shuttle during mission STS-131 in April 2010. Seedlings were grown on nutrient agar in Petri dishes in BRIC hardware under dark conditions and then fixed in flight with paraformaldehyde, glutaraldehyde, or RNAlater. Although the long-term objective was to study the role of the actin cytoskeleton in gravity perception, in this article we focus on the analysis of morphology of seedlings that developed in microgravity. While previous spaceflight studies noted deleterious morphological effects due to the accumulation of ethylene gas, no such effects were observed in seedlings grown with the BRIC system. Seed germination was 89% in the spaceflight experiment and 91% in the ground control, and seedlings grew equally well in both conditions. However, roots of space-grown seedlings exhibited a significant difference (compared to the ground controls) in overall growth patterns in that they skewed to one direction. In addition, a greater number of adventitious roots formed from the axis of the hypocotyls in the flight-grown plants. Our hypothesis is that an endogenous response in plants causes the roots to skew and that this default growth response is largely masked by the normal 1 g conditions on Earth.
Spaceflight Study	European Modular Cultivation System (EMCS) on the ISS(STS-130,STS-131)	SLID-251	Phototropism of Arabidopsis thaliana in microgravity and fractional gravity on the International Space Station	n.a.	Seedling	While there is a great deal of knowledge regarding plant growth and development in microgravity aboard orbiting spacecraft, there is little information available about these parameters in reduced or fractional gravity conditions (less than the nominal 1g on Earth). Thus, in these experiments using the European Modular Cultivation System on the International Space Station, we studied the interaction between phototropism and gravitropism in the WT and mutants of phytochrome A and B of Arabidopis thaliana. Fractional gravity and the 1 g control were provided by centrifuges in the spaceflight hardware, and unidirectional red and blue illumination followed a white light growth period in the time line of the space experiments. The existence of red-light-based positive phototropism in hypocotyls of seedlings that is mediated by phytochrome was confirmed in these microgravity experiments. Fractional gravity studies showed an attenuation of red-light-based phototropism in both roots and hypocotyls of seedlings occurring due to gravitational accelerations ranging from 0.l to 0.3 g. In contrast, blue-light negative phototropism in roots, which was enhanced in microgravity compared with the 1g control, showed a significant attenuation at 0.3 g. In addition, our studies suggest that the well-known red-light enhancement of blue-light-induced phototropism in hypocotyls is likely due to an indirect effect by the attenuation of gravitropism. However, red-light enhancement of root blue-light-based phototropism may occur via a more direct effect on the phototropism system itself, most likely through the phytochrome photoreceptors. To our knowledge, these experiments represent the first to examine the behavior of flowering plants in fractional or reduced gravity conditions.
Spaceflight Study	International Space Station (ISS)	SLID-252	Plant growth strategies are remodeled by spaceflight	15 Days	Seedling	Background: Arabidopsis plants were grown on the International Space Station within specialized hardware that combined a plant growth habitat with a camera system that can capture images at regular intervals of growth. The Imaging hardware delivers telemetric data from the ISS, specifically images received in real-time from experiments on orbit, providing science without sample return. Comparable Ground Controls were grown in a sister unit that is maintained in the Orbital Environment Simulator at Kennedy Space Center. One of many types of biological data that can be analyzed in this fashion is root morphology. Arabidopsis seeds were geminated on orbit on nutrient gel Petri plates in a configuration that encouraged growth along the surface of the gel. Photos were taken every six hours for the 15 days of the experiment. Results: In the absence of gravity, but the presence of directional light, spaceflight roots remained strongly negatively phototropic and grew in the opposite direction of the shoot growth; however, cultivars WS and Col-0 displayed two distinct, marked differences in their growth patterns. First, cultivar WS skewed strongly to the right on orbit, while cultivar Col-0 grew with little deviation away from the light source. Second, the Spaceflight environment also impacted the rate of growth in Arabidopsis. The size of the Flight plants (as measured by primary root and hypocotyl length) was uniformly smaller than comparably aged Ground Control plants in both cultivars. Conclusions: Skewing and waving, thought to be gravity dependent phenomena, occur in spaceflight plants. In the presence of an orienting light source, phenotypic trends in skewing are gravity independent, and the general patterns of directional root growth typified by a given genotype in unit gravity are recapitulated on orbit, although overall growth patterns on orbit are less uniform. Skewing appears independent of axial orientation on the ISS - suggesting that other tropisms (such as for oxygen and temperature) do not influence skewing. An aspect of the spaceflight environment also retards the rate of early Arabidopsis growth.
Spaceflight Study	International Space Station (ISS)	SLID-253	Transcriptome analyses of Arabidopsis thaliana seedlings grown in space: implications for gravity-responsive genes	4 Days	Seedling	The transcriptome of seedlings was analyzed from experiments performed on the International Space Station to study the interacting effects of light and gravity on plant tropisms (project named TROPI-2; Kiss et al. 2012). Seeds of Arabidopsis were germinated in space, and seedlings were then grown in the European Modular Cultivation System for 4 days at ~1g followed by exposure to a range of gravitational accelerations (from microgravity to 1g) and two light treatments (blue light with or without a 1 h pretreatment with red). At the end of the experiments, the cassettes containing the seedlings were frozen in the minus eighty laboratory freezer and returned to Earth on space shuttle mission STS-131. The RNA was extracted from whole seedlings and used for the transcriptome analyses. A comparison of 1g spaceflight samples with 1g ground controls identified 230 genes that were differentially regulated at least twofold, emphasizing the need for "in situ" tissue fixation on a 1g centrifuge as an important control for spaceflight experiments. A further comparison of all spaceflight samples with ground controls identified approximately 280 genes that were differentially regulated at least twofold. Of these genes, several were involved in regulating cell polarity (i.e., auxin, calcium, lipid metabolism), cell-wall development, oxygen status, and cell defense or stress. However, when the transcriptome of the all g-treated spaceflight samples was compared with microgravity samples, only ~130 genes were identified as being differently regulated (P ≤ 0.01). Of this subset, only 27 genes were at least twofold differently regulated between microgravity and 1g space samples and included putative/pseudo/undefined genes (14), transposable elements (5), an expansin (ATEXP24; At1g21240), a cell-wall kinase (WAK3; At1g21240), a laccase-like flavonoid oxidase (TT10; At5g48100), among others.
Spaceflight Study	Space Shuttle Discovery (STS-131)	SLID-254	Transcriptional response of Arabidopsis seedlings during spaceflight reveals peroxidase and cell wall remodeling genes associated with root hair development	2 Weeks	Seedling	Premise of the study: Plants will be an important component of advanced life support systems during space exploration missions. Therefore, understanding their biology in the spacecraft environment will be essential before they can be used for such systems.• Methods: Seedlings of Arabidopsis thaliana were grown for 2 wk in the Biological Research in Canisters (BRIC) hardware on board the second to the last mission of the space shuttle Discovery (STS-131). Transcript profiles between ground controls and space-grown seedlings were compared using stringent selection criteria.• Key results: Expression of transcripts associated with oxidative stress and cell wall remodeling was repressed in microgravity. These downregulated genes were previously shown to be enriched in root hairs consistent with seedling phenotypes observed in space. Mutations in genes that were downregulated in microgravity, including two uncharacterized root hair-expressed class III peroxidase genes (PRX44 and PRX57), led to defective polar root hair growth on Earth. PRX44 and PRX57 mutants had ruptured root hairs, which is a typical phenotype of tip-growing cells with defective cell walls and those subjected to stress.• Conclusions: Long-term exposure to microgravity negatively impacts tip growth by repressing expression of genes essential for normal root hair development. Whereas changes in peroxidase gene expression leading to reduced root hair growth in space are actin-independent, root hair development modulated by phosphoinositides could be dependent on the actin cytoskeleton. These results have profound implications for plant adaptation to microgravity given the importance of tip growing cells such as root hairs for efficient nutrient capture.
Spaceflight Study	Space Shuttle Mission STS-131	SLID-255	Morphometric analyses of petioles of seedlings grown in a spaceflight experiment	14 Days	Seedling	Gravity is a constant unidirectional stimulus on Earth, and gravitropism in plants involves three phases: perception, transduction, and response. In shoots, perception takes place within the endodermis. To investigate the cellular machinery of perception in microgravity, we conducted a spaceflight study with Arabidopsis thaliana seedlings, which were grown in microgravity in darkness using the Biological Research in Canisters (BRIC) hardware during space shuttle mission STS-131. In the 14-day-old etiolated plants, we studied seedling development and the morphological parameters of the endodermal cells in the petiole. Seedlings from the spaceflight experiment (FL) were compared to a ground control (GC), which both were in the BRIC flight hardware. In addition, to assay any potential effects from growth in spaceflight hardware, we performed another control by growing seedlings in Petri dishes in standard laboratory conditions (termed the hardware control, HC). Seed germination was significantly lower in samples grown in flight hardware (FL, GC) compared to the HC. In terms of cellular parameters of endodermal cells, the greatest differences also were between seedlings grown in spaceflight hardware (FL, GC) compared to those grown outside of this hardware (HC). Specifically, the endodermal cells were significantly smaller in seedlings grown in the BRIC system compared to those in the HC. However, a change in the shape of the cell, suggesting alterations in the cell wall, was one parameter that appears to be a true microgravity effect. Taken together, our results suggest that caution must be taken when interpreting results from the increasingly utilized BRIC spaceflight hardware system and that it is important to perform additional ground controls to aid in the analysis of spaceflight experiments.
Spaceflight Study	STS-131	SLID-256	Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight	309 Hours	Seedling	Premise of the study: Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. Methods: We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). Key results: In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. Conclusions: A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants.
Spaceflight Study	International Space Station (ISS)	SLID-257	The combined effects of real or simulated microgravity and red-light photoactivation on plant root meristematic cells	n.a.	Seedling	Red light is able to compensate for deleterious effects of microgravity on root cell growth and proliferation. Partial gravity combined with red light produces differential signals during the early plant development. Light and gravity are environmental cues used by plants throughout evolution to guide their development. We have investigated the cross-talk between phototropism and gravitropism under altered gravity in space. The focus was on the effects on the meristematic balance between cell growth and proliferation, which is disrupted under microgravity in the dark. In our spaceflight experiments, seedlings of three Arabidopsis thaliana genotypes, namely the wild type and mutants of phytochrome A and B, were grown for 6 days, including red-light photoactivation for the last 2 days. Apart from the microgravity and the 1g on-board control conditions, fractional gravity (nominally 0.1g, 0.3g, and 0.5g) was created with on-board centrifuges. In addition, a simulated microgravity (random positioning machine, RPM) experiment was performed on ground, including both dark-grown and photostimulated samples. Photoactivated samples in spaceflight and RPM experiments showed an increase in the root length consistent with phototropic response to red light, but, as gravity increased, a gradual decrease in this response was observed. Uncoupling of cell growth and proliferation was detected under microgravity in darkness by transcriptomic and microscopic methods, but red-light photoactivation produced a significant reversion. In contrast, the combination of red light and partial gravity produced small but consistent variations in the molecular markers of cell growth and proliferation, suggesting an antagonistic effect between light and gravity signals at the early plant development. Understanding these parameters of plant growth and development in microgravity will be important as bioregenerative life support systems for the colonization of the Moon and Mars.
Spaceflight Study	Chinese Recoverable Scientific Satellite SJ-10	SLID-258	Single-base resolution methylome analysis shows epigenetic changes in Arabidopsis seedlings exposed to microgravity spaceflight conditions on board the SJ-10 recoverable satellite	60 Hours	Seedling	DNA methylation is a very important epigenetic modification that participates in many biological functions. Although many studies of DNA methylation have been reported in various plant species, few studies have assessed the global DNA methylation pattern in plants challenged by exposure to microgravity conditions. In this report, we mapped the Arabidopsis genome methylation pattern changes associated with microgravity conditions on board the Chinese recoverable scientific satellite SJ-10 at single-base resolution. Interestingly, we found epigenetic differences in Arabidopsis seedlings exposed to microgravity in that the Arabidopsis genome exhibits lower methylation levels in the CHG, CHH, and CpG contexts under microgravity conditions. Microgravity stimulation was related to altered methylation of a number of genes, including DNA methylation-associated genes, hormone signaling related genes, cell-wall modification genes and transposable elements (TEs). Relatively unstable DNA methylation of TEs was responsible for the induction of active transposons. These observations suggest that DNA demethylation within TEs may affect the transcription of transposons in response to microgravity conditions. In summary, the results of this investigation are beneficial for understanding the mechanism of plant adaptation to microgravity and improve strategies to allow plants to adapt to space.
Spaceflight Study	n.a.	SLID-259	Comparing RNA-Seq and microarray gene expression data in two zones of the Arabidopsis root apex relevant to spaceflight	n.a.	Seedling	Premise of the study: The root apex is an important region involved in environmental sensing, but comprises a very small part of the root. Obtaining root apex transcriptomes is therefore challenging when the samples are limited. The feasibility of using tiny root sections for transcriptome analysis was examined, comparing RNA sequencing (RNA-Seq) to microarrays in characterizing genes that are relevant to spaceflight. Methods: Arabidopsis thaliana Columbia ecotype (Col-0) roots were sectioned into Zone 1 (0.5 mm; root cap and meristematic zone) and Zone 2 (1.5 mm; transition, elongation, and growth-terminating zone). Differential gene expression in each was compared. Results: Both microarrays and RNA-Seq proved applicable to the small samples. A total of 4180 genes were differentially expressed (with fold changes of 2 or greater) between Zone 1 and Zone 2. In addition, 771 unique genes and 19 novel transcriptionally active regions were identified by RNA-Seq that were not detected in microarrays. However, microarrays detected spaceflight-relevant genes that were missed in RNA-Seq. Discussion: Single root tip subsections can be used for transcriptome analysis using either RNA-Seq or microarrays. Both RNA-Seq and microarrays provided novel information. These data suggest that techniques for dealing with small, rare samples from spaceflight can be further enhanced, and that RNA-Seq may miss some spaceflight-relevant changes in gene expression.
Spaceflight Study	SpaceX Mission CRS-4	SLID-260	Variation in the transcriptome of different ecotypes of Arabidopsis thaliana reveals signatures of oxidative stress in plant responses to spaceflight	8 Days	Seedling	Premise of the study: Spaceflight provides a unique environment in which to dissect plant stress response behaviors and to reveal potentially novel pathways triggered in space. We therefore analyzed the transcriptomes of Arabidopsis thaliana plants grown on board the International Space Station to find the molecular fingerprints of these space-related response networks. Methods: Four ecotypes (Col-0, Ws-2, Ler-0 and Cvi-0) were grown on orbit and then their patterns of transcript abundance compared to ground-based controls using RNA sequencing. Key results: Transcripts from heat-shock proteins were upregulated in all ecotypes in spaceflight, whereas peroxidase transcripts were downregulated. Among the shared and ecotype-specific changes, gene classes related to oxidative stress and hypoxia were detected. These spaceflight transcriptional response signatures could be partly mimicked on Earth by a low oxygen environment and more fully by oxidative stress (H2O2 ) treatments. Conclusions: These results suggest that the spaceflight environment is associated with oxidative stress potentially triggered, in part, by hypoxic response. Further, a shared spaceflight response may be through the induction of molecular chaperones (such as heat shock proteins) that help protect cellular machinery from the effects of oxidative damage. In addition, this research emphasizes the importance of considering the effects of natural variation when designing and interpreting changes associated with spaceflight experiments.
Spaceflight Study	International Space Station (ISS)	SLID-261	Epigenomics in an extraterrestrial environment: organ-specific alteration of DNA methylation and gene expression elicited by spaceflight in Arabidopsis thaliana	11 Days	Seedling	Background: Plants adapted to diverse environments on Earth throughout their evolutionary history, and developed mechanisms to thrive in a variety of terrestrial habitats. When plants are grown in the novel environment of spaceflight aboard the International Space Station (ISS), an environment completely outside their evolutionary history, they respond with unique alterations to their gene expression profile. Identifying the genes important for physiological adaptation to spaceflight and dissecting the biological processes and pathways engaged by plants during spaceflight has helped reveal spaceflight adaptation, and has furthered understanding of terrestrial growth processes. However, the underlying regulatory mechanisms responsible for these changes in gene expression patterns are just beginning to be explored. Epigenetic modifications, such as DNA methylation at position five in cytosine, has been shown to play a role in the physiological adaptation to adverse terrestrial environments, and may play a role in spaceflight as well. Results: Whole Genome Bisulfite Sequencing of DNA of Arabidopsis grown on the ISS from seed revealed organ-specific patterns of differential methylation compared to ground controls. The overall levels of methylation in CG, CHG, and CHH contexts were similar between flight and ground DNA, however, thousands of specifically differentially methylated cytosines were discovered, and there were clear organ-specific differences in methylation patterns. Spaceflight leaves had higher methylation levels in CHG and CHH contexts within protein-coding genes in spaceflight; about a fifth of the leaf genes were also differentially regulated in spaceflight, almost half of which were associated with reactive oxygen signaling. Conclusions: The physiological adaptation of plants to spaceflight is likely nuanced by epigenomic modification. This is the first examination of differential genomic methylation from plants grown completely in the spaceflight environment of the ISS in plant growth hardware developed for informing exploration life support strategies. Yet even in this optimized plant habitat, plants respond as if stressed. These data suggest that gene expression associated with physiological adaptation to spaceflight is regulated in part by methylation strategies similar to those engaged with familiar terrestrial stress responses. The differential methylation maps generated here provide a useful reference for elucidating the layers of regulation of spaceflight responses.
Spaceflight Study	n.a.	SLID-262	Spaceflight-induced alternative splicing during seedling development in Arabidopsis thaliana	n.a.	Seedling	Plants grown in spaceflight experience novel environmental signals, including those associated with microgravity and ionizing radiation. Spaceflight triggers a response involving transcriptional re-programming and altered cell morphology, though many aspects of this response remain uncharacterized. We analyzed the spaceflight-induced transcriptome with a focus on genes that undergo alternative splicing to examine differential splicing associated with spaceflight-an unstudied characteristic of the molecular response to spaceflight exposure. RNA sequence data obtained during the APEX03 spaceflight experiment that was collected from two Arabidopsis thaliana ecotypes at two seedling stages grown onboard the International Space Station, or as ground controls at Kennedy Space Center, were re-examined to detect alternative splicing differences induced by spaceflight. Presence/absence variation analysis was used to identify putative expression-level differences in alternatively spliced isoforms between spaceflight and ground controls and was followed by analysis of significant differential alternative splicing. This study provides the first evidence of a role for alternative splicing in the molecular processes of physiological adaptation to the spaceflight environment.
Spaceflight Study	SpaceX Mission CRS-5	SLID-263	Root Skewing-Associated Genes Impact the Spaceflight Response of Arabidopsis thaliana	n.a.	Seedling	The observation that plant roots skew in microgravity recently refuted the long-held conviction that skewing was a gravity-dependent phenomenon. Further, spaceflight root skewing suggests that specific root morphologies and cell wall remodeling systems may be important aspects of spaceflight physiological adaptation. However, connections between skewing, cell wall modification and spaceflight physiology are currently based on inferences rather than direct tests. Therefore, the Advanced Plant Experiments-03-2 (APEX-03-2) spaceflight study was designed to elucidate the contribution of two skewing- and cell wall-associated genes in Arabidopsis to root behavior and gene expression patterns in spaceflight, to assess whether interruptions of different skewing pathways affect the overall spaceflight-associated process. SPIRAL1 is a skewing-related protein implicated in directional cell expansion, and functions by regulating cortical microtubule dynamics. SKU5 is skewing-related glycosylphosphatidylinositol-anchored protein of the plasma membrane and cell wall implicated in stress response signaling. These two genes function in different cellular pathways that affect skewing on the Earth, and enable a test of the relevance of skewing pathways to spaceflight physiological adaptation. In this study, both sku5 and spr1 mutants showed different skewing behavior and markedly different patterns of gene expression in the spaceflight environment. The spr1 mutant showed fewer differentially expressed genes than its Col-0 wild-type, whereas sku5 showed considerably more than its WS wild-type. Developmental age played a substantial role in spaceflight acclimation in all genotypes, but particularly in sku5 plants, where spaceflight 4d seedlings had almost 10-times as many highly differentially expressed genes as the 8d seedlings. These differences demonstrated that the two skewing pathways represented by SKU5 and SPR1 have unique and opposite contributions to physiological adaptation to spaceflight. The spr1 response is less intense than wild type, suggesting that the loss of SPR1 positively impacts spaceflight adaptation. Conversely, the intensity of the sku5 responses suggests that the loss of SKU5 initiates a much more complex, deeper and more stress related response to spaceflight. This suggests that proper SKU5 function is important to spaceflight adaptation.
Spaceflight Study	SpaceX 5	SLID-264	Spaceflight induces novel regulatory responses in Arabidopsis seedling as revealed by combined proteomic and transcriptomic analyses	n.a.	Seedling	Background: Understanding of gravity sensing and response is critical to long-term human habitation in space and can provide new advantages for terrestrial agriculture. To this end, the altered gene expression profile induced by microgravity has been repeatedly queried by microarray and RNA-seq experiments to understand gravitropism. However, the quantification of altered protein abundance in space has been minimally investigated. Results: Proteomic (iTRAQ-labelled LC-MS/MS) and transcriptomic (RNA-seq) analyses simultaneously quantified protein and transcript differential expression of three-day old, etiolated Arabidopsis thaliana seedlings grown aboard the International Space Station along with their ground control counterparts. Protein extracts were fractionated to isolate soluble and membrane proteins and analyzed to detect differentially phosphorylated peptides. In total, 968 RNAs, 107 soluble proteins, and 103 membrane proteins were identified as differentially expressed. In addition, the proteomic analyses identified 16 differential phosphorylation events. Proteomic data delivered novel insights and simultaneously provided new context to previously made observations of gene expression in microgravity. There is a sweeping shift in post-transcriptional mechanisms of gene regulation including RNA-decapping protein DCP5, the splicing factors GRP7 and GRP8, and AGO4,. These data also indicate AHA2 and FERONIA as well as CESA1 and SHOU4 as central to the cell wall adaptations seen in spaceflight. Patterns of tubulin-α 1, 3,4 and 6 phosphorylation further reveal an interaction of microtubule and redox homeostasis that mirrors osmotic response signaling elements. The absence of gravity also results in a seemingly wasteful dysregulation of plastid gene transcription. Conclusions: The datasets gathered from Arabidopsis seedlings exposed to microgravity revealed marked impacts on post-transcriptional regulation, cell wall synthesis, redox/microtubule dynamics, and plastid gene transcription. The impact of post-transcriptional regulatory alterations represents an unstudied element of the plant microgravity response with the potential to significantly impact plant growth efficiency and beyond. What's more, addressing the effects of microgravity on AHA2, CESA1, and alpha tubulins has the potential to enhance cytoskeletal organization and cell wall composition, thereby enhancing biomass production and growth in microgravity. Finally, understanding and manipulating the dysregulation of plastid gene transcription has further potential to address the goal of enhancing plant growth in the stressful conditions of microgravity.
Spaceflight Study	Orbiter Columbia (STS-93)	SLID-265	Arabidopsis gene expression patterns are altered during spaceflight	5 Days	Seedling	The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the b-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K.,Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613–621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman!). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced – but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment opportunities.
Spaceflight Study	International Space Station (ISS)	SLID-266	Operations of a spaceflight experiment to investigate plant tropisms	n.a.	Seedling	Plants will be an important component in bioregenerative systems for long-term missions to the Moon and Mars. Since gravity is reduced both on the Moon and Mars, studies that identify the basic mechanisms of plant growth and development in altered gravity are required to ensure successful plant production on these space colonization missions. To address these issues, we have developed a project on the International Space Station (ISS) to study the interaction between gravitropism and phototropism in Arabidopsis thaliana. These experiments were termed TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS) in 2006. In this paper, we provide an operational summary of TROPI and preliminary results on studies of tropistic curvature of seedlings grown in space. Seed germination in TROPI was lower compared to previous space experiments, and this was likely due to extended storage in hardware for up to 8 months. Video downlinks provided an important quality check on the automated experimental time line that also was monitored with telemetry. Good quality images of seedlings were obtained, but the use of analog video tapes resulted in delays in image processing and analysis procedures. Seedlings that germinated exhibited robust phototropic curvature. Frozen plant samples were returned on three space shuttle missions, and improvements in cold stowage and handing procedures in the second and third missions resulted in quality RNA extracted from the seedlings that was used in subsequent microarray analyses. While the TROPI experiment had technical and logistical difficulties, most of the procedures worked well due to refinement during the project.
Spaceflight Study	Shijian-8	SLID-267	Assessment of genetic diversity and variation of Robinia pseudoacacia seeds induced by short-term spaceflight based on two molecular marker systems and morphological traits	15 Days	Seed	The black locust (Robinia pseudoacacia) is a forest legume that is highly valued as a honey plant and for its wood. We explored the effect of short-term spaceflight on development of R. pseudoacacia seedlings derived from seeds that endured a 15-day flight; the genetic diversity and variation of plants sampled from space-mutagenized seeds were compared to plants from parallel ground-based control seeds using molecular markers and morphological traits. In the morphology analysis, the space-mutagenized group had apparent variation compared with the control group in morphological traits, including plant height, basal diameter, number of branches, branch stipular thorn length, branch stipular thorn middle width, leaflet vertex angle, and tippy leaf vertex angle. Simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular marker analyses showed a slightly higher levels of genetic diversity in the space-mutagenized group compared to the control group. In the SRAP analysis, the space-mutagenized group had 115 polymorphic bands vs 98 in the controls; 91.27% polymorphic loci vs 77.78% in the controls; 1.9127 ± 0.2834 alleles vs 1.7778 ± 0.4174 in the controls; Nei's genetic diversity (h) was 0.2930 ± 0.1631 vs 0.2688 ± 0.1862 in the controls, and the Shannon's information index (I) was 0.4452 ± 0.2177 vs 0.4031 ± 0.2596 in the controls. The number of alleles was significantly higher in the space-mutagenized group. In the SSR analysis, the space-mutagenized group also had more polymorphic bands (51 vs 46), a greater percentage of polymorphic loci (89.47% vs 80.70%); h was also higher (0.2534 ± 0.1533 vs 0.2240 ± 0.1743), as was I (0.3980 ± 0.2069 vs 0.3501 ± 0.2412). These results demonstrated that the range of genetic variation in the populations of R. pseudoacacia increased after spaceflight. It also suggested that the SSR and SRAP markers are effective markers for studying mutations and genetic diversity in R. pseudoacacia. The data provide valuable molecular evidence for the effects of the space environment on R. pseudoacacia and may contribute to future space-breeding programs involving forest trees.
Spaceflight Study	Mir Space Station	SLID-268	Gravity independence of seed-to-seed cycling in Brassica rapa	13 Days	Oil crop	Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Spaceflight Study	Mir Space Station	SLID-268	Gravity independence of seed-to-seed cycling in Brassica rapa	52 Days	Oil crop	Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Spaceflight Study	Mir Space Station	SLID-268	Gravity independence of seed-to-seed cycling in Brassica rapa	54 Days	Oil crop	Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Spaceflight Study	Mir Space Station (NASA-5)	SLID-269	Influence of microgravity on ultrastructure and storage reserves in seeds of Brassica rapa L	n.a.	Vegetable	Successful plant reproduction under spaceflight conditions has been problematic in the past. During a 122 d opportunity on the Mir space station, full life cycles of Brassica rapa L. were completed in microgravity in a series of three experiments in the Svet greenhouse. Ultrastructural and cytochemical analyses of storage reserves in mature dry seeds produced in these experiments were compared with those of seeds produced during a high-fidelity ground control. Additional analyses were performed on developing Brassica embryos, 15 d post pollination, which were produced during a separate experiment on the Shuttle (STS-87). Seeds produced on Mir had less than 20% of the cotyledon cell number found in seeds harvested from the ground control. Cytochemical localization of storage reserves in mature cotyledons showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in ground control seeds. Protein bodies in mature cotyledons produced in space were 44% smaller than those in the ground control seeds. Fifteen days after pollination, cotyledon cells from mature embryos formed in space had large numbers of starch grains, and protein bodies were absent, while in developing ground control seeds at the same stage, protein bodies had already formed and fewer starch grains were evident. These data suggest that both the late stage of seed development and maturation are changed in Brassica by growth in a microgravity environment. While gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Spaceflight Study	STS-87	SLID-269	Influence of microgravity on ultrastructure and storage reserves in seeds of Brassica rapa L	16 Days	Vegetable	Successful plant reproduction under spaceflight conditions has been problematic in the past. During a 122 d opportunity on the Mir space station, full life cycles of Brassica rapa L. were completed in microgravity in a series of three experiments in the Svet greenhouse. Ultrastructural and cytochemical analyses of storage reserves in mature dry seeds produced in these experiments were compared with those of seeds produced during a high-fidelity ground control. Additional analyses were performed on developing Brassica embryos, 15 d post pollination, which were produced during a separate experiment on the Shuttle (STS-87). Seeds produced on Mir had less than 20% of the cotyledon cell number found in seeds harvested from the ground control. Cytochemical localization of storage reserves in mature cotyledons showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in ground control seeds. Protein bodies in mature cotyledons produced in space were 44% smaller than those in the ground control seeds. Fifteen days after pollination, cotyledon cells from mature embryos formed in space had large numbers of starch grains, and protein bodies were absent, while in developing ground control seeds at the same stage, protein bodies had already formed and fewer starch grains were evident. These data suggest that both the late stage of seed development and maturation are changed in Brassica by growth in a microgravity environment. While gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.
Spaceflight Study	n.a.	SLID-270	Analysis of influences of spaceflight on chemical constituents in licorice by HPLC–ESI-MS/MS	18 Days	Seed	Focusing on the variations of chemical constituents in licorice root, influences of exposure to physical factors of spaceflight on licorice (Glycyrrhiza uralensis Fisch.) seeds were investigated. Licorice seeds obtained from two different producing areas were flown on a recoverable satellite for 18 days. After returning to earth, the seeds carried by the satellite and the parallel ground control were cultivated to maturity under the same condition. Chromatographic fingerprint of 1 year licorice root analyzed by high performance liquid chromatography with diode-array detection not only displayed the contents of glycyrrhizic acid and liquiritin increasing in the spaceflight samples but showed the variation of the kinds of chemical constituents. The main components in the root extract were identified by high performance liquid chromatography coupled with electrospray ionization multi-tandem mass spectrometry. The changes in the kind of secondary metabolites of licorice root after spaceflight were firstly reported. A total of 26 components which included 9 flavonoids, 16 triterpene saponins and 1 coumarin were identified according to their mass spectra determined in both negative and positive ion modes. The research provided the scientific data for spaceflight breeding of medicinal plant and indicated that the technology of spaceflight may be a new effective method for the breeding and cultivation of licorice.
Spaceflight Study	Shenzhou 7 Space Flight	SLID-271	Spaceflight environment-induced variation in root yield and active constituents of Salvia miltiorrhiza	68.45 Hours	Seed	Salvia miltiorrhiza is a significant source of bioactive compounds providing human health effects. Here, we surveyed root yield and the active constituents' divergences of second generation S. miltiorrhiza (SP2) responding to a spaceflight environment. High-performance liquid chromatography was conducted for the comprehensive constituents' characterizations of 28 SP2 lines (224 individuals) and the ground control (eight individuals). The results showed that the mean fresh and dry weight of roots ranged from 116 to 172 g and 25 to 119 g, respectively, in SP2 lines. In addition, the mean contents of four tanshinone compounds (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) of 28 SP2 lines varied from 0.32 to 1.04 mg · g(-1), 0.47 to 2.39 mg · g(-1), 0.25 to 1.60 mg · g(-1), and 0.53 to 1.67 mg · g(-1), respectively. Except for salvianolic acid B, which varied drastically from 72 % to 201 % of the ground control treatment, the other six phenolic acid contents of the 28 SP2 lines all increased after spaceflight. Principal component analysis was performed to obtain an overview of the distribution of all samples, and score plots clearly separated the SP2 accessions from ground controls. Moreover, a positive relationship was observed between tanshinone I and tanshinone IIA (r = 0.790, p < 0.01), and rosmarinic acid was positively correlated with salvianolic acid B (r = 0.728, p < 0.01). In conclusion, this study demonstrated that a spaceflight environment induced SP2 accessions remarkably in the variation of root yield and active constituent content.
Spaceflight Study	International Space Station (ISS)	SLID-272	Seed Storage Reserves and Glucosinolates in Brassica rapa L. Grown on the International Space Station	n.a.	Vegetable	Although plants are envisioned to play a central role in life support systems for future long-duration space travel, plant growth in space has been problematic due to horticultural problems of nutrient delivery and gas resupply posed by the weightless environment. Iterative improvement in hardware designed for growth of plants on orbital platforms now provides confidence that plants can perform well in microgravity, enabling investigation of their nutritional characteristics. Plants of B. rapa (cv. Astroplants) were grown in the Biomass Production System on the International Space Station. Flowers were hand-pollinated and seeds were produced prior to harvest at 39 days after planting. The material was frozen or fixed while on orbit and subsequently analyzed in our laboratories. Gross measures of growth, leaf chlorophyll, starch and soluble carbohydrates confirmed comparable performance by the plants in spaceflight and ground control treatments. Analysis of glucosinolate production in the plant stems indicated that 3-butenylglucosinolate concentration was on average 75% greater in flight samples than in ground control samples. Similarly, the biochemical make-up of immature seeds produced during spaceflight and fixed or frozen while in orbit was significantly different from the ground controls. The immature seeds from the spaceflight treatment had higher concentrations of chlorophyll, starch, and soluble carbohydrates than the ground controls. Seed protein was significantly lower in the spaceflight material. Microscopy of immature seeds fixed in flight showed embryos to be at a range of developmental stages, while the ground control embryos had all reached the premature stage of development. Storage reserve deposition was more advanced in the ground control seeds. The spaceflight environment thus influences B. rapa metabolite production in ways that may affect flavor and nutritional quality of potential space produce.
Spaceflight Study	Shuttle Transport System (STS)-110 and International Space Station (ISS)	SLID-272	Seed Storage Reserves and Glucosinolates in Brassica rapa L. Grown on the International Space Station	n.a.	Vegetable	Although plants are envisioned to play a central role in life support systems for future long-duration space travel, plant growth in space has been problematic due to horticultural problems of nutrient delivery and gas resupply posed by the weightless environment. Iterative improvement in hardware designed for growth of plants on orbital platforms now provides confidence that plants can perform well in microgravity, enabling investigation of their nutritional characteristics. Plants of B. rapa (cv. Astroplants) were grown in the Biomass Production System on the International Space Station. Flowers were hand-pollinated and seeds were produced prior to harvest at 39 days after planting. The material was frozen or fixed while on orbit and subsequently analyzed in our laboratories. Gross measures of growth, leaf chlorophyll, starch and soluble carbohydrates confirmed comparable performance by the plants in spaceflight and ground control treatments. Analysis of glucosinolate production in the plant stems indicated that 3-butenylglucosinolate concentration was on average 75% greater in flight samples than in ground control samples. Similarly, the biochemical make-up of immature seeds produced during spaceflight and fixed or frozen while in orbit was significantly different from the ground controls. The immature seeds from the spaceflight treatment had higher concentrations of chlorophyll, starch, and soluble carbohydrates than the ground controls. Seed protein was significantly lower in the spaceflight material. Microscopy of immature seeds fixed in flight showed embryos to be at a range of developmental stages, while the ground control embryos had all reached the premature stage of development. Storage reserve deposition was more advanced in the ground control seeds. The spaceflight environment thus influences B. rapa metabolite production in ways that may affect flavor and nutritional quality of potential space produce.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-273	Transcriptome Analysis of Oryza sativa Calli Under Microgravity	17 Days	Grain	The transcriptome of Oryza sativacalli was analyzed on board the Chinese spaceship “Shenzhou 8” to study the effects of microgravity on plant signal transduction and secondary metabolism (as one of the experiments with SIMBOX on Shenzhou 8). Calli of Oryza sativa were pre-cultured for 4 days on ground and then loaded into the stationary platform or the rotating platform of a biological incubator, called SIMBOX, to grow in space under microgravity conditions or 1g-conditions, respectively. The calli were fixed by RNAlater after grew 324 h under microgravity. After 17 days, Shenzhou 8 returned to Earth carrying SIMBOX. Oryza sativa calli were recovered, and the RNA was extracted for transcriptome analysis. After comparing 1 gspaceflight controls-inflight controls with 1 g-ground controls, 157 probe sets with different expression levels (fold change ≥2, p<0.05) were identified. When comparing spaceflight controls to 1 g-ground controls and to 1 g-inflight controls, 678 probe sets with different expression levels (fold change ≥2, p<0.05) were identified. The fact that the same 678 probe sets were identified in these two comparisons suggests that transcription was affected under microgravity conditions. MapMan analysis was used to classify 627 microgravity responsive (MR) transcripts. The MR transcripts were mainly involved in cell wall structure, the TCA cycle, primary metabolism, transcription, protein modification and degradation, hormone metabolism, calcium regulation, receptor like kinase activity and transport.
Spaceflight Study	STS-51 (Discovery)	SLID-274	Plant reproduction during spaceflight: importance of the gaseous environment	10 Days	Plant	Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.
Spaceflight Study	STS-54 (Endeavour)	SLID-274	Plant reproduction during spaceflight: importance of the gaseous environment	6 Days	Plant	Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.
Spaceflight Study	STS-68 (Endeavour)	SLID-274	Plant reproduction during spaceflight: importance of the gaseous environment	11 Days	Plant	Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.
Spaceflight Study	International Space Station (ISS)	SLID-275	Seed-to-seed-to-seed growth and development of Arabidopsis in microgravity	46 Days	Seed to seed to seed	Arabidopsis thaliana was grown from seed to seed wholly in microgravity on the International Space Station. Arabidopsis plants were germinated, grown, and maintained inside a growth chamber prior to returning to Earth. Some of these seeds were used in a subsequent experiment to successfully produce a second (back-to-back) generation of microgravity-grown Arabidopsis. In general, plant growth and development in microgravity proceeded similarly to those of the ground controls, which were grown in an identical chamber. Morphologically, the most striking feature of space-grown Arabidopsis was that the secondary inflorescence branches and siliques formed nearly perpendicular angles to the inflorescence stems. The branches grew out perpendicularly to the main inflorescence stem, indicating that gravity was the key determinant of branch and silique angle and that light had either no role or a secondary role in Arabidopsis branch and silique orientation. Seed protein bodies were 55% smaller in space seed than in controls, but protein assays showed only a 9% reduction in seed protein content. Germination rates for space-produced seed were 92%, indicating that the seeds developed in microgravity were healthy and viable. Gravity is not necessary for seed-to-seed growth of plants, though it plays a direct role in plant form and may influence seed reserves.
Spaceflight Study	STS-122; STS-128;STS 133;Russian Progress spacecraft	SLID-276	Survival and DNA Damage in Plant Seeds Exposed for 558 and 682 Days outside the International Space Station	558 Days	Seed	For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV<200nm, and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV254nm, ranging as high as 2420 MJ m-2. Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel.
Spaceflight Study	STS-122; STS-128;STS 133;Russian Progress spacecraft	SLID-276	Survival and DNA Damage in Plant Seeds Exposed for 558 and 682 Days outside the International Space Station	682 Days	Seed	For life to survive outside the biosphere, it must be protected from UV light and other radiation by exterior shielding or through sufficient inherent resistance to survive without protection. We tested the plausibility of inherent resistance in plant seeds, reporting in a previous paper that Arabidopsis thaliana and tobacco (Nicotiana tabacum) seeds exposed for 558 days outside the International Space Station (ISS) germinated and developed into fertile plants after return to Earth. We have now measured structural genetic damage in tobacco seeds from this EXPOSE-E experiment by quantitatively amplifying a segment of an antibiotic resistance gene, nptII, inserted into the chloroplast genome. We also assessed the survival of the antibiotic resistance encoded by nptII, using marker rescue in a soil bacterium. Chloroplast DNA damage occurred, but morphological mutants were not detected among the survivors. In a second, longer mission (EXPOSE-R), a nearly lethal exposure was received by Arabidopsis seeds. Comparison between a ground simulation, lacking UV<200nm, and fully exposed seeds in space indicated severe damage from these short wavelengths and again suggested that DNA degradation was not limiting seed survival. To test UV resistance in long-lived, larger seeds, we exposed Arabidopsis, tobacco, and morning glory seeds in the laboratory to doses of UV254nm, ranging as high as 2420 MJ m-2. Morning glory seeds resisted this maximum dose, which killed tobacco and Arabidopsis. We thus confirm that a naked plant seed could survive UV exposures during direct transfer from Mars to Earth and suggest that seeds with a more protective seed coat (e.g., morning glory) should survive much longer space travel.
Spaceflight Study	Shenzhou 3 Space Flight	SLID-277	A crinkly leaf and delay flowering mutant of tobacco obtained from recoverable satellite-flown seeds	162 Hours	Seed	Dry seeds of Nicotiana tabacum (L) cv. K346 were flown with a recoverable satellite, the Chinese “Shen Zhou III” for 162 h. After spaceflight, a crinkly leaf and delay flowering mutant of tobacco (T-cldf), which phenotype differed from the ground control (K346), was obtained from the seedlings after 48 d of the recoverable satellite-flown seeds germination. Major characteristics of T-cldf phenotype included crinkly leaf with outgrowth of the adaxial surface among the secondary veins and delay flowering. Amplified fragment length polymorphism (AFLP) analysis showed that five polymorphic bands were detected between T-cldf and ground control. The results suggested that recoverable satellite-flown condition could bring inheritable mutagenic effects on tobacco seeds and maybe used as a tool for accelerating the progress in tobacco breeding.
Spaceflight Study	Space Shuttle STS-60	SLID-278	Early Root Cap Development and Graviresponse in White Clover (Trifolium repens) Grown in Space and on a Two-Axis Clinostat	n.a.	Seedling	White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.
Spaceflight Study	STS-63	SLID-278	Early Root Cap Development and Graviresponse in White Clover (Trifolium repens) Grown in Space and on a Two-Axis Clinostat	n.a.	Seedling	White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.
Spaceflight Study	International Space Station (ISS)	SLID-279	Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station	12 Days	Seedling	The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.
Spaceflight Study	International Space Station (ISS), (launched to the ISS on STS-129, returned on STS-130)	SLID-280	Spaceflight induces specific alterations in the proteomes of Arabidopsis	12 Days	Seedling	Life in spaceflight demonstrates remarkable acclimation processes within the specialized habitats of vehicles subjected to the myriad of unique environmental issues associated with orbital trajectories. To examine the response processes that occur in plants in space, leaves and roots from Arabidopsis (Arabidopsis thaliana) seedlings from three GFP reporter lines that were grown from seed for 12 days on the International Space Station and preserved on orbit in RNAlater were returned to Earth and analyzed by using iTRAQ broad-scale proteomics procedures. Using stringent criteria, we identified over 1500 proteins, which included 1167 leaf proteins and 1150 root proteins we were able to accurately quantify. Quantification revealed 256 leaf proteins and 358 root proteins that showed statistically significant differential abundance in the spaceflight samples compared to ground controls, with few proteins differentially regulated in common between leaves and roots. This indicates that there are measurable proteomics responses to spaceflight and that the responses are organ-specific. These proteomics data were compared with transcriptome data from similar spaceflight samples, showing that there is a positive but limited relationship between transcriptome and proteome regulation of the overall spaceflight responses of plants. These results are discussed in terms of emergence understanding of plant responses to spaceflight particularly with regard to cell wall remodeling, as well as in the context of deriving multiple omics data sets from a single on-orbit preservation and operations approach.
Spaceflight Study	International Space Station (ISS), GENARA A experiment STS-132	SLID-281	Microsome-associated proteome modifications of Arabidopsis seedlings grown on board the International Space Station reveal the possible effect on plants of space stresses other than microgravity	n.a.	Seedling	Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity.
Spaceflight Study	International Space Station (ISS)	SLID-282	A novel blue-light phototropic response is revealed in roots of Arabidopsis thaliana in microgravity	n.a.	Seedling	Blue-light positive phototropism in roots is masked by gravity and revealed in conditions of microgravity. In addition, the magnitude of red-light positive phototropic curvature is correlated to the magnitude of gravity. Due to their sessile nature, plants utilize environmental cues to grow and respond to their surroundings. Two of these cues, light and gravity, play a substantial role in plant orientation and directed growth movements (tropisms). However, very little is currently known about the interaction between light- (phototropic) and gravity (gravitropic)-mediated growth responses. Utilizing the European Modular Cultivation System on board the International Space Station, we investigated the interaction between phototropic and gravitropic responses in three Arabidopsis thaliana genotypes, Landsberg wild type, as well as mutants of phytochrome A and phytochrome B. Onboard centrifuges were used to create a fractional gravity gradient ranging from reduced gravity up to 1g. A novel positive blue-light phototropic response of roots was observed during conditions of microgravity, and this response was attenuated at 0.1g. In addition, a red-light pretreatment of plants enhanced the magnitude of positive phototropic curvature of roots in response to blue illumination. In addition, a positive phototropic response of roots was observed when exposed to red light, and a decrease in response was gradual and correlated with the increase in gravity. The positive red-light phototropic curvature of hypocotyls when exposed to red light was also confirmed. Both red-light and blue-light phototropic responses were also shown to be affected by directional light intensity. To our knowledge, this is the first characterization of a positive blue-light phototropic response in Arabidopsis roots, as well as the first description of the relationship between these phototropic responses in fractional or reduced gravities.
Spaceflight Study	International Space Station (ISS)	SLID-283	Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight?	n.a.	Seedling	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 establishing the spaceflight responses are being identified, their roles 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 actually 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 in the root tips 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 plants 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 cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could 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 implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.
Spaceflight Study	SpaceX Mission CRS-3	SLID-283	Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight?	11 Days	Seedling	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 establishing the spaceflight responses are being identified, their roles 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 actually 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 in the root tips 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 plants 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 cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could 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 implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.
Spaceflight Study	SpaceX Mission CRS-3	SLID-284	The effect of spaceflight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit	n.a.	Seedling	Our primary aim was to determine whether gravity has a direct role in establishing the auxin-mediated gravity-sensing system in primary roots. Major plant architectures have long been thought to be guided by gravity, including the directional growth of the primary root via auxin gradients that are then disturbed when roots deviate from the vertical as a gravity sensor. However, experiments on the International Space Station (ISS) now allow physical clarity with regard to any assumptions regarding the role of gravity in establishing fundamental root auxin distributions. We examined the spaceflight green fluorescent protein (GFP)-reporter gene expression in roots of transgenic lines of Arabidopsis thaliana: pDR5r::GFP, pTAA1::TAA1-GFP, pSCR::SCR-GFP to monitor auxin and pARR5::GFP to monitor cytokinin. Plants on the ISS were imaged live with the Light Microscopy Module (LMM), and compared with control plants imaged on the ground. Preserved spaceflight and ground control plants were examined post flight with confocal microscopy. Plants on orbit, growing in the absence of any physical reference to the terrestrial gravity vector, displayed typically "vertical" distribution of auxin in the primary root. This confirms that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is gravity independent. The cytokinin distribution in the root tip differs between spaceflight and the ground controls, suggesting spaceflight-induced features of root growth may be cytokinin related. The distribution of auxin in the gravity-sensing portion of the root is not dependent on gravity. Spaceflight appears benign to auxin and its role in the development of the primary root tip, whereas spaceflight may influence cytokinin-associated processes.
Spaceflight Study	SpaceX Mission CRS-5	SLID-284	The effect of spaceflight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit	n.a.	Seedling	Our primary aim was to determine whether gravity has a direct role in establishing the auxin-mediated gravity-sensing system in primary roots. Major plant architectures have long been thought to be guided by gravity, including the directional growth of the primary root via auxin gradients that are then disturbed when roots deviate from the vertical as a gravity sensor. However, experiments on the International Space Station (ISS) now allow physical clarity with regard to any assumptions regarding the role of gravity in establishing fundamental root auxin distributions. We examined the spaceflight green fluorescent protein (GFP)-reporter gene expression in roots of transgenic lines of Arabidopsis thaliana: pDR5r::GFP, pTAA1::TAA1-GFP, pSCR::SCR-GFP to monitor auxin and pARR5::GFP to monitor cytokinin. Plants on the ISS were imaged live with the Light Microscopy Module (LMM), and compared with control plants imaged on the ground. Preserved spaceflight and ground control plants were examined post flight with confocal microscopy. Plants on orbit, growing in the absence of any physical reference to the terrestrial gravity vector, displayed typically "vertical" distribution of auxin in the primary root. This confirms that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is gravity independent. The cytokinin distribution in the root tip differs between spaceflight and the ground controls, suggesting spaceflight-induced features of root growth may be cytokinin related. The distribution of auxin in the gravity-sensing portion of the root is not dependent on gravity. Spaceflight appears benign to auxin and its role in the development of the primary root tip, whereas spaceflight may influence cytokinin-associated processes.
Spaceflight Study	SpaceX Mission CRS-2	SLID-285	RNAseq Analysis of the Response of Arabidopsis thaliana to Fractional Gravity Under Blue-Light Stimulation During Spaceflight	n.a.	Seedling	Introduction: 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. Materials and Methods: 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 h. 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. Results: Differential gene expression analysis revealed only one differentially expressed gene (AT4G21560, VPS28-1 a vacuolar protein) across all gravity conditions using FDR correction (q < 0.05). However, the same 14 genes appeared differentially expressed when comparing either micro-g, low-g level (< 0.1g) or the Moon g-level with 1g control conditions. Apart from these 14-shared genes, the number of differentially expressed genes was similar in microgravity and the Moon g-level and increased in the intermediate g-level (< 0.1g), but it was then progressively reduced as the difference with the Earth gravity became smaller. The GO groups were differentially affected at each g-level: light and photosynthesis GO under microgravity, genes belonged to general stress, chemical and hormone responses under low-g, and a response related to cell wall and membrane structure and function under the Moon g-level. Discussion: Transcriptional analyses of plants under blue light stimulation suggests that root blue-light phototropism may be enough to reduce the gravitational stress response caused by the lack of gravitropism in microgravity. Competition among tropisms induces an intense perturbation at the micro-g level, which shows an extensive stress response that is progressively attenuated. Our results show a major effect on cell wall/membrane remodeling (detected at the interval from the Moon to Mars gravity), which can be potentially related to graviresistance mechanisms.
Spaceflight Study	SpaceX Mission CRS-4	SLID-285	RNAseq Analysis of the Response of Arabidopsis thaliana to Fractional Gravity Under Blue-Light Stimulation During Spaceflight	n.a.	Seedling	Introduction: 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. Materials and Methods: 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 h. 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. Results: Differential gene expression analysis revealed only one differentially expressed gene (AT4G21560, VPS28-1 a vacuolar protein) across all gravity conditions using FDR correction (q < 0.05). However, the same 14 genes appeared differentially expressed when comparing either micro-g, low-g level (< 0.1g) or the Moon g-level with 1g control conditions. Apart from these 14-shared genes, the number of differentially expressed genes was similar in microgravity and the Moon g-level and increased in the intermediate g-level (< 0.1g), but it was then progressively reduced as the difference with the Earth gravity became smaller. The GO groups were differentially affected at each g-level: light and photosynthesis GO under microgravity, genes belonged to general stress, chemical and hormone responses under low-g, and a response related to cell wall and membrane structure and function under the Moon g-level. Discussion: Transcriptional analyses of plants under blue light stimulation suggests that root blue-light phototropism may be enough to reduce the gravitational stress response caused by the lack of gravitropism in microgravity. Competition among tropisms induces an intense perturbation at the micro-g level, which shows an extensive stress response that is progressively attenuated. Our results show a major effect on cell wall/membrane remodeling (detected at the interval from the Moon to Mars gravity), which can be potentially related to graviresistance mechanisms.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-286	Physiological, transcriptional, and metabolic alterations in spaceflight-subjected Senna obtusifolia	17 Days	Seed	Senna obtusifolia is a widely used medicinal herb in Asian countries. To select elite cultivars, S. obtusifolia seeds were carried by "ShenZhou Ⅷ" recoverable satellite to space. Three spaceflight-subjected lines (SP-lines), namely QC10, QC29, QC46, and their ground control line (GC-line) were cultivated on the ground. Previous studies demonstrated that biological traits and secondary metabolites are different between SP-lines and GC-line. Here, we combined physiological, transcriptional, and metabolic studies to compare the differences between SP-lines and GC-line. The results showed that activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and monodehydroascorbate reductase (MDHAR) were dramatically increased in SP-lines as compared to that of GC-line. Transcript levels of SOD, POD, CAT, APX, and MDHAR were significantly up-regulated in SP-lines. Malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents decreased in SP-lines. Seed yields of QC29 and QC46 were considerably higher than that of GC-line. Besides, QC29 had significantly higher aurantio-obtusin content. Pearson correlation coefficient analysis revealed positive relationships between POD and aurantio-obtusin, as well as APX and aurantio-obtusin. In conclusion, SP-lines have higher antioxidant gene expression level and antioxidant enzyme activity as compared to that of GC-line. With higher seed yield and aurantio-obtusin content, QC29 can be used to breed elite S. obtusifolia cultivars. This study provides a new insight in SP-lines and paves the way to breed elite S. obtusifolia cultivars in the future.
Spaceflight Study	n.a.	SLID-287	Survival of plant seeds, their UV screens, and nptII DNA for 18 months outside the International Space Station	1.5 Years	Seed	The plausibility that life was imported to Earth from elsewhere can be tested by subjecting life-forms to space travel. Ultraviolet light is the major liability in short-term exposures (Horneck et al., 2001 ), and plant seeds, tardigrades, and lichens-but not microorganisms and their spores-are candidates for long-term survival (Anikeeva et al., 1990 ; Sancho et al., 2007 ; Jönsson et al., 2008 ; de la Torre et al., 2010 ). In the present study, plant seeds germinated after 1.5 years of exposure to solar UV, solar and galactic cosmic radiation, temperature fluctuations, and space vacuum outside the International Space Station. Of the 2100 exposed wild-type Arabidopsis thaliana and Nicotiana tabacum (tobacco) seeds, 23% produced viable plants after return to Earth. Survival was lower in the Arabidopsis Wassilewskija ecotype and in mutants (tt4-8 and fah1-2) lacking UV screens. The highest survival occurred in tobacco (44%). Germination was delayed in seeds shielded from solar light, yet full survival was attained, which indicates that longer space travel would be possible for seeds embedded in an opaque matrix. We conclude that a naked, seed-like entity could have survived exposure to solar UV radiation during a hypothetical transfer from Mars to Earth. Chemical samples of seed flavonoid UV screens were degraded by UV, but their overall capacity to absorb UV was retained. Naked DNA encoding the nptII gene (kanamycin resistance) was also degraded by UV. A fragment, however, was detected by the polymerase chain reaction, and the gene survived in space when protected from UV. Even if seeds do not survive, components (e.g., their DNA) might survive transfer over cosmic distances.
Spaceflight Study	STS-107	SLID-288	Caenorhabditis elegans survives atmospheric breakup of STS-107, space shuttle Columbia	16 Days	Animals	The nematode Caenorhabditis elegans, a popular organism for biological studies, is being developed as a model system for space biology. The chemically defined liquid medium, C. elegans Maintenance Medium (CeMM), allows axenic cultivation and automation of experiments that are critical for spaceflight research. To validate CeMM for use during spaceflight, we grew animals using CeMM and standard laboratory conditions onboard STS-107, space shuttle Columbia. Tragically, the Columbia was destroyed while reentering the Earth's atmosphere. During the massive recovery effort, hardware that contained our experiment was found. Live animals were observed in four of the five recovered canisters, which had survived on both types of media. These data demonstrate that CeMM is capable of supporting C. elegans during spaceflight. They also demonstrate that animals can survive a relatively unprotected reentry into the Earth's atmosphere, which has implications with regard to the packaging of living material during space flight, planetary protection, and the interplanetary transfer of life.
Spaceflight Study	Russian Soyuz Spacecraft	SLID-289	Genomic response of the nematode Caenorhabditis elegans to spaceflight	10 Days	Animals	On Earth, it is common to employ laboratory animals such as the nematode Caenorhabditis elegans to help understand human health concerns. Similar studies in Earth orbit should help understand and address the concerns associated with spaceflight. The “International Caenorhabditis elegans Experiment FIRST” (ICE FIRST), was carried out onboard the Dutch Taxiflight in April of 2004 by an international collaboration of laboratories in France, Canada, Japan and the United States. With the exception of a slight movement defect upon return to Earth, the result of altered muscle development, no significant abnormalities were detected in spaceflown C. elegans. Work from Japan revealed apoptosis proceeds normally and work from Canada revealed no significant increase in the rate of mutation. These results suggest that C. elegans can be used to study non-lethal responses to spaceflight and can possibly be developed as a biological sensor. To further our understanding of C. elegans response to spaceflight, we examined the gene transcription response to the 10 days in space using a near full genome microarray analysis. The transcriptional response is consistent with the observed normal developmental timing, apoptosis, DNA repair, and altered muscle development. The genes identified as altered in response to spaceflight are enriched for genes known to be regulated, in C. elegans, in response to altered environmental conditions (Insulin and TGF-β regulated). These results demonstrate C. elegans can be used to study the effects of altered gravity and suggest that C. elegans responds to spaceflight by altering the expression of at least some of the same metabolic genes that are altered in response to differing terrestrial environments.
Spaceflight Study	Experiment First Flight (ICE-FIRST)	SLID-290	Genes down-regulated in spaceflight are involved in the control of longevity in Caenorhabditis elegans	11 Days	Animals	How microgravitational space environments affect aging is not well understood. We observed that, in Caenorhabditis elegans, spaceflight suppressed the formation of transgenically expressed polyglutamine aggregates, which normally accumulate with increasing age. Moreover, the inactivation of each of seven genes that were down-regulated in space extended lifespan on the ground. These genes encode proteins that are likely related to neuronal or endocrine signaling: acetylcholine receptor, acetylcholine transporter, choline acetyltransferase, rhodopsin-like receptor, glutamate-gated chloride channel, shaker family of potassium channel, and insulin-like peptide. Most of them mediated lifespan control through the key longevity-regulating transcription factors DAF-16 or SKN-1 or through dietary-restriction signaling, singly or in combination. These results suggest that aging in C. elegans is slowed through neuronal and endocrine response to space environmental cues.
Ground Study	2-D Clinostat SM-X1	SLID-291	Reproductive and locomotory capacities of Caenorhabditis elegans were not affected by simulated variable gravities and spaceflight during the Shenzhou-8 mission	n.a.	Animals	Reproduction and locomotion are essential features of animals that help to facilitate their interaction with the surrounding environment. Previous studies have produced inconsistent results on behavioral response to spaceflight by the model animal Caenorhabditis elegans (C. elegans) in liquid culture. Using standard agar-based nematode growth medium (NGM), we show here that both reproductive and locomotory capacities of C. elegans were not significantly changed by centrifuge-produced hypergravity or clinostat-simulated microgravity. To investigate the effect of actual spaceflight on C. elegans, a nematode test unit was specifically designed to maintain its normal growth on solid NGM slides and to allow automatic RNA fixation on board the Shenzhou-8 spaceflight. We did not detect alteration in either brood size of immediate progenies from postflight nematodes or locomotory behavior, including speed of locomotion, frequency of reversals, and rate of body bends of space-flown nematodes collected directly from nematode test units. Our results provide clear evidence that the nematode test unit is an appropriate apparatus for nematode growth on standard NGM and can be used for on-orbit analysis of C. elegans, including onboard RNA fixation for molecular analysis and real-time video acquisition for behavioral analysis, which are critical for further studies in unmanned spaceflight and outer space exploration.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-291	Reproductive and locomotory capacities of Caenorhabditis elegans were not affected by simulated variable gravities and spaceflight during the Shenzhou-8 mission	16 Days	Animals	Reproduction and locomotion are essential features of animals that help to facilitate their interaction with the surrounding environment. Previous studies have produced inconsistent results on behavioral response to spaceflight by the model animal Caenorhabditis elegans (C. elegans) in liquid culture. Using standard agar-based nematode growth medium (NGM), we show here that both reproductive and locomotory capacities of C. elegans were not significantly changed by centrifuge-produced hypergravity or clinostat-simulated microgravity. To investigate the effect of actual spaceflight on C. elegans, a nematode test unit was specifically designed to maintain its normal growth on solid NGM slides and to allow automatic RNA fixation on board the Shenzhou-8 spaceflight. We did not detect alteration in either brood size of immediate progenies from postflight nematodes or locomotory behavior, including speed of locomotion, frequency of reversals, and rate of body bends of space-flown nematodes collected directly from nematode test units. Our results provide clear evidence that the nematode test unit is an appropriate apparatus for nematode growth on standard NGM and can be used for on-orbit analysis of C. elegans, including onboard RNA fixation for molecular analysis and real-time video acquisition for behavioral analysis, which are critical for further studies in unmanned spaceflight and outer space exploration.
Ground Study	Tailor-made Centrifuge	SLID-291	Reproductive and locomotory capacities of Caenorhabditis elegans were not affected by simulated variable gravities and spaceflight during the Shenzhou-8 mission	n.a.	Animals	Reproduction and locomotion are essential features of animals that help to facilitate their interaction with the surrounding environment. Previous studies have produced inconsistent results on behavioral response to spaceflight by the model animal Caenorhabditis elegans (C. elegans) in liquid culture. Using standard agar-based nematode growth medium (NGM), we show here that both reproductive and locomotory capacities of C. elegans were not significantly changed by centrifuge-produced hypergravity or clinostat-simulated microgravity. To investigate the effect of actual spaceflight on C. elegans, a nematode test unit was specifically designed to maintain its normal growth on solid NGM slides and to allow automatic RNA fixation on board the Shenzhou-8 spaceflight. We did not detect alteration in either brood size of immediate progenies from postflight nematodes or locomotory behavior, including speed of locomotion, frequency of reversals, and rate of body bends of space-flown nematodes collected directly from nematode test units. Our results provide clear evidence that the nematode test unit is an appropriate apparatus for nematode growth on standard NGM and can be used for on-orbit analysis of C. elegans, including onboard RNA fixation for molecular analysis and real-time video acquisition for behavioral analysis, which are critical for further studies in unmanned spaceflight and outer space exploration.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-292	The DNA damage response of C. elegans affected by gravity sensing and radiosensitivity during the Shenzhou-8 spaceflight	17 Days	Animals	Space radiation and microgravity are recognized as primary and inevitable risk factors for humans traveling in space, but the reports regarding their synergistic effects remain inconclusive and vary across studies due to differences in the environmental conditions and intrinsic biological sensitivity. Thus, we studied the synergistic effects on transcriptional changes in the global genome and DNA damage response (DDR) by using dys-1 mutant and ced-1 mutant of C. elegans, which respectively presented microgravity-insensitivity and radiosensitivity when exposure to spaceflight condition (SF) and space radiation (SR). The dys-1 mutation induced similar transcriptional changes under both conditions, including the transcriptional distribution and function of altered genes. The majority of alterations were related to metabolic shift under both conditions, including transmembrane transport, lipid metabolic processes and proteolysis. Under SF and SR conditions, 12/14 and 10/13 altered pathways, respectively, were both grouped in the metabolism category. Out of the 778 genes involved in DDR, except eya-1 and ceh-34, 28 altered genes in dys-1 mutant showed no predicted protein interactions, or anti-correlated miRNAs during spaceflight. The ced-1 mutation induced similar changes under SF and SR; however, these effects were stronger than those of the dys-1 mutant. The additional genes identified were related to phosphorous/phosphate metabolic processes and growth rather than, metabolism, especially for environmental information processing under SR. Although the DDR profiles were significantly changed under both conditions, the ced-1 mutation favored DNA repair under SF and apoptosis under SR. Notably, 37 miRNAs were predicted to be involved in the DDR. Our study indicates that, the dys-1 mutation reduced the transcriptional response to SF, and the ced-1 mutation increased the response to SR, when compared with the wild type C. elegans. Although some effects were due to radiosensitivity, microgravity, depending on the dystrophin, exerts predominant effects on transcription in C. elegans during short-duration spaceflight.
Spaceflight Study	STS-129	SLID-293	Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans	4 Days	Animals	Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.
Spaceflight Study	FOTON-M2 Satellite	SLID-294	Comparative Studies of the Thick-Toed Geckos after the 16 and 12 Days Spaceflight in Foton-M Experiments	16 Days	Animals	In our study we compare the data from analysis of thick-toed geckoes Pachydactylus turneri from 16 and 12 days spaceflights onboard «Foton-M2» (M2) and «Foton-M3» (M3) satellites respectively. These studies were realized in the frames of Russian-American joint experiments. In M2 they were performed on 4 females and 1 male in each of the following groups: flight (F), basal (BC) and delayed synchronous (SC) controls. In M3 there were 5 females in each group. The animals were euthanized and examined using traditional histology, immunohistochemistry and X-ray microtomography. Mallory, Nissl and hematoxylin-eosin staining were used to compare the condition of brain, heart, liver, pancreas, spleen and small intestine. Brain and pancreas were also studied immunohistochemically. Behavior was registered by video camera in F and SC (M3). Thus we confirm the previous assumption that geckoes can preserve in weightlessness their ability to fi x themselves to the surfaces by their toe pads. We did not reveal in liver, pancreas, spleen and small intestine of F-M3 geckoes such evident changes like in F-M2 group. Glial destruction was detected immunohistochemically in the brains of F-M3 geckoes, especially in the cortical structures and epithalamus. Gluckocorticoids level for geckoes' feces in F-M2 was 4 times higher than in SC-M2 whereas the results for M3 were almost the same. Microtomografi c analysis of the femur bones showed some redistribution of the trabeculae in F-M3 group which occured in the direction from the outer compact bone to the bone center. Thus we conclude that in most structures of F-M3 animals the changes were less then in F-M2 ones. It can be explaned by shorter duration of M3 flight, higer temperature and the presence of water source. More prolonged experiments with larger groups of geckoes are necessary to verify the obtained data. Probably geckoes are well preadapted to conditions of spaceflight due to their specific biology.
Spaceflight Study	FOTON-M3 Satellite	SLID-294	Comparative Studies of the Thick-Toed Geckos after the 16 and 12 Days Spaceflight in Foton-M Experiments	12 Days	Animals	In our study we compare the data from analysis of thick-toed geckoes Pachydactylus turneri from 16 and 12 days spaceflights onboard «Foton-M2» (M2) and «Foton-M3» (M3) satellites respectively. These studies were realized in the frames of Russian-American joint experiments. In M2 they were performed on 4 females and 1 male in each of the following groups: flight (F), basal (BC) and delayed synchronous (SC) controls. In M3 there were 5 females in each group. The animals were euthanized and examined using traditional histology, immunohistochemistry and X-ray microtomography. Mallory, Nissl and hematoxylin-eosin staining were used to compare the condition of brain, heart, liver, pancreas, spleen and small intestine. Brain and pancreas were also studied immunohistochemically. Behavior was registered by video camera in F and SC (M3). Thus we confirm the previous assumption that geckoes can preserve in weightlessness their ability to fi x themselves to the surfaces by their toe pads. We did not reveal in liver, pancreas, spleen and small intestine of F-M3 geckoes such evident changes like in F-M2 group. Glial destruction was detected immunohistochemically in the brains of F-M3 geckoes, especially in the cortical structures and epithalamus. Gluckocorticoids level for geckoes' feces in F-M2 was 4 times higher than in SC-M2 whereas the results for M3 were almost the same. Microtomografi c analysis of the femur bones showed some redistribution of the trabeculae in F-M3 group which occured in the direction from the outer compact bone to the bone center. Thus we conclude that in most structures of F-M3 animals the changes were less then in F-M2 ones. It can be explaned by shorter duration of M3 flight, higer temperature and the presence of water source. More prolonged experiments with larger groups of geckoes are necessary to verify the obtained data. Probably geckoes are well preadapted to conditions of spaceflight due to their specific biology.
Spaceflight Study	International Space Station (ISS)	SLID-295	Recovery from 6-month spaceflight at the International Space Station: muscle-related stress into a proinflammatory setting	6 Months	n.a.	The Sarcolab pilot study of 2 crewmembers, investigated before and after a 6‐mo International Space Station mission, has demonstrated the substantial muscle wasting and weakness, along with disruption of muscle's oxidative metabolism. The present work aimed at evaluating the pro/anti‐inflammatory status in the same 2 crewmembers (A, B). Blood circulating (c‐)microRNAs (miRs), c‐proteasome, c‐mitochondrial DNA, and cytokines were assessed by real‐time quantitative PCR or ELISA tests. Time series analysis was performed (i.e., before flight and after landing) at 1 and 15 d of recovery (R+1 and R+15, respectively). C‐biomarkers were compared with an age‐matched control population and with 2‐dimensional proteomic analysis of the 2 crewmembers' muscle biopsies. Striking differences were observed between the 2 crewmembers at R+1, in terms of inflamma‐miRs (c‐miRs‐21‐5p, ‐126‐3p, and ‐146a‐5p), muscle specific (myo)‐miR‐206, c‐proteasome, and IL‐6/leptin, thus making the 2 astronauts dissimilar to each other. Final recovery levels of c‐proteasome, c‐inflamma‐miRs, and c‐myo‐miR‐206 were not reverted to the baseline values in crewmember A. In both crewmembers, myo‐miR‐206 changed significantly after recovery. Muscle biopsy of astronaut A showed an impressive 80% increase of α‐1‐antitrypsin, a target of miR‐126‐3p. These results point to a strong stress response induced by spaceflight involving muscle tissue and the proinflammatory setting, where inflamma‐miRs and myo‐miR‐206 mediate the systemic recovery phase after landing.
Spaceflight Study	International Space Station (ISS)	SLID-296	Effects of spaceflight on the composition and function of the human gut microbiota	n.a.	Gut microbiota composition changes	Interaction between humans and the gut microbiota is important for human physiology. Here, the gut microbiota was analyzed via metagenomic sequencing, and the fluctuations in the gut microbiota under the conditions of spaceflight were characterized. The composition and function of the gut microbiota were substantially affected by spaceflight; however, individual specificity was uncompromised. We further confirmed the species fluctuations and functional genes from both missions. Resistance and virulence genes in the gut microbiota were affected by spaceflight, but the species attributions remained stable. Spaceflight markedly affected the composition and function of the human gut microbiota, implying that the human gut microbiota is sensitive to spaceflight.
Spaceflight Study	n.a.	SLID-297	The influence of spaceflight on the astronaut salivary microbiome and the search for a microbiome biomarker for viral reactivation	n.a.	Salivary microbiome changes	Background: Spaceflight impacts astronauts in many ways but little is known on how spaceflight affects the salivary microbiome and the consequences of these changes on astronaut health, such as viral reactivation. In order to understand this, the salivary microbiome was analyzed with 16S rRNA gene amplicon sequencing, and saliva viral titers were analyzed with quantitative polymerase chain reaction (qPCR) with primers specific for Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella zoster virus (VZV) from 10 astronauts pre-flight, in-flight, and post-flight. Results: Streptococcus was the most abundant organism in the saliva, making up 8% of the total organisms detected, and their diversity decreased during spaceflight. Other organisms that had statistically significant changes were Proteobacteria and Fusobacteria which increased during flight and Actinobacteria which decreased during flight. At the genus level, Catonella, Megasphera, and Actinobacillus were absent in more than half of saliva samples collected pre-flight but were then detected during flight. In those subjects that already had these genera pre-flight, their relative abundances increased during flight. Correlation analyses between the microbiome and viral titers revealed a positive correlation with Gracilibacteria, Absconditabacteria, and Abiotrophia and a negative correlation between Oribacterium, Veillonella, and Haemophilus. There was also a significant positive correlation between microbiome richness and EBV viral titers. Conclusions: This is the first study to look at how the salivary microbiome changes as a result of spaceflight and the search for bacterial biomarkers for viral reactivation. Further studies examining the role of specific organisms that were shown to be correlative and predictive in viral reactivation, a serious problem in astronauts during spaceflight, could lead to mitigation strategies to help prevent disease during both short and long duration space missions. Video abstract.
Spaceflight Study	Spacelab Life Sciences 2 (SLS-2)	SLID-298	The effects of spaceflight on open-loop and closed-loop postural control mechanisms: human neurovestibular studies on SLS-2	14 Days	n.a.	Stabilogram-diffusion analysis was used to examine how prolonged periods in microgravity affect the open-loop and closed-loop postural control mechanisms. It was hypothesized that following spaceflight: (1) the effective stochastic activity of the open-loop postural control schemes in astronauts is increased; (2) the effective stochastic activity and uncorrelated behavior, respectively, of the closed-loop postural control mechanisms in astronauts are increased; and (3) astronauts utilized open-loop postural controls schemes for shorter time intervals and smaller displacements. Four crew members and two alternates from the 14-day Spacelab Life Sciences 2 Mission were included in the study. Each subject was tested under eyes-open, quiet-standing conditions on multiple preflight and postflight days. The subjects' center-of-pressure trajectories were measured with a force platform and analyzed according to stabilogram-diffusion analysis. It was found that the effective stochastic activity of the open-loop postural control schemes in three of the four crew members was increased following spaceflight. This result is interpreted as an indication that there may be in-flight adaptations to higher-level descending postural control pathways, e.g., a postflight increase in the tonic activation of postural muscles. This change may also be the consequence of a compensatory (e.g., "stiffening") postural control strategy that is adopted by astronauts to account for general feeling of postflight unsteadiness. The crew members, as a group, did not exhibit any consistent preflight/postflight differences in the steady-state behavior of their closed-loop postural control mechanisms or in the functional interaction of their open-loop and closed-loop postural control mechanisms. These results are interpreted as indications that although there may be in-flight adaptations to the vestibular system and/or proprioceptive system, input from the visual system can compensate for such changes during undisturbed stance.
Spaceflight Study	Apollo Missions 16	SLID-299	Filamentous fungi exposed to spaceflight stresses including known levels of ultraviolet irradiations	11 Days	Fungi	Chaetomium globosum ascospores and Trichophyton terrestre conidia were exposed to space- flight parameters of Apollo 16 then returned to Earth for studies in hyphal growth dynamics as one of the postflight investigations. Selected phenotypic strains of each species appeared to show changes in growth according to specific ultraviolet irradiations in space.
Spaceflight Study	STS-77	SLID-300	The effects of space flight on the production of monorden by Humicola fuscoatra WC5157 in solid-state fermentation	10 Days	Fungi	The effect of space flight on the production of the antibiotic monorden on two types of agar media, T8 and PG, by Humicola fuscoatra WC5157 was examined on board the US Space Shuttle mission STS-77 in May 1996. Paired space-flight and ground control samples were prepared using identical hardware, protocol, media, and inoculum. Inoculation occurred simultaneously for both groups 2.5 after launch. The flight and ground samples were allowed to grow for the entire 10-day mission in a dark, thermally controlled (22 degrees C) environment. Post-flight HPLC analysis of the flight and ground sample extracts indicated that the production of monorden by H. fuscoatra WC5157 in the flight samples was higher than in the ground samples in both agar media. In the T8 medium, the production of monorden in the flight and ground samples was 11.6 +/- 3.5 micrograms and 8.9 +/- 1.1 micrograms respectively (30% increase). In the PG medium, the production of monorden in the flight and ground samples was 23.8 +/- 3.3 micrograms and 8.2 +/- 2.2 micrograms respectively (190% increase). The production of monorden in the flight and ground control samples was confirmed by HPLC-MS analysis.
Spaceflight Study	S/MM 05 Space Shuttle Mission (STS-81)	SLID-301	Chromosome mechanics of fungi under spaceflight conditions--tetrad analysis of two-factor crosses between spore color mutants of Sordaria macrospora	5 Days	Fungi	Spore color mutants of the fungus Sordaria macrospora Auersw. were crossed under spaceflight conditions on the space shuttle to MIR mission S/MM 05 (STS-81). The arrangement of spores of different colors in the asci allowed conclusions on the influence of spaceflight conditions on sexual recombination in fungi. Experiments on a 1-g centrifuge in space and in parallel on the ground were used for controls. The samples were analyzed microscopically on their return to earth. Each fruiting body was assessed separately. Statistical analysis of the data showed a significant increase in gene recombination frequencies caused by the heavy ion particle stream in space radiation. The lack of gravity did not influence crossing-over frequencies. Hyphae of the flown samples were assessed for DNA strand breaks. No increase in damage was found compared with the ground samples. It was shown that S. macrospora is able to repair radiation-induced DNA strand breaks within hours.
Spaceflight Study	Russian Mir Mission	SLID-302	Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir	40 Days	Fungi	Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.
Spaceflight Study	STS-80	SLID-303	The effect of space flight on the production of actinomycin D by Streptomyces plicatus	17.7 Days	Bacteria	The effect of space flight on production of the antibiotic actinomycin D by Streptomyces plicatus WC56452 was examined onboard the US Space Shuttle mission STS-80. Paired space flight and ground control samples were similarly prepared using identical hardware, media, and inoculum. The cultures were grown in defined and complex media under dark, anaerobic, thermally controlled (20 degrees C) conditions with samples fixed after 7 and 12 days in orbit, and viable residuals maintained through landing at 17 days, 15 h. Postflight analyses indicated that space flight had reduced the colony-forming unit (CFU) per milliliter count of S. plicatus and increased the specific productivity (pg CFU(-1)) of actinomycin D. The antibiotic compound itself was not affected, but its production time course was altered in space. Viable flight samples also maintained their sporulation ability when plated on agar medium postflight, while the residual ground controls did not sporulate.
Ground Study	n.a.	SLID-304	Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli	235 Hours	Bacteria	Cultures of Escherichia coli grown in space reached a 25% higher average final cell population than those in comparably matched ground controls (p<0.05). However, both groups consumed the same quantity of glucose, which suggests that space flight not only stimulated bacterial growth as has been previously reported, but also resulted in a 25% more efficient utilization of the available nutrients. Supporting experiments performed in "simulated weightlessness" under clinorotation produced similar trends of increased growth and efficiency, but to a lesser extent in absolute values. These experiments resulted in increases of 12% and 9% in average final cell population (p<0.05), while the efficiency of substrate utilization improved by 6% and 9% relative to static controls (p=0.12 and p<0.05, respectively). In contrast, hypergravity, produced by centrifugation, predictably resulted in the opposite effect--a decrease of 33% to 40% in final cell numbers with corresponding 29% to 40% lower net growth efficiencies (p<0.01). Collectively, these findings support the hypothesis that the increased bacterial growth observed in weightlessness is a result of reduced extracellular mass transport that occurs in the absence of sedimentation and buoyancy-driven convection, which consequently also improves substrate utilization efficiency in suspended cultures.
Spaceflight Study	STS-95	SLID-304	Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli	235 Hours	Bacteria	Cultures of Escherichia coli grown in space reached a 25% higher average final cell population than those in comparably matched ground controls (p<0.05). However, both groups consumed the same quantity of glucose, which suggests that space flight not only stimulated bacterial growth as has been previously reported, but also resulted in a 25% more efficient utilization of the available nutrients. Supporting experiments performed in "simulated weightlessness" under clinorotation produced similar trends of increased growth and efficiency, but to a lesser extent in absolute values. These experiments resulted in increases of 12% and 9% in average final cell population (p<0.05), while the efficiency of substrate utilization improved by 6% and 9% relative to static controls (p=0.12 and p<0.05, respectively). In contrast, hypergravity, produced by centrifugation, predictably resulted in the opposite effect--a decrease of 33% to 40% in final cell numbers with corresponding 29% to 40% lower net growth efficiencies (p<0.01). Collectively, these findings support the hypothesis that the increased bacterial growth observed in weightlessness is a result of reduced extracellular mass transport that occurs in the absence of sedimentation and buoyancy-driven convection, which consequently also improves substrate utilization efficiency in suspended cultures.
Spaceflight Study	Atlantis Space Flight	SLID-305	Microbial antibiotic production aboard the International Space Station	72 Days	Bacteria	Previous studies examining metabolic characteristics of bacterial cultures have mostly suggested that reduced gravity is advantageous for microbial growth. As a consequence, the question of whether space flight would similarly enhance secondary metabolite production was raised. Results from three prior space shuttle experiments indicated that antibiotic production was stimulated in space for two different microbial systems, albeit under suboptimal growth conditions. The goal of this latest experiment was to determine whether the enhanced productivity would also occur with better growth conditions and over longer durations of weightlessness. Microbial antibiotic production was examined onboard the International Space Station during the 72-day 8A increment. Findings of increased productivity of actinomycin D by Streptomyces plicatus in space corroborated with previous findings for the early sample points (days 8 and 12); however, the flight production levels were lower than the matched ground control samples for the remainder of the mission. The overall goal of this research program is to elucidate the specific mechanisms responsible for the initial stimulation of productivity in space and translate this knowledge into methods for improving efficiency of commercial production facilities on Earth.
Spaceflight Study	Shenzhou 3 Space Flight	SLID-306	Preliminary report on the biological effects of space flight on the producing strain of a new immunosuppressant, Kanglemycin C	162 Hours	Bacteria	Kanglemycin C (K-C) is a new immunosuppressant isolated from the culture broth of Nocardia mediterranei var. kanglensis 1747-64. To improve the productivity of K-C and to study the biological effects of space flight on its producing strain, spores from five K-C producing strains (U-10, U-15, U-7, M-13, γ-33) mutated from the wild strain N. mediterranei var. kanglensis 1747-64 were carried into space by an unmanned spaceship, “Shenzhou III” (Divine Vessel III) on March 25, 2002. Comparatively, the strain U-7 was the highest K-C producing strain among the above five starting strains when cultivated in 500-ml Erlenmeyer flasks. After a 6 day and 18 h flight, the treated spores went through serial screening processes to screen for high-yield K-C mutant strains, using thin layer chromatography and high performance liquid chromatography (HPLC). The K-C yield produced by one mutant strain, designated as F-16, derived from the starting strain U-7 was increased by up to 200% when compared to that produced by the starting strain U-7 in 500-ml Erlenmeyer flasks after careful postflight HPLC analysis. Another mutant strain, designated as F-210, derived from the starting strain M-13 showed reduced productivity of K-C as well as exhibited changes in some morphological and physiological characteristics. For example, the broth color of the strain F-210 changed from yellow to purple after 96 h of culture, but that of the ground control strain M-13 remained yellow. Similarly, the mycelium morphological change from filamentous to coccoid of F-210 occurred later than that of ground control M-13. Examination of the survivability of postflight spores indicated that exposure to radiation, during the 162 h of space flight, plays a critical role in the survival rates of spores such that spores exposed to strong radiation exhibited lower survival rates than spores exposed to weak radiation.
Ground Study	Rotating Wall Vessel (RWV)	SLID-307	Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq	n.a.	Bacteria	A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.
Spaceflight Study	STS-115	SLID-307	Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq	25 Hours	Bacteria	A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.
Spaceflight Study	n.a.	SLID-308	Mold species in dust from the International Space Station identified and quantified by mold-specific quantitative PCR	n.a.	Fungi	Dust was collected over a period of several weeks in 2007 from HEPA filters in the U.S. Laboratory Module of the International Space Station (ISS). The dust was returned on the Space Shuttle Atlantis, mixed, sieved and the DNA was extracted. Using a DNA-based method called mold-specific quantitative PCR (MSQPCR), 39 molds were measured in the dust. Potential opportunistic pathogens Aspergillus flavus and Aspergillus niger and potential moderate toxin producers Penicillium chrysogenum and Penicillium brevicompactum were noteworthy. No cells of the potential opportunistic pathogens Aspergillus fumigatus, Aspergillus terreus, Fusarium solani or Candida albicans were detected.
Spaceflight Study	n.a.	SLID-309	Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall	15 Days	Fungi	Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite’s landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and β-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of β-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of β-glucan production.
Spaceflight Study	STS-115	SLID-310	Media ion composition controls regulatory and virulence response of Salmonella in spaceflight	25 Hours	Bacteria	The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.
Spaceflight Study	STS-123	SLID-310	Media ion composition controls regulatory and virulence response of Salmonella in spaceflight	25 Hours	Bacteria	The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.
Ground Study	n.a.	SLID-311	Isolation of rpoB mutations causing rifampicin resistance in Bacillus subtilis spores exposed to simulated Martian surface conditions	21 Days	Bacteria	ABSTRACT Bacterial spores are considered prime candidates for Earth-to-Mars transport by natural processes and human spaceflight activities. Previous studies have shown that exposure of Bacillus subtilis spores to ultrahigh vacuum (UHV) characteristic of space both increased the spontaneous mutation rate and altered the spectrum of mutation in various marker genes; but, to date, mutagenesis studies have not been performed on spores exposed to milder low pressures encountered in the martian environment. Mutations to rifampicin-resistance (Rif(R)) were isolated in B. subtilis spores exposed to simulated martian atmosphere (99.9% CO(2), 710 Pa) for 21 days in a Mars Simulation Chamber (MSC) and compared to parallel Earth controls. Exposure in the MSC reduced spore viability by approximately 67% compared to Earth controls, but this decrease was not statistically significant (P = 0.3321). The frequency of mutation to Rif(R) was also not significantly increased in the MSC compared to Earth-exposed spores (P = 0.479). Forty-two and 51 Rif(R) mutant spores were isolated from the MSC- and Earth-exposed controls, respectively. Nucleotide sequencing located the Rif(R) mutations in the rpoB gene encoding the beta subunit of RNA polymerase at residue V135F of the N-cluster and at residues Q469K/L, H482D/P/R/Y, and S487L in Cluster I. No mutations were found in rpoB Clusters II or III. Two new alleles, Q469L and H482D, previously unreported in B. subtilis rpoB, were isolated from spores exposed in the MSC; otherwise, only slight differences were observed in the spectra of spontaneous Rif(R) mutations from spores exposed to Earth vs. the MSC. However, both spectra are distinctly different from Rif(R) mutations previously reported arising from B. subtilis spores exposed to simulated space vacuum.
Ground Study	Random Position Machine (RPM)	SLID-312	Response of Pseudomonas aeruginosa PAO1 to low shear modelled microgravity involves AlgU regulation	24 Hours	Bacteria	As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immunocompromised astronauts during long‐term missions. Therefore, insights into the behaviour of P. aeruginosa under spaceflight conditions were gained using two spaceflight‐analogue culture systems: the rotating wall vessel (RWV) and the random position machine (RPM). Microarray analysis of P. aeruginosa PAO1 grown in the low shear modelled microgravity (LSMMG) environment of the RWV, compared with the normal gravity control (NG), revealed an apparent regulatory role for the alternative sigma factor AlgU (RpoE‐like). Accordingly, P. aeruginosa cultured in LSMMG exhibited increased alginate production and upregulation of AlgU‐controlled transcripts, including those encoding stress‐related proteins. The LSMMG increased heat and oxidative stress resistance and caused a decrease in the oxygen transfer rate of the culture. This study also showed the involvement of the RNA‐binding protein Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG and spaceflight response. The global transcriptional response of P. aeruginosa grown in the RPM was highly similar to that in NG. Fluid mixing was assessed in both systems and is believed to be a pivotal factor contributing to transcriptional differences between RWV‐ and RPM‐grown P. aeruginosa. This study represents the first step towards the identification of virulence mechanisms of P. aeruginosa activated in response to spaceflight‐analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections during spaceflight and in immunocompromised patients.
Ground Study	Rotating Wall Vessel (RWV)	SLID-312	Response of Pseudomonas aeruginosa PAO1 to low shear modelled microgravity involves AlgU regulation	24 Hours	Bacteria	As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immunocompromised astronauts during long‐term missions. Therefore, insights into the behaviour of P. aeruginosa under spaceflight conditions were gained using two spaceflight‐analogue culture systems: the rotating wall vessel (RWV) and the random position machine (RPM). Microarray analysis of P. aeruginosa PAO1 grown in the low shear modelled microgravity (LSMMG) environment of the RWV, compared with the normal gravity control (NG), revealed an apparent regulatory role for the alternative sigma factor AlgU (RpoE‐like). Accordingly, P. aeruginosa cultured in LSMMG exhibited increased alginate production and upregulation of AlgU‐controlled transcripts, including those encoding stress‐related proteins. The LSMMG increased heat and oxidative stress resistance and caused a decrease in the oxygen transfer rate of the culture. This study also showed the involvement of the RNA‐binding protein Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG and spaceflight response. The global transcriptional response of P. aeruginosa grown in the RPM was highly similar to that in NG. Fluid mixing was assessed in both systems and is believed to be a pivotal factor contributing to transcriptional differences between RWV‐ and RPM‐grown P. aeruginosa. This study represents the first step towards the identification of virulence mechanisms of P. aeruginosa activated in response to spaceflight‐analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections during spaceflight and in immunocompromised patients.
Spaceflight Study	STS-115	SLID-313	Transcriptional and proteomic responses of Pseudomonas aeruginosa PAO1 to spaceflight conditions involve Hfq regulation and reveal a role for oxygen	25 Hours	Bacteria	Assessing bacterial behavior in microgravity is important for risk assessment and prevention of infectious diseases during spaceflight missions. Furthermore, this research field allows the unveiling of novel connections between low-fluid-shear regions encountered by pathogens during their natural infection process and bacterial virulence. This study is the first to characterize the spaceflight-induced global transcriptional and proteomic responses of Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat. P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq as a global transcriptional regulator. Since Hfq was also differentially regulated in spaceflight-grown Salmonella enterica serovar Typhimurium, Hfq represents the first spaceflight-induced regulator acting across bacterial species. The major P. aeruginosa virulence-related genes induced in spaceflight were the lecA and lecB lectin genes and the gene for rhamnosyltransferase (rhlA), which is involved in rhamnolipid production. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data for this organism grown in microgravity analogue conditions using the rotating wall vessel (RWV) bioreactor. Interesting similarities were observed, including, among others, similarities with regard to Hfq regulation and oxygen metabolism. While RWV-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight presumably adopted an anaerobic mode of growth, in which denitrification was most prominent. Whether the observed changes in pathogenesis-related gene expression in response to spaceflight culture could lead to an alteration of virulence in P. aeruginosa remains to be determined and will be important for infectious disease risk assessment and prevention, both during spaceflight missions and for the general public.
Spaceflight Study	Chinese FSW-3 Recoverable Satellite	SLID-314	Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation	18 Days	Fungi	Aims: S-adenosyl-l-methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and results: Through spaceflight culture, a SAM-accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one-factor-at-a-time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l(-1) of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l(-1) of SAM was produced in fermenter cultivation. Conclusions: A new SAM-accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and impact of the study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l(-1) of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.
Spaceflight Study	EXPOSE-E Spaceflight	SLID-315	Mutagenesis in bacterial spores exposed to space and simulated martian conditions: data from the EXPOSE-E spaceflight experiment PROTECT	559 Days	Bacteria	As part of the PROTECT experiment of the EXPOSE-E mission on board the International Space Station (ISS), the mutagenic efficiency of space was studied in spores of Bacillus subtilis 168. After 1.5 years' exposure to selected parameters of outer space or simulated martian conditions, the rates of induced mutations to rifampicin resistance (Rif(R)) and sporulation deficiency (Spo(-)) were quantified. In all flight samples, both mutations, Rif(R) and Spo(-), were induced and their rates increased by several orders of magnitude. Extraterrestrial solar UV radiation (>110 nm) as well as simulated martian UV radiation (>200 nm) led to the most pronounced increase (up to nearly 4 orders of magnitude); however, mutations were also induced in flight samples shielded from insolation, which were exposed to the same conditions except solar irradiation. Nucleotide sequencing located the Rif(R) mutations in the rpoB gene encoding the β-subunit of RNA polymerase. Mutations isolated from flight and parallel mission ground reference (MGR) samples were exclusively localized to Cluster I. The 21 Rif(R) mutations isolated from the flight experiment showed all a C to T transition and were all localized to one hotspot: H482Y. In mutants isolated from the MGR, the spectrum was wider with predicted amino acid changes at residues Q469K/L/R, H482D/P/R/Y, and S487L. The data show the unique mutagenic power of space and martian surface conditions as a consequence of DNA injuries induced by solar UV radiation and space vacuum or the low pressure of Mars.
Spaceflight Study	EXPOSE-E Spaceflight	SLID-316	Transcriptomic responses of germinating Bacillus subtilis spores exposed to 1.5 years of space and simulated martian conditions on the EXPOSE-E experiment PROTECT	559 Days	Bacteria	Because of their ubiquity and resistance to spacecraft decontamination, bacterial spores are considered likely potential forward contaminants on robotic missions to Mars. Thus, it is important to understand their global responses to long-term exposure to space or martian environments. As part of the PROTECT experiment, spores of B. subtilis 168 were exposed to real space conditions and to simulated martian conditions for 559 days in low-Earth orbit mounted on the EXPOSE-E exposure platform outside the European Columbus module on the International Space Station. Upon return, spores were germinated, total RNA extracted, fluorescently labeled, and used to probe a custom Bacillus subtilis microarray to identify genes preferentially activated or repressed relative to ground control spores. Increased transcript levels were detected for a number of stress-related regulons responding to DNA damage (SOS response, SPβ prophage induction), protein damage (CtsR/Clp system), oxidative stress (PerR regulon), and cell envelope stress (SigV regulon). Spores exposed to space demonstrated a much broader and more severe stress response than spores exposed to simulated martian conditions. The results are discussed in the context of planetary protection for a hypothetical journey of potential forward contaminant spores from Earth to Mars and their subsequent residence on Mars.
Spaceflight Study	Shenzhou 7 Space Flight	SLID-317	Spaceflight-induced enhancement of 2-keto-L-gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis	68 Hours	Bacteria	Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l−1 h−1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m3 fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.
Spaceflight Study	Atlantis Space Flight	SLID-318	Spaceflight promotes biofilm formation by Pseudomonas aeruginosa	4 Days,5 Days	Bacteria	Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.
Spaceflight Study	Atlantis Space Flight	SLID-319	Effect of spaceflight on Pseudomonas aeruginosa final cell density is modulated by nutrient and oxygen availability	72 Days	Bacteria	Background Abundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses. Results Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested. Conclusions These results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.
Spaceflight Study	International Space Station (ISS), National Lab Pathfinder Program	SLID-320	Effects of microgravity on the virulence of Listeria monocytogenes, Enterococcus faecalis, Candida albicans, and methicillin-resistant Staphylococcus aureus	96 Hours	Bacteria	To evaluate effects of microgravity on virulence, we studied the ability of four common clinical pathogens—Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, and Candida albicans—to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation in a 2-D clinorotation device, and static ground controls. The feeding rate of worms for killed E. coli was unaffected by spaceflight or clinorotation. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Spaceflight was associated with reduced virulence for Listeria, Enterococcus, MRSA, and Candida for both larval and adult C. elegans. These are the first data acquired with a direct in vivo assay system in space to demonstrate virulence. Clinorotation reproduced the effects of spaceflight in some, but not all, virulence assays: Candida and Enterococcus were less virulent for larval worms but not adult worms, whereas virulence of MRSA and Listeria were unaffected by clinorotation in tests with both adult and larval worms. We conclude that four common clinical microorganisms are all less virulent in space.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-321	A multi-omic analysis of an Enterococcus faecium mutant reveals specific genetic mutations and dramatic changes in mRNA and protein expression	16 Days	Bacteria	Background: For a long time, Enterococcus faecium was considered a harmless commensal of the mammalian gastrointestinal (GI) tract and was used as a probiotic in fermented foods. In recent decades, E. faecium has been recognised as an opportunistic pathogen that causes diseases such as neonatal meningitis, urinary tract infections, bacteremia, bacterial endocarditis and diverticulitis. E. faecium could be taken into space with astronauts and exposed to the space environment. Thus, it is necessary to observe the phenotypic and molecular changes of E. faecium after spaceflight. Results: An E. faecium mutant with biochemical features that are different from those of the wild-type strain was obtained from subculture after flight on the SHENZHOU-8 spacecraft. To understand the underlying mechanism causing these changes, the whole genomes of both the mutant and the WT strains were sequenced using Illumina technology. The genomic comparison revealed that dprA, a recombination-mediator gene, and arpU, a gene associated with cell wall growth, were mutated. Comparative transcriptomic and proteomic analyses showed that differentially expressed genes or proteins were involved with replication, recombination, repair, cell wall biogenesis, glycometabolism, lipid metabolism, amino acid metabolism, predicted general function and energy production/conversion. Conclusion: This study analysed the comprehensive genomic, transcriptomic and proteomic changes of an E. faecium mutant from subcultures that were loaded on the SHENZHOU-8 spacecraft. The implications of these gene mutations and expression changes and their underlying mechanisms should be investigated in the future. We hope that the current exploration of multiple "-omics" analyses of this E. faecium mutant will provide clues for future studies on this opportunistic pathogen.
Ground Study	n.a.	SLID-322	Protein patterns of black fungi under simulated Mars-like conditions	7 Days	Fungi	Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-323	Comparative genomic analysis of Klebsiella pneumonia (LCT-KP214) and a mutant strain (LCT-KP289) obtained after spaceflight	17 Days	Bacteria	Background: With the development of space science, it is important to analyze the relationship between the space environment and genome variations that might cause phenotypic changes in microbes. Klebsiella pneumoniae is commonly found on the human body and is resistant to multiple drugs. To study space-environment-induced genome variations and drug resistance changes, K. pneumoniae was carried into outer space by the Shenzhou VIII spacecraft. Results: The K. pneumoniae strain LCT-KP289 was selected after spaceflight based on its phenotypic differences compared to the ground-control strain. Analysis of genomic structural variations revealed one inversion, 25 deletions, fifty-nine insertions, two translocations and six translocations with inversions. In addition, 155 and 400 unique genes were observed in LCT-KP214 and LCT-KP289, respectively, including the gene encoding dihydroxyacetone kinase, which generates the ATP and NADH required for microbial growth. Furthermore, a large number of mutant genes were related to transport and metabolism. Phylogenetic analysis revealed that most genes in these two strains had a dN/dS value greater than 1, indicating that the strain diversity increased after spaceflight. Analysis of drug-resistance phenotypes revealed that the K. pneumoniae strain LCT-KP289 was resistant to sulfamethoxazole, whereas the control strain, LCT-KP214, was not; both strains were resistant to benzylpenicillin, ampicillin, lincomycin, vancomycin, chloramphenicol and streptomycin. The sulfamethoxazole resistance may be associated with sequences in Scaffold7 in LCT-KP289, which were not observed in LCT-K214; this scaffold contained the gene sul1. In the strain LCT-KP289, we also observed a drug-resistance integron containing emrE (confers multidrug resistance) and ant (confers resistance to spectinomycin, streptomycin, tobramycin, kanamycin, sisomicin, dibekacin, and gentamicin). The gene ampC (confers resistance to penicillin, cephalosporin-ii and cephalosporin-i) was present near the integron. In addition, 30 and 26 drug-resistance genes were observed in LCT-KP289 and LCT-KP214, respectively. Conclusions: Comparison of a K. pneumoniae strain obtained after spaceflight with the ground-control strain revealed genome variations and phenotypic changes and elucidated the genomic basis of the acquired drug resistance. These data pave the way for future studies on the effects of spaceflight.
Ground Study	n.a.	SLID-324	Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability	31 Days	Bacteria	The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.
Spaceflight Study	Shenzhou 10 Space Flight	SLID-324	Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability	15 Days	Bacteria	The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.
Ground Study	n.a.	SLID-325	Use of genome sequencing to assess nucleotide structure variation of Staphylococcus aureus strains cultured in spaceflight on Shenzhou-X, under simulated microgravity and on the ground	15 Days	Bacteria	The extreme environment of space could affect microbial behavior and may increase the risk of infectious disease during spaceflight. However, the molecular genetic changes of methicillin-resistant Staphylococcus aureus (MRSA) in response to the spaceflight environment have not been fully clarified. In the present study, we determined the draft genome sequences for an ancestral S. aureus strain (LCT-SAO) isolated from a clinical sample and three derivative strains, LCT-SAS, LCT-SAM and LCT-SAG, cultured in parallel during the spaceflight Shenzhou-X, under simulated microgravity and on the ground, respectively. To evaluate the impact of short-term spaceflight on the MRSA strains, comparative genomic analysis was implemented. Genome-based mapping of toxin genes and antibiotic resistance genes confirmed that these strains have the conventional pathogenicity and resistance to drugs, as none of the strains showed significant changes in these regions after culturing in the three different environments; this result suggests that spaceflight may not change bacterial virulence or drug resistance. Thirty-nine strain-specific sequence variants (SVs) were identified throughout the genomes, and the three derivatives exhibited almost the same mutation rates. Fifty-nine percent of SVs were located in the intergenic regions of the genomes, indicating that S. aureus may have an extremely robust repair mechanism responsible for recognizing and repairing DNA replication mismatches. It is noteworthy that strain LCT-SAS, cultured in space, presented the most unique SVs (n=9) and shared the fewest SVs with LCT-SAM (n=5) and LCT-SAG (n=4). Furthermore, we identified 10 potential deletion regions and 2 potential insertion regions, with LCT-SAS appearing more fragile than other strains by this measure. These results suggest that the environment of space is inherently complicated, with multiple variables, and cannot be simulated in a simple manner. Our results represent the first analysis of nucleotide structure variation of S. aureus strains in a spaceflight environment and also provide a valuable insight for understanding the mutation strategies of MRSA on earth.
Spaceflight Study	Shenzhou 10 Space Flight	SLID-325	Use of genome sequencing to assess nucleotide structure variation of Staphylococcus aureus strains cultured in spaceflight on Shenzhou-X, under simulated microgravity and on the ground	15 Days	Bacteria	The extreme environment of space could affect microbial behavior and may increase the risk of infectious disease during spaceflight. However, the molecular genetic changes of methicillin-resistant Staphylococcus aureus (MRSA) in response to the spaceflight environment have not been fully clarified. In the present study, we determined the draft genome sequences for an ancestral S. aureus strain (LCT-SAO) isolated from a clinical sample and three derivative strains, LCT-SAS, LCT-SAM and LCT-SAG, cultured in parallel during the spaceflight Shenzhou-X, under simulated microgravity and on the ground, respectively. To evaluate the impact of short-term spaceflight on the MRSA strains, comparative genomic analysis was implemented. Genome-based mapping of toxin genes and antibiotic resistance genes confirmed that these strains have the conventional pathogenicity and resistance to drugs, as none of the strains showed significant changes in these regions after culturing in the three different environments; this result suggests that spaceflight may not change bacterial virulence or drug resistance. Thirty-nine strain-specific sequence variants (SVs) were identified throughout the genomes, and the three derivatives exhibited almost the same mutation rates. Fifty-nine percent of SVs were located in the intergenic regions of the genomes, indicating that S. aureus may have an extremely robust repair mechanism responsible for recognizing and repairing DNA replication mismatches. It is noteworthy that strain LCT-SAS, cultured in space, presented the most unique SVs (n=9) and shared the fewest SVs with LCT-SAM (n=5) and LCT-SAG (n=4). Furthermore, we identified 10 potential deletion regions and 2 potential insertion regions, with LCT-SAS appearing more fragile than other strains by this measure. These results suggest that the environment of space is inherently complicated, with multiple variables, and cannot be simulated in a simple manner. Our results represent the first analysis of nucleotide structure variation of S. aureus strains in a spaceflight environment and also provide a valuable insight for understanding the mutation strategies of MRSA on earth.
Ground Study	n.a.	SLID-326	Effects of simulated microgravity and spaceflight on morphological differentiation and secondary metabolism of Streptomyces coelicolor A3(2)	16.5 Days	Bacteria	As well-known antibiotic-producing and filamentous bacteria, streptomycetes can be an ideal model to study the effects of microgravity on microbial development and antibiotic-production. In this study, the model organism Streptomyces coelicolor A3(2) was exposed to simulated microgravity (SMG) on a rotating clinostat and microgravity (μg) on the Shenzhou-8 spacecraft. The strain exhibited some similar responses under both conditions. Compared with the controls, its life cycle in agar medium was shortened relatively, and the sporulation process was accelerated with higher accumulation of the gray spore pigment; the liquid cultures yielded more cell biomass, coupled with thicker, more fragmented, and well-dispersed hyphae of the μg spaceflight samples. Global transcriptional analysis verified that most of the differentially expressed genes involved in morphological differentiation of S. coelicolor were upregulated during days 4–6 under SMG conditions, notably the whi genes (whiD, sigF, and whiE). Production of actinorhodin (ACT) in agar cultures decreased under both conditions while undecylprodigiosin (RED) was produced earlier, which were consistent with the transcriptional levels of act and red gene clusters. Meanwhile, expression of the gene clusters for calcium-dependent antibiotic (CDA), methylenomycin (MMY), and a cryptic polyketide (CPK) was unchanged, downregulated, and upregulated, respectively, the latter of which might contribute to the enhanced activity of S. coelicolor against Bacillus subtilis under microgravity. Our study provides new insights into the morphological and secondary metabolic responses of streptomycetes to microgravity.
Ground Study	n.a.	SLID-326	Effects of simulated microgravity and spaceflight on morphological differentiation and secondary metabolism of Streptomyces coelicolor A3(2)	16.5 Days	Bacteria	As well-known antibiotic-producing and filamentous bacteria, streptomycetes can be an ideal model to study the effects of microgravity on microbial development and antibiotic-production. In this study, the model organism Streptomyces coelicolor A3(2) was exposed to simulated microgravity (SMG) on a rotating clinostat and microgravity (μg) on the Shenzhou-8 spacecraft. The strain exhibited some similar responses under both conditions. Compared with the controls, its life cycle in agar medium was shortened relatively, and the sporulation process was accelerated with higher accumulation of the gray spore pigment; the liquid cultures yielded more cell biomass, coupled with thicker, more fragmented, and well-dispersed hyphae of the μg spaceflight samples. Global transcriptional analysis verified that most of the differentially expressed genes involved in morphological differentiation of S. coelicolor were upregulated during days 4–6 under SMG conditions, notably the whi genes (whiD, sigF, and whiE). Production of actinorhodin (ACT) in agar cultures decreased under both conditions while undecylprodigiosin (RED) was produced earlier, which were consistent with the transcriptional levels of act and red gene clusters. Meanwhile, expression of the gene clusters for calcium-dependent antibiotic (CDA), methylenomycin (MMY), and a cryptic polyketide (CPK) was unchanged, downregulated, and upregulated, respectively, the latter of which might contribute to the enhanced activity of S. coelicolor against Bacillus subtilis under microgravity. Our study provides new insights into the morphological and secondary metabolic responses of streptomycetes to microgravity.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-326	Effects of simulated microgravity and spaceflight on morphological differentiation and secondary metabolism of Streptomyces coelicolor A3(2)	16.5 Days	Bacteria	As well-known antibiotic-producing and filamentous bacteria, streptomycetes can be an ideal model to study the effects of microgravity on microbial development and antibiotic-production. In this study, the model organism Streptomyces coelicolor A3(2) was exposed to simulated microgravity (SMG) on a rotating clinostat and microgravity (μg) on the Shenzhou-8 spacecraft. The strain exhibited some similar responses under both conditions. Compared with the controls, its life cycle in agar medium was shortened relatively, and the sporulation process was accelerated with higher accumulation of the gray spore pigment; the liquid cultures yielded more cell biomass, coupled with thicker, more fragmented, and well-dispersed hyphae of the μg spaceflight samples. Global transcriptional analysis verified that most of the differentially expressed genes involved in morphological differentiation of S. coelicolor were upregulated during days 4–6 under SMG conditions, notably the whi genes (whiD, sigF, and whiE). Production of actinorhodin (ACT) in agar cultures decreased under both conditions while undecylprodigiosin (RED) was produced earlier, which were consistent with the transcriptional levels of act and red gene clusters. Meanwhile, expression of the gene clusters for calcium-dependent antibiotic (CDA), methylenomycin (MMY), and a cryptic polyketide (CPK) was unchanged, downregulated, and upregulated, respectively, the latter of which might contribute to the enhanced activity of S. coelicolor against Bacillus subtilis under microgravity. Our study provides new insights into the morphological and secondary metabolic responses of streptomycetes to microgravity.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-326	Effects of simulated microgravity and spaceflight on morphological differentiation and secondary metabolism of Streptomyces coelicolor A3(2)	16.5 Days	Bacteria	As well-known antibiotic-producing and filamentous bacteria, streptomycetes can be an ideal model to study the effects of microgravity on microbial development and antibiotic-production. In this study, the model organism Streptomyces coelicolor A3(2) was exposed to simulated microgravity (SMG) on a rotating clinostat and microgravity (μg) on the Shenzhou-8 spacecraft. The strain exhibited some similar responses under both conditions. Compared with the controls, its life cycle in agar medium was shortened relatively, and the sporulation process was accelerated with higher accumulation of the gray spore pigment; the liquid cultures yielded more cell biomass, coupled with thicker, more fragmented, and well-dispersed hyphae of the μg spaceflight samples. Global transcriptional analysis verified that most of the differentially expressed genes involved in morphological differentiation of S. coelicolor were upregulated during days 4–6 under SMG conditions, notably the whi genes (whiD, sigF, and whiE). Production of actinorhodin (ACT) in agar cultures decreased under both conditions while undecylprodigiosin (RED) was produced earlier, which were consistent with the transcriptional levels of act and red gene clusters. Meanwhile, expression of the gene clusters for calcium-dependent antibiotic (CDA), methylenomycin (MMY), and a cryptic polyketide (CPK) was unchanged, downregulated, and upregulated, respectively, the latter of which might contribute to the enhanced activity of S. coelicolor against Bacillus subtilis under microgravity. Our study provides new insights into the morphological and secondary metabolic responses of streptomycetes to microgravity.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-327	Genomic and Proteomic Analysis of Escherichia coli After Spaceflight Reveals Changes Involving Metabolic Pathways	398 Hours	Bacteria	Background and aims: The space environment could have impacts on a variety of characteristics of microorganism such as cell metabolism, drug resistance, and virulence. However, relevant mechanisms need to be clarified. In the present study, the effect of a space environment on Escherichia coli was investigated. Methods: E. coli strains were sent to space for 398 h on the Shenzhou VIII and ground simulation was conducted as control. After the flight, a mutant strain LCT-EC67 was selected for further analysis. Results: Although no changes in hemolysis, morphology or antibiotic sensitivity were observed, the mutant strain showed elevated carbon source utilization compared with the control group. Genomic and proteomic analyses showed that 801 genes were upregulated and 825 genes were downregulated. In addition, 167 proteins were overexpressed and 92 proteins were downregulated using a cut-off fold-change value of 1.4 and a p < 0.05. The changed proteins were associated with metabolic functions such as alanine and glutamate metabolism, arginine and proline metabolism, and fatty acid and propanoate metabolism. Conclusions: E. coli showed alterations at gene and protein levels mainly regarding biochemical metabolism after spaceflight.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-328	Effects of Space Environment on Genome, Transcriptome, and Proteome of Klebsiella pneumoniae	17 Days	Bacteria	Background and Aims The aim of this study was to explore the effects of space flight on Klebsiella pneumoniae. Methods A strain of K. pneumoniae was sent to space for 398 h aboard the ShenZhou VIII spacecraft during November 1, 2011–November 17, 2011. At the same time, a ground simulation with similar temperature conditions during the space flight was performed as a control. After the space mission, the flight and control strains were analyzed using phenotypic, genomic, transcriptomic and proteomic techniques. Results The flight strains LCT-KP289 exhibited a higher cotrimoxazole resistance level and changes in metabolism relative to the ground control strain LCT-KP214. After the space flight, 73 SNPs and a plasmid copy number variation were identified in the flight strain. Based on the transcriptomic analysis, there are 232 upregulated and 1879 downregulated genes, of which almost all were for metabolism. Proteomic analysis revealed that there were 57 upregulated and 125 downregulated proteins. These differentially expressed proteins had several functions that included energy production and conversion, carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, posttranslational modification, protein turnover, and chaperone functions. At a systems biology level, the ytfG gene had a synonymous mutation that resulted in significantly downregulated expression at both transcriptomic and proteomic levels. Conclusions The mutation of the ytfG gene may influence fructose and mannose metabolic processes of K. pneumoniae during space flight, which may be beneficial to the field of space microbiology, providing potential therapeutic strategies to combat or prevent infection in astronauts.
Spaceflight Study	EXPOSE-E Spaceflight	SLID-329	Survival of Antarctic Cryptoendolithic Fungi in Simulated Martian Conditions On Board the International Space Station	559 Days	Fungi	Dehydrated Antarctic cryptoendolithic communities and colonies of the rock inhabitant black fungi Cryomyces antarcticus (CCFEE 515) and Cryomyces minteri (CCFEE 5187) were exposed as part of the Lichens and Fungi Experiment (LIFE) for 18 months in the European Space Agency's EXPOSE-E facility to simulated martian conditions aboard the International Space Station (ISS). Upon sample retrieval, survival was proved by testing colony-forming ability, and viability of cells (as integrity of cell membrane) was determined by the propidium monoazide (PMA) assay coupled with quantitative PCR tests. Although less than 10% of the samples exposed to simulated martian conditions were able to proliferate and form colonies, the PMA assay indicated that more than 60% of the cells and rock communities had remained intact after the "Mars exposure." Furthermore, a high stability of the DNA in the cells was demonstrated. The results contribute to assessing the stability of resistant microorganisms and biosignatures on the surface of Mars, data that are valuable information for further search-for-life experiments on Mars.
Spaceflight Study	KIBO of the ISS	SLID-330	Characterization of fungi isolated from the equipment used in the International Space Station or Space Shuttle	n.a.	Fungi	As a part of a series of studies regarding the microbial biota in manned space environments, fungi were isolated from six pieces of equipment recovered from the Japanese Experimental Module "KIBO" of the International Space Station and from a space shuttle. Thirty-seven strains of fungi were isolated, identified and investigated with regard to morphological phenotypes and antifungal susceptibilities. The variety of fungi isolated in this study was similar to that of several previous reports. The dominant species belonged to the genera Penicillium, Aspergillus and Cladosporium, which are potential causative agents of allergy and opportunistic infections. The morphological phenotypes and antifungal susceptibilities of the strains isolated from space environments were not significantly different from those of reference strains on Earth.
Spaceflight Study	STS-133	SLID-331	Fungal Spores Viability on the International Space Station	14 Days	Fungi	In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.
Spaceflight Study	STS-133	SLID-331	Fungal Spores Viability on the International Space Station	150 Days	Fungi	In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.
Spaceflight Study	BRIC-18 Space Flight	SLID-332	Cultivation of Staphylococcus epidermidis in the Human Spaceflight Environment Leads to Alterations in the Frequency and Spectrum of Spontaneous Rifampicin-Resistance Mutations in the rpoB Gene	122 Hours	Bacteria	Bacteria of the genus Staphylococcus are persistent inhabitants of human spaceflight habitats and represent potential opportunistic pathogens. The effect of the human spaceflight environment on the growth and the frequency of mutations to antibiotic resistance in the model organism Staphylococcus epidermidis strain ATCC12228 was investigated. Six cultures of the test organism were cultivated in biological research in canisters-Petri dish fixation units for 122 h on orbit in the International Space Station (ISS) as part of the SpaceX-3 resupply mission. Asynchronous ground controls (GCs) consisted of identical sets of cultures cultivated for 122 h in the ISS Environmental Simulator at Kennedy Space Center. S. epidermidis exhibited significantly lower viable counts but significantly higher frequencies of mutation to rifampicin (Rif) resistance in space vs. GC cultures. The spectrum of mutations in the rpoB gene leading to Rif(R) was altered in S. epidermidis isolates cultivated in the ISS compared to GCs. The results suggest that the human spaceflight environment induces unique physiologic stresses on growing bacterial cells leading to changes in mutagenic potential.
Spaceflight Study	STS-115	SLID-333	A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions	25 Hours	Bacteria	Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level.
Spaceflight Study	STS-123	SLID-333	A Systems Biology Analysis Unfolds the Molecular Pathways and Networks of Two Proteobacteria in Spaceflight and Simulated Microgravity Conditions	25 Hours	Bacteria	Bacteria are important organisms for space missions due to their increased pathogenesis in microgravity that poses risks to the health of astronauts and for projected synthetic biology applications at the space station. We understand little about the effect, at the molecular systems level, of microgravity on bacteria, despite their significant incidence. In this study, we proposed a systems biology pipeline and performed an analysis on published gene expression data sets from multiple seminal studies on Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium under spaceflight and simulated microgravity conditions. By applying gene set enrichment analysis on the global gene expression data, we directly identified a large number of new, statistically significant cellular and metabolic pathways involved in response to microgravity. Alteration of metabolic pathways in microgravity has rarely been reported before, whereas in this analysis metabolic pathways are prevalent. Several of those pathways were found to be common across studies and species, indicating a common cellular response in microgravity. We clustered genes based on their expression patterns using consensus non-negative matrix factorization. The genes from different mathematically stable clusters showed protein-protein association networks with distinct biological functions, suggesting the plausible functional or regulatory network motifs in response to microgravity. The newly identified pathways and networks showed connection with increased survival of pathogens within macrophages, virulence, and antibiotic resistance in microgravity. Our work establishes a systems biology pipeline and provides an integrated insight into the effect of microgravity at the molecular systems level.
Spaceflight Study	Cygnus Spacecraft	SLID-334	A Molecular Genetic Basis Explaining Altered Bacterial Behavior in Space	49 Hours	Bacteria	Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity-all of which can be associated with reduced extracellular mass transport.
Spaceflight Study	n.a.	SLID-335	Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station	n.a.	Fungi	One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments.As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.
Spaceflight Study	FOTON-M4 Capsule	SLID-336	The Impact of Space Flight on Survival and Interaction of Cupriavidus metallidurans CH34 with Basalt, a Volcanic Moon Analog Rock	90 Days	Bacteria	Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions.
Spaceflight Study	Expedition 19	SLID-337	Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station	n.a.	Bacteria	In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.
Spaceflight Study	Soyuz TMA-3/expedition 8, Delta Mission	SLID-337	Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station	n.a.	Bacteria	In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.
Spaceflight Study	STS-134/ULF6	SLID-337	Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station	n.a.	Bacteria	In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.
Spaceflight Study	International Space Station (ISS)	SLID-338	Whole metagenome profiles of particulates collected from the International Space Station	n.a.	Bacteria	Background: The built environment of the International Space Station (ISS) is a highly specialized space in terms of both physical characteristics and habitation requirements. It is unique with respect to conditions of microgravity, exposure to space radiation, and increased carbon dioxide concentrations. Additionally, astronauts inhabit a large proportion of this environment. The microbial composition of ISS particulates has been reported; however, its functional genomics, which are pertinent due to potential impact of its constituents on human health and operational mission success, are not yet characterized. Methods: This study examined the whole metagenome of ISS microbes at both species- and gene-level resolution. Air filter and dust samples from the ISS were analyzed and compared to samples collected in a terrestrial cleanroom environment. Furthermore, metagenome mining was carried out to characterize dominant, virulent, and novel microorganisms. The whole genome sequences of select cultivable strains isolated from these samples were extracted from the metagenome and compared. Results: Species-level composition in the ISS was found to be largely dominated by Corynebacterium ihumii GD7, with overall microbial diversity being lower in the ISS relative to the cleanroom samples. When examining detection of microbial genes relevant to human health such as antimicrobial resistance and virulence genes, it was found that a larger number of relevant gene categories were observed in the ISS relative to the cleanroom. Strain-level cross-sample comparisons were made for Corynebacterium, Bacillus, and Aspergillus showing possible distinctions in the dominant strain between samples. Conclusion: Species-level analyses demonstrated distinct differences between the ISS and cleanroom samples, indicating that the cleanroom population is not necessarily reflective of space habitation environments. The overall population of viable microorganisms and the functional diversity inherent to this unique closed environment are of critical interest with respect to future space habitation. Observations and studies such as these will be important to evaluating the conditions required for long-term health of human occupants in such environments.
Spaceflight Study	International Space Station (ISS)	SLID-338	Whole metagenome profiles of particulates collected from the International Space Station	n.a.	Bacteria	Background: The built environment of the International Space Station (ISS) is a highly specialized space in terms of both physical characteristics and habitation requirements. It is unique with respect to conditions of microgravity, exposure to space radiation, and increased carbon dioxide concentrations. Additionally, astronauts inhabit a large proportion of this environment. The microbial composition of ISS particulates has been reported; however, its functional genomics, which are pertinent due to potential impact of its constituents on human health and operational mission success, are not yet characterized. Methods: This study examined the whole metagenome of ISS microbes at both species- and gene-level resolution. Air filter and dust samples from the ISS were analyzed and compared to samples collected in a terrestrial cleanroom environment. Furthermore, metagenome mining was carried out to characterize dominant, virulent, and novel microorganisms. The whole genome sequences of select cultivable strains isolated from these samples were extracted from the metagenome and compared. Results: Species-level composition in the ISS was found to be largely dominated by Corynebacterium ihumii GD7, with overall microbial diversity being lower in the ISS relative to the cleanroom samples. When examining detection of microbial genes relevant to human health such as antimicrobial resistance and virulence genes, it was found that a larger number of relevant gene categories were observed in the ISS relative to the cleanroom. Strain-level cross-sample comparisons were made for Corynebacterium, Bacillus, and Aspergillus showing possible distinctions in the dominant strain between samples. Conclusion: Species-level analyses demonstrated distinct differences between the ISS and cleanroom samples, indicating that the cleanroom population is not necessarily reflective of space habitation environments. The overall population of viable microorganisms and the functional diversity inherent to this unique closed environment are of critical interest with respect to future space habitation. Observations and studies such as these will be important to evaluating the conditions required for long-term health of human occupants in such environments.
Ground Study	n.a.	SLID-339	Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress	131 Hours	Bacteria	Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.
Ground Study	n.a.	SLID-339	Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress	131 Hours	Bacteria	Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.
Ground Study	n.a.	SLID-339	Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress	75 Hours	Bacteria	Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.
Ground Study	n.a.	SLID-339	Exposure of Mycobacterium marinum to low-shear modeled microgravity: effect on growth, the transcriptome and survival under stress	75 Hours	Bacteria	Waterborne pathogenic mycobacteria can form biofilms, and certain species can cause hard-to-treat human lung infections. Astronaut health could therefore be compromised if the spacecraft environment or water becomes contaminated with pathogenic mycobacteria. This work uses Mycobacterium marinum to determine the physiological changes in a pathogenic mycobacteria grown under low-shear modeled microgravity (LSMMG). M. marinum were grown in high aspect ratio vessels (HARVs) using a rotary cell culture system subjected to LSMMG or the control orientation (normal gravity, NG) and the cultures used to determine bacterial growth, bacterium size, transcriptome changes, and resistance to stress. Two exposure times to LSMMG and NG were examined: bacteria were grown for ~40 h (short), or 4 days followed by re-dilution and growth for ~35 h (long). M. marinum exposed to LSMMG transitioned from exponential phase earlier than the NG culture. They were more sensitive to hydrogen peroxide but showed no change in resistance to gamma radiation or pH 3.5. RNA-Seq detected significantly altered transcript levels for 562 and 328 genes under LSMMG after short and long exposure times, respectively. Results suggest that LSMMG induced a reduction in translation, a downregulation of metabolism, an increase in lipid degradation, and increased chaperone and mycobactin expression. Sigma factor H (sigH) was the only sigma factor transcript induced by LSMMG after both short and long exposure times. In summary, transcriptome studies suggest that LSMMG may simulate a nutrient-deprived environment similar to that found within macrophage during infection. SigH is also implicated in the M. marinum LSMMG transcriptome response.
Spaceflight Study	n.a.	SLID-340	Draft Genome Sequences from a Novel Clade of Bacillus cereus Sensu Lato Strains, Isolated from the International Space Station	n.a.	Bacteria	The draft genome sequences of six Bacillus strains, isolated from the International Space Station and belonging to the Bacillus anthracis-B. cereus-B. thuringiensis group, are presented here. These strains were isolated from the Japanese Experiment Module (one strain), U.S. Harmony Node 2 (three strains), and Russian Segment Zvezda Module (two strains).
Spaceflight Study	BRIC Space Flight	SLID-341	Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds	31 Days	Bacteria	Past results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion, Bacillus subtilis cells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth_x0002_normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system. Only 9 compounds were identified by PM screening as exhibiting significant differences (P＜0.05, Student’s t test) in FLversus GC samples: 6-mercaptopurine, cesium chloride, enoxacin, lomefloxacin, manganese(II) chloride, nalidixic acid, penimepicycline, rolitetracycline, and trifluopera_x0002_zine. Testing of the same compounds by standard broth dilution assay did not re_x0002_veal statistically significant differences in the 50% inhibitory concentrations (IC50s) between FL and GC samples. The results indicate that the susceptibility of B. subtilis cells to a wide range of antibiotics and growth inhibitors is not dramatically altered by space flight.
Spaceflight Study	Cygnus Spacecraft	SLID-342	Phenotypic Changes Exhibited by E. coli Cultured in Space	49 Hours	Bacteria	Bacteria will accompany humans in our exploration of space, making it of importance to study their adaptation to the microgravity environment. To investigate potential phenotypic changes for bacteria grown in space, Escherichia coli was cultured onboard the International Space Station with matched controls on Earth. Samples were challenged with different concentrations of gentamicin sulfate to study the role of drug concentration on the dependent variables in the space environment. Analyses included assessments of final cell count, cell size, cell envelope thickness, cell ultrastructure, and culture morphology. A 13-fold increase in final cell count was observed in space with respect to the ground controls and the space flight cells were able to grow in the presence of normally inhibitory levels of gentamicin sulfate. Contrast light microscopy and focused ion beam/scanning electron microscopy showed that, on average, cells in space were 37% of the volume of their matched controls, which may alter the rate of molecule–cell interactions in a diffusion-limited mass transport regime as is expected to occur in microgravity. TEM imagery showed an increase in cell envelope thickness of between 25 and 43% in space with respect to the Earth control group. Outer membrane vesicles were observed on the spaceflight samples, but not on the Earth cultures. While E. coli suspension cultures on Earth were homogenously distributed throughout the liquid medium, in space they tended to form a cluster, leaving the surrounding medium visibly clear of cells. This cell aggregation behavior may be associated with enhanced biofilm formation observed in other spaceflight experiments.
Spaceflight Study	E. coli Antimicrobial Satellite (EcAMSat)	SLID-343	Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant	156.5 Hours	Bacteria	Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.
Ground Study	Low-shear Modeled Microgravity (LSMMG)	SLID-343	Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant	n.a.	Bacteria	Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.
Spaceflight Study	SpX-5/TMA-14A	SLID-344	Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces	n.a.	Bacteria	Antimicrobial resistance (AMR) is a global health issue. In an effort to minimize this threat to astronauts, who may be immunocompromised and thus at a greater risk of infection from antimicrobial resistant pathogens, a comprehensive study of the ISS “resistome’ was conducted. Using whole genome sequencing (WGS) and disc diffusion antibiotic resistance assays, 9 biosafety level 2 organisms isolated from the ISS were assessed for their antibiotic resistance. Molecular analysis of AMR genes from 24 surface samples collected from the ISS during 3 different sampling events over a span of a year were analyzed with Ion AmpliSeq™ and metagenomics. Disc diffusion assays showed that Enterobacter bugandensis strains were resistant to all 9 antibiotics tested and Staphylococcus haemolyticus being resistant to none. Ion AmpliSeq™ revealed that 123 AMR genes were found, with those responsible for beta-lactam and trimethoprim resistance being the most abundant and widespread. Using a variety of methods, the genes involved in antimicrobial resistance have been examined for the first time from the ISS. This information could lead to mitigation strategies to maintain astronaut health during long duration space missions when return to Earth for treatment is not possible.
Spaceflight Study	SpX-6	SLID-344	Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces	n.a.	Bacteria	Antimicrobial resistance (AMR) is a global health issue. In an effort to minimize this threat to astronauts, who may be immunocompromised and thus at a greater risk of infection from antimicrobial resistant pathogens, a comprehensive study of the ISS “resistome’ was conducted. Using whole genome sequencing (WGS) and disc diffusion antibiotic resistance assays, 9 biosafety level 2 organisms isolated from the ISS were assessed for their antibiotic resistance. Molecular analysis of AMR genes from 24 surface samples collected from the ISS during 3 different sampling events over a span of a year were analyzed with Ion AmpliSeq™ and metagenomics. Disc diffusion assays showed that Enterobacter bugandensis strains were resistant to all 9 antibiotics tested and Staphylococcus haemolyticus being resistant to none. Ion AmpliSeq™ revealed that 123 AMR genes were found, with those responsible for beta-lactam and trimethoprim resistance being the most abundant and widespread. Using a variety of methods, the genes involved in antimicrobial resistance have been examined for the first time from the ISS. This information could lead to mitigation strategies to maintain astronaut health during long duration space missions when return to Earth for treatment is not possible.
Spaceflight Study	SpX-8	SLID-344	Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces	n.a.	Bacteria	Antimicrobial resistance (AMR) is a global health issue. In an effort to minimize this threat to astronauts, who may be immunocompromised and thus at a greater risk of infection from antimicrobial resistant pathogens, a comprehensive study of the ISS “resistome’ was conducted. Using whole genome sequencing (WGS) and disc diffusion antibiotic resistance assays, 9 biosafety level 2 organisms isolated from the ISS were assessed for their antibiotic resistance. Molecular analysis of AMR genes from 24 surface samples collected from the ISS during 3 different sampling events over a span of a year were analyzed with Ion AmpliSeq™ and metagenomics. Disc diffusion assays showed that Enterobacter bugandensis strains were resistant to all 9 antibiotics tested and Staphylococcus haemolyticus being resistant to none. Ion AmpliSeq™ revealed that 123 AMR genes were found, with those responsible for beta-lactam and trimethoprim resistance being the most abundant and widespread. Using a variety of methods, the genes involved in antimicrobial resistance have been examined for the first time from the ISS. This information could lead to mitigation strategies to maintain astronaut health during long duration space missions when return to Earth for treatment is not possible.
Spaceflight Study	BRIC-18 Space Flight	SLID-345	Alterations in the Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Bacillus subtilis rpoB Gene after Cultivation in the Human Spaceflight Environment	122 Hours	Bacteria	The effect of Bacillus subtilis exposure to the human spaceflight environment on growth, mutagenic frequency, and spectrum of mutations to rifampicin resistance (RifR) was investigated. B. subtilis cells were cultivated in Biological Research in Canister-Petri Dish Fixation Units (BRIC-PDFUs) on two separate missions to the International Space Station (ISS), dubbed BRIC-18 and BRIC-21, with matching asynchronous ground controls. No statistically significant difference in either growth or in the frequency of mutation to RifR was found in either experiment. However, nucleotide sequencing of the RifR regions of the rpoB gene from RifR mutants revealed dramatic differences in the spectrum of mutations between flight (FL) and ground control (GC) samples, including two newly discovered rpoB alleles in the FL samples (Q137R and L489S). The results strengthen the idea that exposure to the human spaceflight environment causes unique stresses on bacteria, leading to alterations in their mutagenic potential.
Spaceflight Study	BRIC-21 Space Flight	SLID-345	Alterations in the Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Bacillus subtilis rpoB Gene after Cultivation in the Human Spaceflight Environment	25 Hours	Bacteria	The effect of Bacillus subtilis exposure to the human spaceflight environment on growth, mutagenic frequency, and spectrum of mutations to rifampicin resistance (RifR) was investigated. B. subtilis cells were cultivated in Biological Research in Canister-Petri Dish Fixation Units (BRIC-PDFUs) on two separate missions to the International Space Station (ISS), dubbed BRIC-18 and BRIC-21, with matching asynchronous ground controls. No statistically significant difference in either growth or in the frequency of mutation to RifR was found in either experiment. However, nucleotide sequencing of the RifR regions of the rpoB gene from RifR mutants revealed dramatic differences in the spectrum of mutations between flight (FL) and ground control (GC) samples, including two newly discovered rpoB alleles in the FL samples (Q137R and L489S). The results strengthen the idea that exposure to the human spaceflight environment causes unique stresses on bacteria, leading to alterations in their mutagenic potential.
Spaceflight Study	SpaceX-8 Spacecraft	SLID-346	Probiotics into outer space: feasibility assessments of encapsulated freeze-dried probiotics during 1 month's storage on the International Space Station	30 Days	Bacteria	Suppression of immune function during long spaceflights is an issue that needs to be overcome. The well-established probiotic Lactobacillus casei strain Shirota (LcS) could be a promising countermeasure, and we have launched a project to investigate the efficacy of its use on the International Space Station (ISS). As a first step, we developed a specialist probiotic product for space experiments, containing freeze-dried LcS in capsule form (Probiotics Package), and tested its stability through 1 month of storage on the ISS. The temperature inside the ISS ranged from 20.0 to 24.5 °C. The absorbed dose rate of the flight sample was 0.26 mGy/day and the dose equivalent rate was 0.52 mSv/day. The number of live LcS was 1.05 × 1011 colony-forming units/g powder (49.5% of the initial value) 6 months after the start of the study; this value was comparable to those in the two ground controls. Profiles of randomly amplified polymorphic DNA, sequence variant frequency, carbohydrate fermentation, reactivity to LcS-specific antibody, and the cytokine-inducing ability of LcS in the flight sample did not differ from those of the ground controls. We can therefore maintain the viability and basic probiotic properties of LcS stored as a Probiotics Package on the ISS.
Spaceflight Study	n.a.	SLID-347	Characterization of Aspergillus niger Isolated from the International Space Station	n.a.	Fungi	The initial characterization of the Aspergillus niger isolate JSC-093350089, collected from U.S. segment surfaces of the International Space Station (ISS), is reported, along with a comparison to the extensively studied strain ATCC 1015. Whole-genome sequencing of the ISS isolate enabled its phylogenetic placement within the A. niger/welwitschiae/lacticoffeatus clade and revealed that the genome of JSC-093350089 is within the observed genetic variance of other sequenced A. niger strains. The ISS isolate exhibited an increased rate of growth and pigment distribution compared to a terrestrial strain. Analysis of the isolate’s proteome revealed significant differences in the molecular phenotype of JSC-093350089, including increased abundance of proteins involved in the A. niger starvation response, oxidative stress resistance, cell wall modulation, and nutrient acquisition. Together, these data reveal the existence of a distinct strain of A. niger on board the ISS and provide insight into the characteristics of melanized fungal species inhabiting spacecraft environments.
Spaceflight Study	BASE A;BRIC-21 Space Flight;BRIC-23 Space Flight;MESSAGE 2;STS-115;STS-123;	SLID-348	Meta-analysis of data from spaceflight transcriptome experiments does not support the idea of a common bacterial "spaceflight response"	12 Days;25 Hours;48 Hours;10 Days;25 Hours;25 Hours	Bacteria	Several studies have been undertaken with the goal of understanding how bacterial transcriptomes respond to the human spaceflight environment. However, these experiments have been conducted using a variety of organisms, media, culture conditions, and spaceflight hardware, and to date no cross-experiment analyses have been performed to uncover possible commonalities in their responses. In this study, eight bacterial transcriptome datasets deposited in NASA’s GeneLab Data System were standardized through a common bioinformatics pipeline then subjected to meta-analysis to identify among the datasets (i) individual genes which might be significantly differentially expressed, or (ii) gene sets which might be significantly enriched. Neither analysis resulted in identification of responses shared among all datasets. Principal Component Analysis of the data revealed that most of the variation in the datasets derived from differences in the experiments themselves.
Spaceflight Study	BASE A;BRIC-21 Space Flight;BRIC-23 Space Flight;MESSAGE 2;STS-115;STS-124;	SLID-348	Meta-analysis of data from spaceflight transcriptome experiments does not support the idea of a common bacterial "spaceflight response"	12 Days;25 Hours;48 Hours;10 Days;25 Hours;25 Hours	Bacteria	Several studies have been undertaken with the goal of understanding how bacterial transcriptomes respond to the human spaceflight environment. However, these experiments have been conducted using a variety of organisms, media, culture conditions, and spaceflight hardware, and to date no cross-experiment analyses have been performed to uncover possible commonalities in their responses. In this study, eight bacterial transcriptome datasets deposited in NASA’s GeneLab Data System were standardized through a common bioinformatics pipeline then subjected to meta-analysis to identify among the datasets (i) individual genes which might be significantly differentially expressed, or (ii) gene sets which might be significantly enriched. Neither analysis resulted in identification of responses shared among all datasets. Principal Component Analysis of the data revealed that most of the variation in the datasets derived from differences in the experiments themselves.
Spaceflight Study	BASE A;BRIC-21 Space Flight;BRIC-23 Space Flight;MESSAGE 2;STS-115;STS-125;	SLID-348	Meta-analysis of data from spaceflight transcriptome experiments does not support the idea of a common bacterial "spaceflight response"	12 Days;25 Hours;48 Hours;10 Days;25 Hours;25 Hours	Bacteria	Several studies have been undertaken with the goal of understanding how bacterial transcriptomes respond to the human spaceflight environment. However, these experiments have been conducted using a variety of organisms, media, culture conditions, and spaceflight hardware, and to date no cross-experiment analyses have been performed to uncover possible commonalities in their responses. In this study, eight bacterial transcriptome datasets deposited in NASA’s GeneLab Data System were standardized through a common bioinformatics pipeline then subjected to meta-analysis to identify among the datasets (i) individual genes which might be significantly differentially expressed, or (ii) gene sets which might be significantly enriched. Neither analysis resulted in identification of responses shared among all datasets. Principal Component Analysis of the data revealed that most of the variation in the datasets derived from differences in the experiments themselves.
Spaceflight Study	Shenzhou 11 Space Flight	SLID-349	Decreased biofilm formation ability of Acinetobacter baumannii after spaceflight on China's Shenzhou 11 spacecraft	31 Days	Bacteria	China has prepared for construction of a space station by the early 2020s. The mission will require astronauts to stay on the space station for at least 180 days. Microbes isolated from the International Space Station (ISS) have shown profound resistance to clinical antibiotics and environmental stresses. Previous studies have demonstrated that the space environment could affect microbial survival, growth, virulence, biofilms, metabolism, as well as their antibiotic‐resistant phenotypes. Furthermore, several studies have reported that astronauts experience a decline in their immunity during long‐duration spaceflights. Monitoring microbiomes in the ISS or the spacecraft will be beneficial for the prevention of infection among the astronauts during spaceflight. The development of a manned space program worldwide not only provides an opportunity to investigate the impact of this extreme environment on opportunistic pathogenic microbes, but also offers a unique platform to detect mutations in pathogenic bacteria. Various microorganisms have been carried on a spacecraft for academic purposes. Acinetobacter baumannii is a common multidrug‐resistant bacterium often prevalent in hospitals. Variations in the ability to cope with environmental hazards increase the chances of microbial survival. Our study aimed to compare phenotypic variations and analyze genomic and transcriptomic variations in A. baumannii among three different groups: SS1 (33 days on the Shenzhou 11 spacecraft), GS1 (ground control), and Aba (reference strain). Consequently, the biofilm formation ability of the SS1 strain decreased after 33 days of spaceflight. Furthermore, high‐throughput sequencing revealed that some differentially expressed genes were downregulated in the SS1 strain compared with those in the GS1 strain. In conclusion, this present study provides insights into the environmental adaptation of A. baumannii and might be useful for understanding changes in the opportunistic pathogenic microbes on our spacecraft and on China's future ISS.
Spaceflight Study	SpaceX Mission CRS-8	SLID-350	International Space Station conditions alter genomics, proteomics, and metabolomics in Aspergillus nidulans	23 Days，26 Days	Fungi	The first global genomic, proteomic, and secondary metabolomic characterization of the filamentous fungus Aspergillus nidulans following growth onboard the International Space Station (ISS) is reported. The investigation included the A. nidulans wild-type and three mutant strains, two of which were genetically engineered to enhance secondary metabolite production. Whole genome sequencing revealed that ISS conditions altered the A. nidulans genome in specific regions. In strain CW12001, which features overexpression of the secondary metabolite global regulator laeA, ISS conditions induced the loss of the laeA stop codon. Differential expression of proteins involved in stress response, carbohydrate metabolic processes, and secondary metabolite biosynthesis was also observed. ISS conditions significantly decreased prenyl xanthone production in the wild-type strain and increased asperthecin production in LO1362 and CW12001, which are deficient in a major DNA repair mechanism. These data provide valuable insights into the adaptation mechanism of A. nidulans to spacecraft environments.
Spaceflight Study	n.a.	SLID-351	Proteomic characterization of Aspergillus fumigatus isolated from air and surfaces of the International Space Station	n.a.	Fungi	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 suite 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 well-studied 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 that showed increased abundance in ISS isolates were overall involved in stress responses, and carbohydrate and secondary metabolism. Among the most abundant proteins were Pst2 and ArtA involved in oxidative stress response, PdcA and AcuE responsible for ethanol fermentation and glyoxylate cycle, respectively, TpcA, TpcF, and TpcK that are part of trypacidin biosynthetic pathway, and a toxin Asp-hemolysin. This report provides insight into possible molecular adaptation of filamentous fungi to the unique ISS environment.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-352	LCT-EF258 with S17I Mutation in DprA Exhibits Horizontal Gene Transfer Deficiency After Spaceflight	17 Days	Bacteria	BACKGROUND: Space is a special environment in which microgravity and cosmic rays are the primary factors that induce gene mutations of microorganisms. In our previous studies, a single point mutation in the gene dprA was found in an Enterococcus faecium strain of LCT-EF258 after spaceflight. DNA processing protein A (DprA) plays a prominent role in the horizontal transfer of genes among bacteria (such as Streptococcus pneumoniae, Helicobacter pylori, Bacillus subtilis, and Rhodobacter capsulatus). However, the function of DprA in E. faecium remains unknown. Furthermore, E. faecium could acquire antibiotic resistance through the horizontal transfer of antibiotic resistance genes, but it is unclear whether dprA mutants could affect this process in E. faecium.METHODS: In this study, we constructed a plasmid containing the vancomycin resistance gene vanA and then transferred the gene vanA into the dprA-mutant strain LCT-EF258 and the control strain LCT-EF90 using the electroporation technique. We then used Discovery StudioTM software to construct the 3D protein structure.RESULTS: The results showed that the horizontal transfer efficiency of the vancomycin resistance gene vanA in the dprA-mutant E. faecium decreased. And the hydrophobic core of the mutant DprA became stable and the binding affinity between the mutant DprA and ssDNA reduced.DISCUSSION: This study is an exploration of bacterial gene mutation after spaceflight. The dprA mutant could affect the ability of E. faecium to acquire exogenous resistance gene vanA, which offered us an interesting path to block the dissemination of resistance genes between strains.
Spaceflight Study	n.a.	SLID-353	Microbial community composition of water samples stored inside the International Space Station	n.a.	Bacteria	During the VIABLE ISS project (eValuatIon And monitoring of microBiofiLms insidE International Space Station), water samples subjected to two different silver treatments were sent and kept on board the International Space Station (ISS) from 2011 to 2016. In this note we report data on the viable and total bacterial load and on the composition of the microbial communities of the VIABLE ISS samples.
Spaceflight Study	n.a.	SLID-354	Reactivation of Microbial Strains and Synthetic Communities After a Spaceflight to the International Space Station: Corroborating the Feasibility of Essential Conversions in the MELiSSA Loop	9.84 Days	Bacteria	To sustain human deep space exploration or extra-terrestrial settlements where no resupply from the Earth or other planets is possible, technologies for in situ food production, water, air, and waste recovery need to be developed. The Micro-Ecological Life Support System Alternative (MELiSSA) is such a Regenerative Life Support System (RLSS) and it builds on several bacterial bioprocesses. However, alterations in gravity, temperature, and radiation associated with the space environment can affect survival and functionality of the microorganisms. In this study, representative strains of different carbon and nitrogen metabolisms with application in the MELiSSA were selected for launch and Low Earth Orbit (LEO) exposure. An edible photoautotrophic strain (Arthrospira sp. PCC 8005), a photoheterotrophic strain (Rhodospirillum rubrum S1H), a ureolytic heterotrophic strain (Cupriavidus pinatubonensis 1245), and combinations of C. pinatubonensis 1245 and autotrophic ammonia and nitrite oxidizing strains (Nitrosomonas europaea ATCC19718, Nitrosomonas ureae Nm10, and Nitrobacter winogradskyi Nb255) were sent to the International Space Station (ISS) for 7 days. There, the samples were exposed to 2.8 mGy, a dose 140 times higher than on the Earth, and a temperature of 22°C ± 1°C. On return to the Earth, the cultures were reactivated and their growth and activity were compared with terrestrial controls stored under refrigerated (5°C ± 2°C) or room temperature (22°C ± 1°C and 21°C ± 0°C) conditions. Overall, no difference was observed between terrestrial and ISS samples. Most cultures presented lower cell viability after the test, regardless of the type of exposure, indicating a harsher effect of the storage and sample preparation than the spaceflight itself. Postmission analysis revealed the successful survival and proliferation of all cultures except for Arthrospira, which suffered from the premission depressurization test. These observations validate the possibility of launching, storing, and reactivating bacteria with essential functionalities for microbial bioprocesses in RLSS.
Ground Study	n.a.	SLID-355	Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations	30 Days	Fungi	Baker’s yeast (Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or ‘giant’ colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.
Spaceflight Study	SpaceX-8 Spacecraft	SLID-355	Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations	30 Days	Fungi	Baker’s yeast (Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or ‘giant’ colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.
Spaceflight Study	n.a.	SLID-356	Decreased metabolism and increased tolerance to extreme environments in Staphylococcus warneri during long-term spaceflight	64 Days	Bacteria	Many studies have shown that the space environment can affect bacteria by causing a range of mutations. However, to date, few studies have explored the effects of long‐term spaceflight (>1 month) on bacteria. In this study, a Staphylococcus warneri strain that was isolated from the Shenzhou‐10 spacecraft and had experienced a spaceflight (15 days) was carried into space again. After a 64‐day flight, combined phenotypic, genomic, transcriptomic, and proteomic analyses were performed to compare the influence of the two spaceflights on this bacterium. Compared with short‐term spaceflight, long‐term spaceflight increased the biofilm formation ability of S. warneri and the cell wall resistance to external environmental stress but reduced the sensitivity to chemical stimulation. Further analysis showed that these changes might be associated with the significantly upregulated gene expression of the phosphotransferase system, which regulates the metabolism of sugars, including glucose, mannose, fructose, and cellobiose. The mutation of S. warneri caused by the 15‐day spaceflight was limited at the phenotype and gene level after cultivation on the ground. After 79 days of spaceflight, significant changes in S. warneri were observed. The phosphotransferase system of S. warneri was upregulated by long‐term space stimulation, which resulted in a series of changes in the cell wall, biofilm, and chemical sensitivity, thus enhancing the resistance and adaptability of the bacterium to the external environment.
Spaceflight Study	Shenzhou 10 Space Flight	SLID-356	Decreased metabolism and increased tolerance to extreme environments in Staphylococcus warneri during long-term spaceflight	15 Days	Bacteria	Many studies have shown that the space environment can affect bacteria by causing a range of mutations. However, to date, few studies have explored the effects of long‐term spaceflight (>1 month) on bacteria. In this study, a Staphylococcus warneri strain that was isolated from the Shenzhou‐10 spacecraft and had experienced a spaceflight (15 days) was carried into space again. After a 64‐day flight, combined phenotypic, genomic, transcriptomic, and proteomic analyses were performed to compare the influence of the two spaceflights on this bacterium. Compared with short‐term spaceflight, long‐term spaceflight increased the biofilm formation ability of S. warneri and the cell wall resistance to external environmental stress but reduced the sensitivity to chemical stimulation. Further analysis showed that these changes might be associated with the significantly upregulated gene expression of the phosphotransferase system, which regulates the metabolism of sugars, including glucose, mannose, fructose, and cellobiose. The mutation of S. warneri caused by the 15‐day spaceflight was limited at the phenotype and gene level after cultivation on the ground. After 79 days of spaceflight, significant changes in S. warneri were observed. The phosphotransferase system of S. warneri was upregulated by long‐term space stimulation, which resulted in a series of changes in the cell wall, biofilm, and chemical sensitivity, thus enhancing the resistance and adaptability of the bacterium to the external environment.
Spaceflight Study	E. coli Antimicrobial Satellite (EcAMSat)	SLID-357	EcAMSat spaceflight measurements of the role of σs in antibiotic resistance of stationary phase Escherichia coli in microgravity	156.5 Hours	Bacteria	We report the results of the EcAMSat (Escherichia coli Antimicrobial Satellite) autonomous space flight experiment, investigating the role of σs in the development of antibiotic resistance in uropathogenic E. coli (UPEC) in microgravity (µ-g). The presence of σs, encoded by the rpoS gene, has been shown to increase antibiotic resistance in Earth gravity, but it was unknown if this effect occurs in µ-g. Two strains, wildtype (WT) UPEC and its isogenic ΔrpoS mutant, were grown to stationary phase aboard EcAMSat, an 11-kg small satellite, and in a parallel ground-based control experiment; cell growth rates for the two strains were found to be unaltered by µ-g. After starvation for over 24 h, stationary-phase cells were incubated with three doses of gentamicin (Gm), a common treatment for urinary tract infections (which have been reported in astronauts). Cellular metabolic activity was measured optically using the redox-based indicator alamarBlue (aB): both strains exhibited slower metabolism in µ-g, consistent with results from previous smallsat missions. The results also showed that µ-g did not enhance UPEC resistance to Gm; in fact, both strains were more susceptible to Gm in µ-g. It was also found, via a second ground-control experiment, that multi-week storage in the payload hardware stressed the cells, potentially obscuring small differential effects of the antibiotic between WT and mutant and/or between µ-g and ground. Overall, results showed that the ∆rpoS mutant was 34-37% less metabolically active than the WT for four different sets of conditions: ground without Gm, ground with Gm; µ-g without Gm, µ-g with Gm. We conclude therefore that the rpoS gene and its downstream products are important therapeutic targets for treating bacterial infections in space, much as they are on the ground.
Spaceflight Study	SpaceX-CRS 5 Space Flight	SLID-358	Spaceflight and simulated microgravity conditions increase virulence of Serratia marcescens in the Drosophila melanogaster infection model	30 Days	Bacteria	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 the increased virulence phenotype of S. marcescens did not persist after the bacterial cultures were passaged on the ground. Increased virulence was also observed in bacteria that were grown in simulated microgravity conditions on the ground using the rotating wall vessel. Increased virulence of the space-flown bacteria was similar in magnitude between wild-type flies and those that were mutants for the well-characterized immune pathways Imd and Toll, suggesting that changes to the host immune system after infection are likely not a major factor contributing towards increased susceptibility of ground-reared flies infected with space-flown bacteria. Characterization of the bacteria shows that at later timepoints spaceflight bacteria grew at a greater rate than ground controls in vitro, and in the host. These results suggest complex physiological changes occurring in pathogenic bacteria in space environments, and there may be novel mechanisms mediating these physiological effects that need to be characterized.
Spaceflight Study	n.a.	SLID-359	Metabolomic Analysis of Aspergillus niger Isolated From the International Space Station Reveals Enhanced Production Levels of the Antioxidant Pyranonigrin A	n.a.	Fungi	Secondary metabolite (SM) production in Aspergillus niger JSC-093350089, isolated from the International Space Station (ISS), is reported, along with a comparison to the experimentally established strain ATCC 1015. The analysis revealed enhanced production levels of naphtho-γ-pyrones and therapeutically relevant SMs, including bicoumanigrin A, aurasperones A and B, and the antioxidant pyranonigrin A. Genetic variants that may be responsible for increased SM production levels in JSC-093350089 were identified. These findings include INDELs within the predicted promoter region of flbA, which encodes a developmental regulator that modulates pyranonigrin A production via regulation of Fum21. The pyranonigrin A biosynthetic gene cluster was confirmed in A. niger, which revealed the involvement of a previously undescribed gene, pyrE, in its biosynthesis. UVC sensitivity assays enabled characterization of pyranonigrin A as a UV resistance agent in the ISS isolate.
Spaceflight Study	BRIC-21 Space Flight	SLID-360	Comparisons of Transcriptome Profiles from Bacillus subtilis Cells Grown in Space versus High Aspect Ratio Vessel (HARV) Clinostats Reveal a Low Degree of Concordance	25 Hours	Bacteria	Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data.
Spaceflight Study	BRIC-23 Space Flight	SLID-360	Comparisons of Transcriptome Profiles from Bacillus subtilis Cells Grown in Space versus High Aspect Ratio Vessel (HARV) Clinostats Reveal a Low Degree of Concordance	36 Hours	Bacteria	Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data.
Ground Study	n.a.	SLID-360	Comparisons of Transcriptome Profiles from Bacillus subtilis Cells Grown in Space versus High Aspect Ratio Vessel (HARV) Clinostats Reveal a Low Degree of Concordance	25 Hours, 36 Hours	Bacteria	Although clinostats have long been used in space microbiology studies as ground-based analogs of spaceflight, few studies to date have systematically compared -omics data from clinostats versus spaceflight. This study compared the transcriptomic response of the Gram-positive bacterium Bacillus subtilis flown in space with corresponding transcriptomes derived from 2-D clinostat (High Aspect Ratio Vessel: HARV) experiments performed under the same conditions of bacterial strain, growth medium, temperature, and incubation time. High-quality total RNA (RNA Integrity Number >9.6) was isolated from multiple biological replicates from each treatment, transcripts were quantified by RNA-seq, and raw data was processed through a previously described standardized bioinformatics pipeline. Transcriptome data sets from spaceflight-grown and corresponding clinostat-grown cells were compared by using three different methods: (i) principal component analysis, (ii) analysis of differentially expressed genes, and (iii) gene set enrichment analysis of KEGG pathways. All three analyses found a low degree of concordance between the spaceflight and corresponding clinostat transcriptome data sets, ranging from 0.9% to 5.3% concordance. These results are in agreement with prior studies that also revealed low concordances between spaceflight and clinostat transcriptomes of the Gram-negative bacteria Rhodospirillum rubrum and Pseudomonas aeruginosa. The results are discussed from the perspective of several potential confounding factors, and suggestions are offered with the aim of achieving increased concordance between clinostat and spaceflight data.
Spaceflight Study	Practice Eight Recoverable Satellite	SLID-361	The postmitotic Saccharomyces cerevisiae after spaceflight showed higher viability	15 Days	Fungi	The budding yeast Saccharomyces cerevisiae has been proposed as an ideal model organism for clarifying the biological effects caused by spaceflight conditions. The postmitotic S. cerevisiae cells onboard Practice eight recoverable satellite were subjected to spaceflight for 15 days. After recovery, the viability, the glycogen content, the activities of carbohydrate metabolism enzymes, the DNA content and the lipid peroxidation level in yeast cells were analyzed. The viability of the postmitotic yeast cells after spaceflight showed a three-fold increase as compared with that of the ground control cells. Compared to the ground control cells, the lipid peroxidation level in the spaceflight yeast cells markedly decreased. The spaceflight yeast cells also showed an increase in G2/M cell population and a decrease in Sub-G1 cell population. The glycogen content and the activities of hexokinase and succinate dehydrogenase significantly decreased in the yeast cells after spaceflight. In contrast, the activity of malate dehydrogenase showed an obvious increase after spaceflight. These results suggested that microgravity or spaceflight could promote the survival of postmitotic S. cerevisiae cells through regulating carbohydrate metabolism, ROS level and cell cycle progression.
Spaceflight Study	Shenzhou 8 Space Flight	SLID-362	Transcriptomic and proteomic responses of Serratia marcescens to spaceflight conditions involve large-scale changes in metabolic pathways	398 Hours	Bacteria	The microgravity environment of spaceflight expeditions has been associated with altered microbial responses. This study explores the characterization of Serratia marcescensis grown in a spaceflight environment at the phenotypic, transcriptomic and proteomic levels. From November 1, 2011 to November 17, 2011, a strain of S. marcescensis was sent into space for 398 h on the Shenzhou VIII spacecraft, and ground simulation was performed as a control (LCT-SM213). After the flight, two mutant strains (LCT-SM166 and LCT-SM262) were selected for further analysis. Although no changes in the morphology, post-culture growth kinetics, hemolysis or antibiotic sensitivity were observed, the two mutant strains exhibited significant changes in their metabolic profiles after exposure to spaceflight. Enrichment analysis of the transcriptome showed that the differentially expressed genes of the two spaceflight strains and the ground control strain mainly included those involved in metabolism and degradation. The proteome revealed that changes at the protein level were also associated with metabolic functions, such as glycolysis/gluconeogenesis, pyruvate metabolism, arginine and proline metabolism and the degradation of valine, leucine and isoleucine. In summary S. marcescens showed alterations primarily in genes and proteins that were associated with metabolism under spaceflight conditions, which gave us valuable clues for future research.
Spaceflight Study	International Space Station (ISS)	SLID-363	The risk of Staphylococcus skin infection during space travel and mitigation strategies	n.a.	Fungi	Among numerous challenges facing space travellers, microbial infection is one of the unknown risks associated with human spaceflight. Prevention and control of microbial infections are of critical concern during space missions. The objective of this research is to develop a quantitative microbial risk assessment (QMRA) to model the risk of Staphylococcus aureus skin infection and mitigation strategies that may effectively reduce skin infection risks. QMRA was carried out by incorporating the level of S. aureus contamination from International Space Station Microbial Observatory Experiment, bacterial transfer rate to skin, growth pattern of S. aureus on skin, space travellers’ daily behaviour and dose-response model. The results demonstrate that a daily skin cleaning regimen has a significant effect on reducing the skin infection risks. Once a day skin cleaning reduces infection risk by 84.2% and twice a day skin cleaning can reduce the risk of skin infection by 96.1% during a seven-day space mission. Frequency of contact with contaminated surfaces and time elapsed between cleaning events are the most important input parameters that contribute to the overall risk outcome. There are degrees of uncertainties associated with the predicted outcomes when interpreted by itself due to the limitation of microbial data and the dose-response model that derived from a short-term clinical study on Earth. The comparative risk analysis as used in this study offers a scientific basis regarding the effectiveness of interventions (skin cleaning regimens) in mitigating skin infection risks during spaceflight.
Spaceflight Study	The Chinese 18th Recoverable Satellite	SLID-364	Space-flight Mutation of Streptomyces gilvosporeus for Enhancing Natamycin Production	18 Days	Bacteria	Mutants of the strain producing natamycin, Streptomyces gilvosporeus, were obtained after space-flight mutation. With respect to the sand spores and slant spores, the mutation ratios were up to 67.6% and 78.3% and the survival ratio was 43.1% and 3.0%, respectively. An improved mutant producing natamycin, S. gilvosporeus LK-45, was screened, which showed natamycin productivity of 1420mg · L−1. A mutant resistant to 2-deoxy glucose, S. gilvosporeus LK-119, was further obtained using a rational screening procedure. The natamycin productivity of 1940mg · L−1 was achieved when glucose was used as the carbon source.
Spaceflight Study	n.a.	SLID-365	Effects of Microgravity on the Virulence of Salmonella Toward Caenorhabditis elegans	15 Days	Bacteria	To evaluate the effects of microgravity on virulence genes in Salmonella, we studied the ability of various Salmonella deletion mutants to kill wild-type Caenorhabditis elegans nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation, and static ground controls. Nematodes, Salmonella, and growth media were separated until exposed to true or simulated microgravity, and then mixed and grown for 48 h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Prior flight in space led to reduced virulence of wild-type Salmonella when subsequently evaluated in a ground-based virulence assay with carefully matched inocula for never-flown Salmonella controls. However, when the virulence assay was conducted in spaceflight, there was only a minimal change in the virulence of wild-type Salmonella toward C. elegans. Deletion of pipA, a gene in Salmonella pathogenicity island-5, reduced Salmonella virulence toward wild-type and Tol1-deletion L2 larvae in spaceflight but had no effect on virulence for Tol1-deletion adult worms in spaceflight. PipA-deletion Salmonella were also less virulent toward wild-type L2 larvae in clinorotation, but showed a paradoxical increased virulence toward Tol1-deletion L2 larvae in clinorotation.
Ground Study	n.a.	SLID-365	Effects of Microgravity on the Virulence of Salmonella Toward Caenorhabditis elegans	15 Days	Bacteria	To evaluate the effects of microgravity on virulence genes in Salmonella, we studied the ability of various Salmonella deletion mutants to kill wild-type Caenorhabditis elegans nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, clinorotation, and static ground controls. Nematodes, Salmonella, and growth media were separated until exposed to true or simulated microgravity, and then mixed and grown for 48 h. Experiments were terminated by paraformaldehyde fixation, and optical density measurements were used to assay residual microorganisms. Prior flight in space led to reduced virulence of wild-type Salmonella when subsequently evaluated in a ground-based virulence assay with carefully matched inocula for never-flown Salmonella controls. However, when the virulence assay was conducted in spaceflight, there was only a minimal change in the virulence of wild-type Salmonella toward C. elegans. Deletion of pipA, a gene in Salmonella pathogenicity island-5, reduced Salmonella virulence toward wild-type and Tol1-deletion L2 larvae in spaceflight but had no effect on virulence for Tol1-deletion adult worms in spaceflight. PipA-deletion Salmonella were also less virulent toward wild-type L2 larvae in clinorotation, but showed a paradoxical increased virulence toward Tol1-deletion L2 larvae in clinorotation.
Spaceflight Study	Shenzhou 1 Space Flight	SLID-366	Analysis of genetic variation in Ganoderma Lucidum after space flight	21 Hours	Fungi	A modified CTAB method was used in the extraction of total cellular DNA of Ganoderma lucidum. Four strains Cx, Ch, C3 and C4, and their counterparts, four space flown strains Sx, Xh, S3 and S4, were analysed by amplified fragment length polymorphism (AFLP) with several primer combinations. Polymorphic bands were detected between Sx and Cx, S3 and C3, respectively. Somatic incompatibility tests further confirmed their heterogeneity. However, no disparity between Sh and Ch, S4 and C4 was detectable. The results suggest that spaceflight may be used to accelerate breeding of Ganoderma lucidum strains for commercial cultivation.
Spaceflight Study	Shuttle Mission STS-51	SLID-367	Modification of reproductive development in Arabidopsis thaliana under spaceflight conditions	10 Days	Seedling	Reproductive development in Arabidopsis thaliana (L.) Heynh. cv. Columbia plants was investigated under spaceflight conditions on shuttle mission STS-51. Plants launched just prior to initiation of the reproductive phase developed flowers and siliques during the 10-d flight. Approximately 500 flowers were produced in total by the 12 plants in both the ground control and spaceflight material, and there was no significant difference in the number of flowers in each size class. The flower buds and siliques of the spaceflight plants were not morphologically different from the ground controls. Pollen viability tests immediately post-flight using fluorescein diacetate indicated that about 35% of the pollen was viable in the spaceflight material. Light-microscopy observations on this material showed that the female gametophytes also had developed normally to maturity. However, siliques from the spaceflight plants contained empty, shrunken ovules, and no evidence of pollen transfer to stigmatic papillae was found by light microscopy immediately post-flight or by scanning electron microscopy on fixed material. Short stamen length and indehiscent anthers were observed in the spaceflight material, and a film-like substance inside the anther that connected to the tapetum appeared to restrict the release of pollen from the anthers. These observations indicate that given appropriate growing conditions, early reproductive development in A. thaliana can occur normally under spaceflight conditions. On STS-51, reproductive development aborted due to obstacles in pollination or fertilization.
Spaceflight Study	n.a.	SLID-368	Changes in plant medium composition after a spaceflight experiment: Potassium levels are of special interest	5 Days	Plant	We present results on the analysis of 100 mL medium samples extracted from sterilized foam (Smithers-Oasis, Kent OH) used to support the growth of a representative dicotyledon (Haplopappus gracilis) and a representative monocotyledon (Hemerocallis cv Autumn Blaze) in NASA’s Plant Growth Unit (PGU) during a 5-day Space Shuttle flight and ground experiments. At recovery, the media remaining within replicate (n = 5) foam blocks (for both the spaceflight and ground experiments) were extracted under vacuum, filtered and subjected to elemental analyses. A unique aspect of this experiment was that all plants were either aseptically-generated tissue culture propagated plantlets or aseptic seedling clones. The design of the PGU facilitated the maintenance of asepsis throughout the mission (confirmed by post-flight microbial sampling) and thus any possible impact of microorganisms on medium composition was eliminated. Concentration levels of some elements remained the same, while some decreased and others increased. There was a significant two-fold difference between the final concentrations of potassium when the Earth-based and microgravity experiments were contrasted.
Spaceflight Study	n.a.	SLID-368	Changes in plant medium composition after a spaceflight experiment: Potassium levels are of special interest	5 Days	Plant	We present results on the analysis of 100 mL medium samples extracted from sterilized foam (Smithers-Oasis, Kent OH) used to support the growth of a representative dicotyledon (Haplopappus gracilis) and a representative monocotyledon (Hemerocallis cv Autumn Blaze) in NASA’s Plant Growth Unit (PGU) during a 5-day Space Shuttle flight and ground experiments. At recovery, the media remaining within replicate (n = 5) foam blocks (for both the spaceflight and ground experiments) were extracted under vacuum, filtered and subjected to elemental analyses. A unique aspect of this experiment was that all plants were either aseptically-generated tissue culture propagated plantlets or aseptic seedling clones. The design of the PGU facilitated the maintenance of asepsis throughout the mission (confirmed by post-flight microbial sampling) and thus any possible impact of microorganisms on medium composition was eliminated. Concentration levels of some elements remained the same, while some decreased and others increased. There was a significant two-fold difference between the final concentrations of potassium when the Earth-based and microgravity experiments were contrasted.
Spaceflight Study	A Biocosmos Satellite	SLID-369	Effect on a Flour Beetle of Irradiation during Space Flight	n.a.	Animals	n.a.
Spaceflight Study	International Space Station (ISS)	SLID-370	Pharmacokinetics of acetaminophen administered in tablets and capsules under long-term space flight conditions	n.a.	Therapeutic drug	The pharmacokinetics of acetaminophen in two medicinal forms, tablets and capsules, have been studied in healthy volunteers after single peroral administration at a dose of 499 mg under usual living conditions and during long-term space flight. The rate of drug absorption from tablets decreases significantly whereas the relative bioavailability increases substantially under microgravity conditions (compared with usual conditions). For the encapsulated medicinal form, the time of absorption decreases statistically reliably and the half-elimination time, the average retention time, and the distribution volume increase considerably whereas the bioavailability changes insignificantly.
Spaceflight Study	International Space Station (ISS)/STS	SLID-371	Evaluation of physical and chemical changes in pharmaceuticals flown on space missions	n.a.	Therapeutic drug	Efficacy and safety of medications used for the treatment of astronauts in space may be compromised by altered stability in space. We compared physical and chemical changes with time in 35 formulations contained in identical pharmaceutical kits stowed on the International Space Station (ISS) and on Earth. Active pharmaceutical content (API) was determined by ultra- and high-performance liquid chromatography after returning to Earth. After stowage for 28 months in space, six medications aboard the ISS and two of matching ground controls exhibited changes in physical variables; nine medications from the ISS and 17 from the ground met the United States Pharmacopeia (USP) acceptance criteria for API content after 28 months of storage. A higher percentage of medications from each flight kit had lower API content than the respective ground controls. The number of medications failing API requirement increased as a function of time in space, independent of expiration date. The rate of degradation was faster in space than on the ground for many of the medications, and most solid dosage forms met USP standard for dissolution after storage in space. Cumulative radiation dose was higher and increased with time in space, whereas temperature and humidity remained similar to those on the ground. Exposure to the chronic low dose of ionizing radiation aboard the spacecraft as well as repackaging of solid dosage forms in flight-specific dispensers may adversely affect stability of pharmaceuticals. Characterization of degradation profiles of unstable formulations and identification of chemical attributes of stability in space analog environments on Earth will facilitate development of space-hardy medications.
Spaceflight Study	International Space Station (ISS)	SLID-372	Stability of vitamin B complex in multivitamin and multimineral supplement tablets after space flight	4 months	Health Supplement	The effect of storage in space on the stability of vitamin B complex in two commercial vitamin tablets was examined. Multiple vitamin samples returned after storage on the space shuttle and International Space Station (ISS) along with two ground control and three positive control groups were included in the study. Content of vitamin B(3) in the tablets and in vitro dissolution rate were determined using a modified high performance liquid chromatographic assay from USP/NF 2010. Results indicate that vitamin B(3) in one of the brands tested (#2) may be subject to marginal degradation after storage on ISS for 4 months as indicated by the chromatograms for all six tablets showing a split peak appearing as a notch at the peak tip. Chromatograms were not different for ground and flight samples for Brand #1 suggesting that this may be more suitable for use in space.
Spaceflight Study	n.a.	SLID-372	Stability of vitamin B complex in multivitamin and multimineral supplement tablets after space flight	n.a.	Health Supplement	The effect of storage in space on the stability of vitamin B complex in two commercial vitamin tablets was examined. Multiple vitamin samples returned after storage on the space shuttle and International Space Station (ISS) along with two ground control and three positive control groups were included in the study. Content of vitamin B(3) in the tablets and in vitro dissolution rate were determined using a modified high performance liquid chromatographic assay from USP/NF 2010. Results indicate that vitamin B(3) in one of the brands tested (#2) may be subject to marginal degradation after storage on ISS for 4 months as indicated by the chromatograms for all six tablets showing a split peak appearing as a notch at the peak tip. Chromatograms were not different for ground and flight samples for Brand #1 suggesting that this may be more suitable for use in space.
Spaceflight Study	International Space Station (ISS)	SLID-373	Medication use by U.S. crewmembers on the International Space Station	average, 159 ± 36 days	Therapeutic drug	The environment on the International Space Station (ISS) includes a variety of potential physiologic stressors, including low gravity, elevated exposure to radiation, confined living and working quarters, a heavy workload, and high public visibility. This retrospective study examined medication use during long-duration spaceflights (>30 d). Medication records from 24 crewmembers on 20 missions longer than 30 d over a 10 yr period were examined for trends in usage rates, efficacy, and indication, as well as adverse event quality, frequency, and severity. Results were compared with those from crewmembers on shorter space shuttle missions (>16 d) and other reports of medication use by healthy adults. The most frequently used medications on the ISS were for sleep problems, pain, congestion, or allergy. Medication use during spaceflight missions was similar to that noted on the Space Shuttle and in adult ambulatory medicine, except that usage of sleep aids was about 10 times higher during spaceflight missions. There were also 2 apparent treatment failures in cases of skin rash, raising questions about the efficacy or suitability of the treatments used. Many spaceflight-related medication uses (at least 10%) were linked to extravehicular activities, exercise protocols, or equipment and operationally driven schedule changes. It seems likely that alterations in spaceflight mission operations (schedule-shifting and lighting) or hardware (extravehicular activity suits and exercise equipment) could reduce the need for a sizable fraction of medication uses.
Spaceflight Study	International Space Station (ISS)	SLID-374	Chemical Potency and Degradation Products of Medications Stored Over 550 Earth Days at the International Space Station	550 days	Therapeutic drug	Medications degrade over time, and degradation is hastened by extreme storage conditions. Current procedures ensure that medications aboard the International Space Station (ISS) are restocked before their expiration dates, but resupply may not be possible on future long-duration exploration missions. For this reason, medications stored on the ISS were returned to Earth for analysis. This was an opportunistic, observational pilot-scale investigation to test the hypothesis that ISS-aging does not cause unusual degradation. Nine medications were analyzed for active pharmaceutical ingredient (API) content and degradant amounts; results were compared to 2012 United States Pharmacopeia (USP) requirements. The medications were two sleep aids, two antihistamines/decongestants, three pain relievers, an antidiarrheal, and an alertness medication. Because the samples were obtained opportunistically from unused medical supplies, each medication was available at only 1 time point and no control samples (samples aged for a similar period on Earth) were available. One medication met USP requirements 5 months after its expiration date. Four of the nine (44% of those tested) medications tested met USP requirements 8 months post expiration. Another three medications (33%) met USP guidelines 2–3 months before expiration. One compound, a dietary supplement used as a sleep aid, failed to meet USP requirements at 11 months post expiration. No unusual degradation products were identified. Limited, evidence-based extension of medication shelf-lives may be possible and would be useful in preparation for lengthy exploration missions. Only analysis of flight-aged samples compared to appropriately matched ground controls will permit determination of the spaceflight environment on medication stability.
Spaceflight Study	n.a.	SLID-375	Principles of Clinical Medicine for Space Flight	n.a.	Therapeutic drug, Medical Supply, Health Supplement	Humans who have travelled in space have used medications to ease adaptation to their new environment (like anti-nausea medications) and to prevent adaptations that could prove deleterious to their long-term well-being (e.g., anti-resorptives to maintain bone mineral density). They have also treated the ordinary illnesses that humans experience and made certain that they have medication stocks available for the treatment of medical emergencies. A medical system for any space flight will be heavily reliant on medications, since surgical treatment options may not be feasible during a mission. For exploration missions, duration is a critical consideration. Longer journey length means increased likelihood of medical events occurring, which increase the supplies required; this must be balanced against the mass and volume limits inherent in a vehicle of limited size. Stability during storage is a crucial consideration for missions longer than 1 year. More research is required to understand the degradation of pharmaceutical products over time, with special attention to minimizing harmful degradation and determining how older products might be used safely. New manufacturing methods like 3D printing or expression by bioengineered microorganisms might 1 day enable crewmembers to produce fresh new supplies during the course of their mission, but there is much research and testing required to ensure safety and efficacy of the finished products.
Spaceflight Study	International Space Station (ISS)/STS	SLID-376	Prevalence of sleep deficiency and use of hypnotic drugs in astronauts before, during, and after spaceflight: an observational study	n.a.	Therapeutic drug	[Background]: Sleep deprivation and fatigue are common subjective complaints among astronauts. Previous studies of sleep and hypnotic drug use in space have been limited to post-flight subjective survey data or in-flight objective data collection from a small number of crew members. We aimed to characterise representative sleep patterns of astronauts on both short-duration and long-duration spaceflight missions. [Methods]: For this observational study, we recruited crew members assigned to Space Transportation System shuttle flights with in-flight experiments between July 12, 2001, and July 21, 2011, or assigned to International Space Station (ISS) expeditions between Sept 18, 2006, and March 16, 2011. We assessed sleep-wake timing objectively via wrist actigraphy, and subjective sleep characteristics and hypnotic drug use via daily logs, in-flight and during Earth-based data-collection intervals: for 2 weeks scheduled about 3 months before launch, 11 days before launch until launch day, and for 7 days upon return to Earth. [Findings]: We collected data from 64 astronauts on 80 space shuttle missions (26 flights, 1063 in-flight days) and 21 astronauts on 13 ISS missions (3248 in-flight days), with ground-based data from all astronauts (4014 days). Crew members attempted and obtained significantly less sleep per night as estimated by actigraphy during space shuttle missions (7·35 h [SD 0·47] attempted, 5·96 h [0·56] obtained), in the 11 days before spaceflight (7·35 h [0·51], 6·04 h [0·72]), and about 3 months before spaceflight (7·40 h [0·59], 6·29 h [0·67]) compared with the first week post-mission (8·01 h [0·78], 6·74 h [0·91]; p<0·0001 for both measures). Crew members on ISS missions obtained significantly less sleep during spaceflight (6·09 h [0·67]), in the 11 days before spaceflight (5·86 h [0·94]), and during the 2-week interval scheduled about 3 months before spaceflight (6·41 h [SD 0·65]) compared with in the first week post-mission (6·95 h [1·04]; p<0·0001). 61 (78%) of 78 shuttle-mission crew members reported taking a dose of sleep-promoting drug on 500 (52%) of 963 nights; 12 (75%) of 16 ISS crew members reported using sleep-promoting drugs. [Interpretation]: Sleep deficiency in astronauts was prevalent not only during space shuttle and ISS missions, but also throughout a 3 month preflight training interval. Despite chronic sleep curtailment, use of sleep-promoting drugs was pervasive during spaceflight. Because chronic sleep loss leads to performance decrements, our findings emphasise the need for development of effective countermeasures to promote sleep.
Spaceflight Study	n.a.	SLID-377	Spaceflight medical countermeasures: a strategic approach for mitigating effects from solar particle events	n.a.	Therapeutic drug, Medical Supply	NASA was recently charged with returning humans to the lunar surface within the next five years. This will require preparation for spaceflight missions of longer distance and duration than ever performed in the past. Protecting the crew and mission from the hazards associated with spaceflight will be a priority. One of the primary hazards to address is the challenging radiation environment. Space is unforgiving when it comes to radiation. There is galactic cosmic radiation (GCR) that is pervasive in space and the possibility of solar particle events (SPE) that release high energy particles from the sun that can result in high doses of radiation to the crew if unprotected. NASA has been preparing and evaluating several means of ensuring that crew health is not compromised during these missions. Physical shielding, space weather monitoring, and more recently storm shelters are all possible means of protecting crew during a SPE. Medical countermeasures have not been necessary for operations in low Earth orbit; however, future human exploration missions should consider including therapies onboard to address radiation-induced health effects. While the likelihood of experiencing a significant SPE is very low, serious adverse health effects or even death could occur if no medical countermeasures were available. Having a Food and Drug Administration (FDA) approved medical countermeasure on board that could mitigate acute radiation-induced hematopoietic syndrome due to a SPE could provide life saving measures for the crew. This paper discusses the mitigation strategies that can be implemented for Artemis missions and identifies numerous areas of research for future improvements.
Spaceflight Study	n.a.	SLID-378	Physiological, pharmacokinetic, and pharmacodynamic changes in space	116 days	Therapeutic drug	The objectives of this investigation were to 1) determine changes in GI motility during space flight using a noninvasive lactulose breath-hydrogen test, 2) determine absorption, bioavailability, and elimination of acetaminophen during space flight, 3) determine hepatic metabolic activity during space flight by measuring the clearance of antipyrine after an oral dose, and 4) correlate functional changes in the GI tract and liver with the absorption and metabolism of acetaminophen. (Both acetaminophen and antipyrine are substances used to reduce fever and relieve pain.). [Conclusion]: The limited inflight data collected to date, although indicating trends of GI and hepatic function changes during flight, are inadequate to characterize the degree and magnitude of such changes. The mechanisms underlying these changes are difficult to identify because of the complexity of these changes as well as the large number of variables that may influence disposition profiles and kinetic parameters during flight. Further investigation is required to generate information that will be useful for the development of pharmaceutical and nutritional countermeasures for microgravity-induced deconditioning.
Spaceflight Study	n.a.	SLID-379	Bioavailability of Promethazine during Spaceflight	n.a.	Therapeutic drug	Promethazine (PMZ) is the choice anti-motion sickness medication for treating space motion sickness (SMS) during flight. The side effects associated with PMZ include dizziness, drowsiness, sedation, and impaired psychomotor performance which could impact crew performance and mission operations. Early anecdotal reports from crewmembers indicate that these central nervous system side effects of PMZ are absent or greatly attenuated in microgravity, potentially due to changes in pharmacokinetics (PK) and pharmacodynamics in microgravity. These changes could also affect the therapeutic effectiveness of drugs in general and PMZ, in particular. In this investigation, we examined bioavailability and associated pharmacokinetics of PMZ in astronauts during and after space flight. Methods. Nine astronauts received, per their preference, PMZ (25 or 50 mg as intramuscular injection, oral tablet, or rectal suppository) on flight day one for the treatment of SMS and subsequently collected saliva samples and completed sleepiness scores for 72 h post dose. Thirty days after the astronauts returned to Earth, they repeated the protocol. Bioavailability and PK parameters were calculated and compared between flight and ground. Results. Maximum concentration (Cmax) was lower and time to reach Cmax (tmax) was longer in flight than on the ground. Area under the curve (AUC), a measure of bioavailability, was lower and biological half-life (t1/2) was longer in flight than on the ground. Conclusion. Results indicate that bioavailability of PMZ is reduced during spaceflight. Number of samples, sampling method, and sampling schedule significantly affected PK parameter estimates.
Spaceflight Study	Apollo Missions	SLID-380	Spaceflight Medical Systems	n.a.	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Spaceflight Study	Gemini spacecraft	SLID-380	Spaceflight Medical Systems	n.a.	Therapeutic drug, Medical Supply, Health Supplement	Providing adequate medical care for spaceflight crews requires that appropriate diagnostic tools and treatment modalities be available to them throughout their mission. The challenge for mission planners is deciding what medical capability to provide and then packaging it in a way that meets the many unique constraints of space flight. Crews also must receive adequate training that will help them to make correct diagnoses and administer the appropriate level of care to an ill or injured crewmember. Identification of appropriate levels of medical care is driven by the risks that have been identified in space flight. One practical way of identifying such risks is by studying risks among analogous populations, such as military pilots, submarine crews, and Antarctic winter-over research teams. From these groups, which undergo medical screening processes similar to those of spaceflight crews, the probabilities and risks of illness occurring during a mission can be estimated. Review of reported illnesses in U.S. and Russian spaceflight crews also can be useful, although such data were not available to medical mission planners in the earliest days of space flight. The duration of a space mission and the number of high-risk activities associated with it (e.g., extravehicular activities) will also influence decisions concerning the content of onboard medical systems. Mission planners must also consider environmental factors that are unique to the space environment—factors that include microgravity, radiation, toxicology, microbiology, and purity of reclaimed water. Finally, the unique physiological responses to space flight must also be examined—space adaptation syndrome, cardiovascular deconditioning, and bone demineralization, among others. Only by accounting for all of these factors can the best possible care and facilities be provided to spaceflight crews.
Spaceflight Study	International Space Station (ISS)	SLID-381	Aquatic invertebrate protein sources for long-duration space travel	n.a.	Food Attribute	During the summer of 2020, NASA returned to launching astronauts to the International Space Station (ISS) from American soil. By 2024, NASA's mission is to return to the Moon, and by 2028 create a sustainable presence. Long duration missions come with obstacles, especially when trying to create a sustainable environment in a location where "living off the land" is impossible. Some resources on the Moon can be recovered or resupplied; however, many resources such as those needed for sustaining life must be recycled or grown to support humans. To achieve sustainability, food and water must be grown and recycled using elements found within the habitat. NASA's current work focuses on food resupply and growing plants as supplemental nutrient content. This paper examines the possibility for using aquaculture systems to purify water while growing nutrient-rich species as food sources, which aquatic food sources would be ideal for a habitat environment, and which species might provide an ideal test case for future studies aboard ISS. The aquatic species should be rapidly grown with high protein content and low launch mass requirements. Although there are numerous challenges and unknown technology gaps for maintaining aquaculture systems in reduced gravity environments, the benefit of employing such systems would be of great advantage towards creating a sustainable presence beyond Earth's orbit for sustainable aquaculture.
Spaceflight Study	International Space Station (ISS)	SLID-381	Aquatic invertebrate protein sources for long-duration space travel	n.a.	Food Usage	During the summer of 2020, NASA returned to launching astronauts to the International Space Station (ISS) from American soil. By 2024, NASA's mission is to return to the Moon, and by 2028 create a sustainable presence. Long duration missions come with obstacles, especially when trying to create a sustainable environment in a location where "living off the land" is impossible. Some resources on the Moon can be recovered or resupplied; however, many resources such as those needed for sustaining life must be recycled or grown to support humans. To achieve sustainability, food and water must be grown and recycled using elements found within the habitat. NASA's current work focuses on food resupply and growing plants as supplemental nutrient content. This paper examines the possibility for using aquaculture systems to purify water while growing nutrient-rich species as food sources, which aquatic food sources would be ideal for a habitat environment, and which species might provide an ideal test case for future studies aboard ISS. The aquatic species should be rapidly grown with high protein content and low launch mass requirements. Although there are numerous challenges and unknown technology gaps for maintaining aquaculture systems in reduced gravity environments, the benefit of employing such systems would be of great advantage towards creating a sustainable presence beyond Earth's orbit for sustainable aquaculture.
Spaceflight Study	International Space Station (ISS)	SLID-381	Aquatic invertebrate protein sources for long-duration space travel	n.a.	Food Storage	During the summer of 2020, NASA returned to launching astronauts to the International Space Station (ISS) from American soil. By 2024, NASA's mission is to return to the Moon, and by 2028 create a sustainable presence. Long duration missions come with obstacles, especially when trying to create a sustainable environment in a location where "living off the land" is impossible. Some resources on the Moon can be recovered or resupplied; however, many resources such as those needed for sustaining life must be recycled or grown to support humans. To achieve sustainability, food and water must be grown and recycled using elements found within the habitat. NASA's current work focuses on food resupply and growing plants as supplemental nutrient content. This paper examines the possibility for using aquaculture systems to purify water while growing nutrient-rich species as food sources, which aquatic food sources would be ideal for a habitat environment, and which species might provide an ideal test case for future studies aboard ISS. The aquatic species should be rapidly grown with high protein content and low launch mass requirements. Although there are numerous challenges and unknown technology gaps for maintaining aquaculture systems in reduced gravity environments, the benefit of employing such systems would be of great advantage towards creating a sustainable presence beyond Earth's orbit for sustainable aquaculture.
Spaceflight Study	International Space Station (ISS)	SLID-382	Assessment of nutrient stability in foods from the space food system after long-duration spaceflight on the ISS	n.a.	Food Attribute	Maintaining an intact nutrient supply in the food system flown on spacecraft is a critical issue for mission success and crew health. Ground-based evidence indicates that some vitamins may be altered and fatty acids oxidized (and therefore rendered useless, or even dangerous) by long-term storage and by exposure to radiation, both of which will be issues for long-duration exploration missions in space. In this study, the stability of nutrients was investigated in food samples exposed to spaceflight on the Intl. Space Station (ISS). A total of 6 replicates of 5 different space food items, a multivitamin, and a vitamin D supplement were packaged into 4 identical kits and were launched in 2006 on the space shuttle. After 13, 353, 596, and 880 d of spaceflight aboard the ISS, the kits were returned to Earth. Nine replicates of each food item and vitamin, from the same lots as those sent into space, remained in an environmental chamber on Earth to serve as controls at each time point. Vitamins, hexanal, oxygen radical absorbance capacity, and amino acids were measured in identical-lot food samples at each time point. After 596 d of spaceflight, differences in intact vitamin concentrations due to duration of storage were observed for most foodstuffs, but generally, nutrients from flight samples did not degrade any faster than ground controls. This study provided the 1st set of spaceflight data for investigation of nutrient stability in the food system, and the results will help NASA design food systems for both ISS and space exploration missions.
Spaceflight Study	n.a.	SLID-383	Development of Spaceflight Foods with High Microbial Concentrations	n.a.	Food Storage	The use of bioregenerative food systems has been proposed for long duration spaceflight missions, though the impact of this type of system on microbial contamination and crew health is greatly understudied. Current spaceflight food requirements are inadequate for microbial monitoring associated with crop production, the use of bulk foods, and subsequent processing and handling, as well as foods with inherently high microbial concentrations, such as probiotics, which may be beneficial for crew health. This review provides an initial investigation into (a) the potential of microbial load from bioregenerative produce, (b) the current microbial strains and uses of live cultures in shelf stable foods, (c) a comparison of levels of microbial load in these foods/supplements to current requirements and an assessment of additional risk, and (d) an investigation of current literature and commercial operations to provide guidance in establishing microbiological requirements and testing methods for foods with high and diversified microbial concentrations. The evidence from this review indicates a need for further research to determine the procedures necessary for growing and processing regenerative produce and development of specific cleaning methods to mitigate microbiological risks. In addition, data mining performed during this review identified potential benefits for the use of food products with high microbial concentrations. Specifically, this review has performed an extensive market survey to generate a comprehensive list of potential probiotic products, which should be evaluated in future research to determine preferred probiotic formulations for spaceflight. Future research would also determine the specific microbial testing procedures for those formulations and assess the risk of adding the formulation to the food system.
Spaceflight Study	n.a.	SLID-383	Development of Spaceflight Foods with High Microbial Concentrations	n.a.	Food Storage	The use of bioregenerative food systems has been proposed for long duration spaceflight missions, though the impact of this type of system on microbial contamination and crew health is greatly understudied. Current spaceflight food requirements are inadequate for microbial monitoring associated with crop production, the use of bulk foods, and subsequent processing and handling, as well as foods with inherently high microbial concentrations, such as probiotics, which may be beneficial for crew health. This review provides an initial investigation into (a) the potential of microbial load from bioregenerative produce, (b) the current microbial strains and uses of live cultures in shelf stable foods, (c) a comparison of levels of microbial load in these foods/supplements to current requirements and an assessment of additional risk, and (d) an investigation of current literature and commercial operations to provide guidance in establishing microbiological requirements and testing methods for foods with high and diversified microbial concentrations. The evidence from this review indicates a need for further research to determine the procedures necessary for growing and processing regenerative produce and development of specific cleaning methods to mitigate microbiological risks. In addition, data mining performed during this review identified potential benefits for the use of food products with high microbial concentrations. Specifically, this review has performed an extensive market survey to generate a comprehensive list of potential probiotic products, which should be evaluated in future research to determine preferred probiotic formulations for spaceflight. Future research would also determine the specific microbial testing procedures for those formulations and assess the risk of adding the formulation to the food system.
Spaceflight Study	Different missions	SLID-384	Factors affecting flavor perception in space: Does the spacecraft environment influence food intake by astronauts?	n.a.	Food Intake	The intention to send a crewed mission to Mars involves a huge amount of planning to ensure a safe and successful mission. Providing adequate amounts of food for the crew is a major task, but 20 years of feeding astronauts on the International Space Station (ISS) have resulted in a good knowledge base. A crucial observation from the ISS is that astronauts typically consume only 80% of their daily calorie requirements when in space. This is despite daily exercise regimes that keep energy usage at very similar levels to those found on Earth. This calorie deficit seems to have little effect on astronauts who spend up to 12 months on the ISS, but given that a mission to Mars would take 30 to 36 months to complete, there is concern that a calorie deficit over this period may lead to adverse effects in crew members. The key question is why astronauts undereat when they have a supply of food designed to fully deliver their nutritional needs. This review focuses on evidence from astronauts that foods taste different in space, compared to on Earth. The underlying hypothesis is that conditions in space may change the perceived flavor of the food, and this flavor change may, in turn, lead to underconsumption by astronauts. The key areas investigated in this review for their potential impact on food intake are the effects of food shelf life, physiological changes, noise, air and water quality on the perception of food flavor, as well as the link between food flavor and food intake.
Ground Study	n.a.	SLID-385	Initial assessment of the nutritional quality of the space food system over three years of ambient storage	n.a.	Food Attribute	Processed and prepackaged space food is the main source of nutrition for crew aboard the International Space Station, and likely will continue to be the main source of nutrition for future exploration missions. However, very little information is available on the nutritional stability of space foods. To better understand their nutritional stability, 24 micronutrients were measured in 109 space foods stored over 3 years at room temperature. Our analysis indicated that potassium, calcium, vitamin D, and vitamin K concentrations in the food may not be adequate to meet the recommended daily intake requirements even before storage. Decreases in vitamins A, C, B1, and B6 were observed during storage. Notably, vitamins B1 and C may degrade to inadequate levels after 1 year and 3 years, respectively. This assessment suggests that different technological approaches will be required to stabilize processed foods to enable spaceflight missions over 1 year.
Ground Study	n.a.	SLID-386	Kinetic parameters of thiamine degradation in NASA spaceflight foods determined by the endpoints method for long-term storage	n.a.	Food Storage	Retention of labile vitamins such as thiamine (vitamin B1) in NASA spaceflight foods intended for extended-duration missions is critical for the health of the crew. In this study, the degradation kinetics of thiamine in three NASA spaceflight foods (brown rice, split pea soup, BBQ beef brisket) during storage was determined for the first time, using an interactive isothermal model developed by our group. Results showed that brown rice and split pea soup demonstrated resistance to thiamine degradation, while thiamine in beef brisket was less stable. Model-predicted thiamine retention in brown rice stored at 20 °C for 720 days was 55% of the original thiamine content after thermal processing, 42% for split pea soup, and 3% for beef brisket. Water activity, moisture content, and pH differences did not sufficiently explain the variation in the degradation kinetics of thiamine among these foods.
Spaceflight Study	n.a.	SLID-386	Kinetic parameters of thiamine degradation in NASA spaceflight foods determined by the endpoints method for long-term storage	n.a.	Food Storage	Retention of labile vitamins such as thiamine (vitamin B1) in NASA spaceflight foods intended for extended-duration missions is critical for the health of the crew. In this study, the degradation kinetics of thiamine in three NASA spaceflight foods (brown rice, split pea soup, BBQ beef brisket) during storage was determined for the first time, using an interactive isothermal model developed by our group. Results showed that brown rice and split pea soup demonstrated resistance to thiamine degradation, while thiamine in beef brisket was less stable. Model-predicted thiamine retention in brown rice stored at 20 °C for 720 days was 55% of the original thiamine content after thermal processing, 42% for split pea soup, and 3% for beef brisket. Water activity, moisture content, and pH differences did not sufficiently explain the variation in the degradation kinetics of thiamine among these foods.
Spaceflight Study	International Space Station (ISS)	SLID-387	Mission to Mars: food production and processing for the final frontier	n.a.	Food Storage	The food systems of the National Aeronautics and Space Administration (NASA) have evolved tremendously since the early manned spaceflights of the 1960s. To date, NASA's mission focus has been limited to exploration of low Earth orbit (LEO), and the agency's prepackaged food systems have been adequate to enable success of their parent programs. With NASA's mission focus increasing to achieve manned space exploration of the Martian surface, the agency is considering a significant departure from the prepackaged food systems of current and past space programs. NASA's Advanced Food Technology (AFT) project is presently investigating the introduction of a bioregenerative food system to support long duration habitat missions to the Martian surface. A bioregenerative food system is expected to impart less of a burden on critical mission resources, such as mass and volume, than a prepackaged, shelf-stable system. This review provides an introduction to past and present spaceflight food systems, and provides a broad examination of the research conducted to date to enable crop production and food processing on the Martian surface.
Spaceflight Study	International Space Station (ISS)	SLID-387	Mission to Mars: food production and processing for the final frontier	n.a.	Food Storage	The food systems of the National Aeronautics and Space Administration (NASA) have evolved tremendously since the early manned spaceflights of the 1960s. To date, NASA's mission focus has been limited to exploration of low Earth orbit (LEO), and the agency's prepackaged food systems have been adequate to enable success of their parent programs. With NASA's mission focus increasing to achieve manned space exploration of the Martian surface, the agency is considering a significant departure from the prepackaged food systems of current and past space programs. NASA's Advanced Food Technology (AFT) project is presently investigating the introduction of a bioregenerative food system to support long duration habitat missions to the Martian surface. A bioregenerative food system is expected to impart less of a burden on critical mission resources, such as mass and volume, than a prepackaged, shelf-stable system. This review provides an introduction to past and present spaceflight food systems, and provides a broad examination of the research conducted to date to enable crop production and food processing on the Martian surface.
Spaceflight Study	International Space Station (ISS)	SLID-388	Space food and bacterial infections: Realities of the risk and role of science	n.a.	Food Storage	Background:Space food has evolved remarkably from a simple toothpaste-like tube to ready-to-eat Earth-like cuisine. Currently, the major mission of space food development is to provide a safe, nutritious, and acceptable food system that can function for a long-duration spaceflight, such as a human Mars mission in the 2030s.Scope and approach:Ensuring food safety during the spaceflight is considered a large health challenge for crews because there are potential hazards of bacterial contamination and infection through food, which have consequences in the confined system of a spacecraft or space station. Key findings and conclusions: Despite the efforts invested in the microbial quality control of the environment and recycling systems during spaceflight, microorganisms inevitably accompany all space habitats occupied by crew members and can be transmitted everywhere, including food, other locations, and even humans. Opportunistic pathogens have been isolated from air, surfaces, water systems, and crew members; moreover, various studies have documented the stronger stress resistance or virulence of pathogenic bacteria in response to the space environment. The current study is therefore intended to provide a comprehensive review of the current status of space food and the potential hazard of bacterial infections during a manned space mission, which could be used for future research on space food.
Spaceflight Study	International Space Station (ISS)	SLID-389	Spaceflight Metabolism and Nutritional Support	n.a.	Food Intake	Adequate nutritional status is critical to maintaining crew health during extended-duration space flight and postflight rehabilitation. Nutrition issues relate to intake of required nutrients, physiological adaptation to microgravity, psychological adaptation to extreme environments, and countermeasures to ameliorate the negative effects of space flight. Our ability to define the nutrient requirements for space flight and to ensure the provision and intake of those nutrients by spaceflight crews is thus critical for crew health and mission success.Specialized nutritional requirements have only been considered for extended-duration flights—those lasting longer than 30 days. Although adequate nutrition is important on the 1- to 3-week Space Shuttle flights, intake of specific nutrients above or below space-specific requirements for such periods is not thought to be cause for concern. Thus, planning menus for Space Shuttle flights has always used recognized nutritional requirements for adult males and females [1,2]. In this chapter, we will further classify nutritional requirements for long-duration space flight into those for orbital missions, such as on the International Space Station, and those for exploration- class missions.
Spaceflight Study	International Space Station (ISS)	SLID-390	The Spaceflight Food System: A Case Study in Long Duration Preservation	n.a.	Food Storage	NASA's food system is a unique case study for many of the preservation methods and chemistry principles that extend the shelf life of foods. The NASA food system must be safe, nutritious, and acceptable in room temperature storage for two years for missions to the International Space Station and five years for the Mars missions. The food system is constrained by the limitations of microgravity (no cooking, limited food transfer, no high-crumb producing foods), and vehicle resources (mass, power, crew time, water, waste disposal). Although preservation strategies such as retort thermostabilization, freeze-drying, and irradiation have been adequate for all NASA missions to date, they will not meet the shelf life requirements of a Mars mission. This article will focus on the preservation methods currently used for spaceflight foods and the potential of alternatives, such as high pressure processing and microwave sterilization, to meet the requirements of a Mars mission.
Spaceflight Study	n.a.	SLID-391	Spaceflight Metabolism and Nutritional Support	n.a.	Food Intake	Nutrition plays a multifaceted role during space flight. Although its most obvious function is maintaining general health through the consumption of required nutrients, the most important specific functions of proper nutrition are maintaining endocrine and immune system function, skeletal and muscle integrity, and hydration status of spaceflight crews. In addition, interpersonal interactions during mealtimes build team morale and enhance productivity. Providing high-quality, palatable foods is imperative for ensuring adequate nutritional intake, and careful assessment is required to monitor the success or failure of the food system and to ensure crew health. We believe that acknowledging the full role of nutrition will be of paramount importance to the success of extended-duration space missions.
Spaceflight Study	n.a.	SLID-392	Inflight Salivary Pharmacokinetics of Scopolamine and Dextroamphetamine	n.a.	Therapeutic drug	n.a.
Spaceflight Study	n.a.	SLID-393	In-flight Pharmacokinetics of Acetaminophen in Saliva	n.a.	Therapeutic drug	n.a.
Spaceflight Study	International Space Station (ISS)	SLID-394	A Psychiatric Formulary for Deep Space	n.a.	Therapeutic drug	n.a.
Spaceflight Study	Apollo Missions 17	SLID-395	Apollo food technology apollo food technology	n.a.	Food Menu	n.a.
Spaceflight Study	Apollo Missions 11-16	SLID-395	Apollo food technology apollo food technology	n.a.	Food Menu	n.a.
Spaceflight Study	Apollo Missions 7-10	SLID-395	Apollo food technology apollo food technology	n.a.	Food Menu	n.a.
Spaceflight Study	International Space Station (ISS)	SLID-396	Risk of Performance Decrement and Crew Illness Due to an Inadequate Food System	n.a.	Food Intake	NASA is preparing for long duration manned missions beyond low-Earth orbit that will be challenged with long-term exposure to the space environment and very limited resupply. Productive, reliable, and safe human space exploration depends on an adequate food system to provide the crew with safe, nutritious, and acceptable foods for up to 5 years with minimal impact to mission resources.The food system is the sole source of nutrition to the crew. A significant loss in nutrition, either through loss of nutrients in the food during processing and storage or inadequate food intake due to low acceptability, variety, or usability, may significantly compromise crew health and performance. Recent research has indicated that the current food system will not meet the nutrition, acceptability, or resource requirements of a long duration mission beyond low-Earth orbit. The current shelf life is only 1.5 years and several key nutrients degrade in many foods prior to the targeted 5 year shelf life. Additionally mass, volume, waste, and disposal issues presented by the current packaging must be addressed. Alternative provisioning strategies, such as inclusion of a bioregenerative system, reduce initial resource use and add fresh foods that may benefit crew health but also increase infrastructure and crew time requirements. A bioregenerative system also introduces the possibility of food borne illness and food scarcity, which may compromise mission success. Current preflight procedures and the use of prepackaged provisions have ensured food safety so far, but there is currently no technology to enable efficient testing of a bioregenerative system in a resource constrained environment.Current research is investigating strategies to increase the shelf life of a prepackaged food system, decrease use of vehicle resources, and determine the most effective way to balance resource use with provisioning of an adequate food system. The paramount importance of the food system in a long-duration manned exploration mission must not be underestimated. The food system provides not only the nutrients needed for the survival of the crew, but also enhances their psychological well being by being a familiar element in an unfamiliar and hostile environment. This document presents the evidence for the Risk of Performance Decrement and Crew Illness Due to an Inadequate Food System and the gaps that remain.
Spaceflight Study	n.a.	SLID-397	Effect of spaceflight on cardiovascular responses to upright posture in a 77-year-old astronaut	10 Days	Cardiovascular responses	No abstract available
Ground Study	n.a.	SLID-398	Space Food and Nutrition	n.a.	Food Menu	From John Glenn s mission to orbit Earth to the International Space Station program, space food research has met the challenge of providing food that tastes good and travels well in space. To better understand this process, we can look back through history. Explorers have always had to face the problem of how to carry enough food for their journeys. Whether those explorers are onboard a sailing ship or on the Space Shuttle, adequate storage space has been a problem. Food needs to remain edible throughout the voyage, and it also needs to provide all the nutrients required to avoid vitamin-deficiency diseases such as scurvy.
Ground Study	n.a.	SLID-399	Assessment of the Long-Term Stability of Retort Pouch Foods to Support Extended Duration Spaceflight	n.a.	Food Storage	To determine the suitability of retort processed foods to support long-duration spaceflight, a series of 36-mo accelerated shelf life studies were performed on 13 representative retort pouch products. Combined sensory evaluations, physical properties assessments, and nutritional analyses were employed to determine shelf life endpoints for these foods, which were either observed during the analysis or extrapolated via mathematical projection. Data obtained through analysis of these 13 products were later used to estimate the shelf life values of all retort-processed spaceflight foods. In general, the major determinants of shelf life appear to be the development of off-flavor and off-color in products over time. These changes were assumed to be the result of Maillard and oxidation reactions, which can be initiated or accelerated as a result of the retort process and product formulation. Meat products and other vegetable entrées are projected to maintain their quality the longest, between 2 and 8 y, without refrigeration. Fruit and dessert products (1.5 to 5 y), dairy products (2.5 to 3.25 y), and starches, vegetable, and soup products (1 to 4 y) follow. Aside from considerable losses in B and C vitamin content, nutritional value of most products was maintained throughout shelf life. Fortification of storage-labile vitamins was proposed as a countermeasure to ensure long-term nutritive value of these products. The use of nonthermal sterilization technologies was also recommended, as a means to improve initial quality of these products and extend their shelf life for use in long-duration missions. Data obtained also emphasize the importance of low temperature storage in maintaining product quality.
Spaceflight Study	International Space Station (ISS)	SLID-400	Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station	14 Days	DNA damage	Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37°C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37°C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.
Ground Study	n.a.	SLID-401	A harness for enhanced comfort and loading during treadmill exercise in space	n.a.	Muscle strength changes	Introduction:Locomotor and some resistance exercises in space require a gravity replacement force in order to allow 1g-like ground reaction forces to be generated. Currently bungee cords, or other loading devices, interface with the crew member through a harness with a waist belt and shoulder straps. Crew members often find the application of the required loads to be uncomfortable, particularly at the hips. Methods:An experimental harness was built that differed from previous in-flight designs by having a wider, moldable waist belt and contoured shoulder straps with additional padding. Eight subjects ran at 100% body weight (BW) loading for a total duration of 30 min per day on 12 days over a 3-week period in simulated 0-g conditions using horizontal suspension. A 100 mm Visual Analog Scale (VAS)1 was used to assess harness-related and lower extremity discomfort at the end of each run. Results:The overall rating of harness discomfort decreased from 27 mm on the 100 mm scale on day 1 to 10 mm on day 12, with significant decreases recorded for the back and hip regions as well as the overall harness. Discussion:The experimental harness allows for repeated exposure to 30-minute bouts of 100% BW loaded simulated 0-g running with levels of discomfort less than 30 mm on a VAS scale of 0–100 mm. We believe that the use of such a harness during on-orbit exercise countermeasures may allow exercise to be performed at levels which are more effective in preventing bone and muscle loss.
Ground Study	n.a.	SLID-402	Binocular misalignments elicited by altered gravity provide evidence for nonlinear central compensation	n.a.	Binocular misalignments	Increased ocular positioning misalignments upon exposure to altered gravity levels (g-levels) have been strongly correlated with space motion sickness (SMS) severity, possibly due to underlying otolith asymmetries uncompensated in novel gravitational environments. We investigated vertical and torsional ocular positioning misalignments elicited by the 0 and 1.8 g g-levels of parabolic flight and used these data to develop a computational model to describe how such misalignments might arise. Ocular misalignments were inferred through two perceptual nulling tasks: Vertical Alignment Nulling (VAN) and Torsional Alignment Nulling (TAN). All test subjects exhibited significant differences in ocular misalignments in the novel g-levels, which we postulate to be the result of healthy individuals with 1 g-tuned central compensatory mechanisms unadapted to the parabolic flight environment. Furthermore, the magnitude and direction of ocular misalignments in hypo-g and hyper-g, in comparison to 1 g, were nonlinearand nonmonotonic. Previous linear models of central compensation do not predict this. Here we show that a single model of the form a + bgε, where a, b, and ε are the model parameters and g is the current g-level, accounts for both the vertical and torsional ocular misalignment data observed inflight. Furthering our understanding of oculomotor control is critical for the development of interventions that promote adaptation in spaceflight (e.g., countermeasures for novel g-level exposure) and terrestrial (e.g., rehabilitation protocols for vestibular pathology) environments.
Spaceflight Study	International Space Station (ISS)	SLID-403	CCISS, Vascular and BP Reg Canadian space life science research on ISS	6 months	Cardiovascular changes	A comprehensive goal of the Canadian Space Agency studies (CCISS, Vascular and BP Reg) has been to investigate the efficacy of current exercise countermeasures to maintain cardiovascular and cerebrovascular health on return to Earth after up to 6-months in space. Results from the CCISS experiments revealed no significant change of in-flight heart rate during daily activities or sleep, and small, but variable between astronauts, post-flight elevation. The between astronaut differences were exaggerated during measurement of spontaneous baroreflex slope, which was reduced post-flight (P<0.05) during paced breathing with 3 astronauts having significant correlations between reduced baroreflex and reduced RR-interval (consistent with reduced fitness). Cerebrovascular autoregulation and CO2 response were mildly impaired after flight. Some loss of in-flight fitness of astronauts in Vascular was reflected by the increase in HR at a work rate of 161±46 W of 12.3±10.5 bpm, 10.4±5.9 bpm and 13.4±5.7 bpm for early-flight, late-flight and R+1, respectively. On return to gravity, changes in resting heart rate for supine (5.9±3.5 bpm), sit (8.1±3.3 bpm) and stand (10.3±10.0 bpm) were small but variable between individuals (from −5 bpm to +20 bpm in post-flight standing) and not related to the change in exercise heart rate. In Vascular astronauts, pulse wave transit time measured to the finger tended to be reduced post-flight and carotid artery distensibility was significantly reduced (P=0.03, and n=6). The heart rate and baroreflex data suggest that some astronauts return with cardiovascular deconditioning in spite of the exercise regimes. However, greater arterial stiffness is common among all astronauts studied to date. The new CSA project, BP Reg, will monitor inflight blood pressure in an attempt to identify astronauts in greater need for countermeasures. Future research should focus on whether Vascular changes in astronauts might make them an appropriate model to study the mechanisms of arterial aging on Earth.
Spaceflight Study	International Space Station (ISS)	SLID-404	Fluid shifts, vasodilatation and ambulatory blood pressure eduction during long duration spaceflight	6 months	Cardiovascular changes	Acute weightlessness in space induces a fluid shift leading to central volume expansion. Simultaneously, blood pressure is either unchanged or decreased slightly. Whether these effects persist for months in space is unclear. Twenty-four hour ambulatory brachial arterial pressures were automatically recorded at 1–2 h intervals with portable equipment in eight male astronauts: once before launch, once between 85 and 192 days in space on the International Space Station and, finally, once at least 2 months after flight. During the same 24 h, cardiac output (rebreathing method) was measured two to five times (on the ground seated), and venous blood was sampled once (also seated on the ground) for determination of plasma catecholamine concentrations. The 24 h average systolic, diastolic and mean arterial pressures (mean ± se) in space were reduced by 8 ± 2 mmHg (P = 0.01; ANOVA), 9 ± 2 mmHg (P < 0.001) and 10 ± 3 mmHg (P = 0.006), respectively. The nightly blood pressure dip of 8 ± 3 mmHg (P = 0.015) was maintained. Cardiac stroke volume and output increased by 35 ± 10% and 41 ± 9% (P < 0.001); heart rate and catecholamine concentrations were unchanged; and systemic vascular resistance was reduced by 39 ± 4% (P < 0.001). The increase in cardiac stroke volume and output is more than previously observed during short duration flights and might be a precipitator for some of the vision problems encountered by the astronauts. The spaceflight vasodilatation mechanism needs to be explored further.
Spaceflight Study	International Space Station (ISS)	SLID-405	Hemolysis contributes to anemia during long-duration space flight	6 months	Cardiovascular changes	Anemia in astronauts has been noted since the first space missions, but the mechanisms contributing to anemia in space flight have remained unclear. Here, we show that space flight is associated with persistently increased levels of products of hemoglobin degradation, carbon monoxide in alveolar air and iron in serum, in 14 astronauts throughout their 6-month missions onboard the International Space Station. One year after landing, erythrocytic effects persisted, including increased levels of hemolysis, reticulocytosis and hemoglobin. These findings suggest that the destruction of red blood cells, termed hemolysis, is a primary effect of microgravity in space flight and support the hypothesis that the anemia associated with space flight is a hemolytic condition that should be considered in the screening and monitoring of both astronauts and space tourists.
Spaceflight Study	International Space Station (ISS)	SLID-406	Impact of communication delays to and from the International Space Station on self-reported individual and team behavior and performance: A mixed-methods study	166 Days	Mental performance	Deep space explorations will involve significant delays in communication to and from Earth that will likely impact individual and team outcomes. However, the extent of these impacts and the appropriate countermeasures for their mitigation remain largely unknown. This study utilized the International Space Station (ISS), a high-fidelity analog for deep space, as a research platform to assess the impact of communication delays on individual and team performance, mood, and behavior. Three astronauts on the ISS and 18 mission support personnel performed tasks with and without communication delays (50-s one-way) during a mission lasting 166 days. Self-reported assessments of individual and team performance and mood were obtained after each task. Secondary outcomes included communication quality and task autonomy. Qualitative data from post-mission interviews with astronauts were used to validate and expand on quantitative data, and to elicit recommendations for countermeasures. Crew well-being and communication quality were significantly reduced in communication delay tasks compared to control. Communication delays were also significantly associated with increased individual stress/frustration. Qualitative data suggest communication delays impacted operational outcomes (i.e. task efficiency), teamwork processes (i.e. team/task coordination) and mood (i.e. stress/frustration), particularly when tasks involved high task-related communication demands, either because of poor communication strategies or low crew autonomy. Training, teamwork, and technology-focused countermeasures were identified to mitigate or prevent adverse impacts.
Spaceflight Study	International Space Station (ISS)	SLID-407	Physiological and Functional Alterations after Spaceflight and Bed Rest		Muscle function changes	INTRODUCTION:Exposure to microgravity causes alterations in multiple physiological systems, potentially impacting the ability of astronauts to perform critical mission tasks. The goal of this study was to determine the effects of spaceflight on functional task performance and to identify the key physiological factors contributing to their deficits. METHODS:A test battery comprised of seven functional tests and 15 physiological measures was used to investigate the sensorimotor, cardiovascular, and neuromuscular adaptations to spaceflight. Astronauts were tested before and after 6-month spaceflights. Subjects were also tested before and after 70 d of 6° head-down bed rest, a spaceflight analog, to examine the role of axial body unloading on the spaceflight results. These subjects included control and exercise groups to examine the effects of exercise during bed rest. RESULTS:Spaceflight subjects showed the greatest decrement in performance during functional tasks that required the greatest demand for dynamic control of postural equilibrium which was paralleled by similar decrements in sensorimotor tests that assessed postural and dynamic gait control. Other changes included reduced lower limb muscle performance and increased HR to maintain blood pressure. Exercise performed during bed rest prevented detrimental change in neuromuscular and cardiovascular function; however, both bed rest groups experienced functional and balance deficits similar to spaceflight subjects. CONCLUSION:Bed rest data indicate that body support unloading experienced during spaceflight contributes to postflight postural control dysfunction. Further, the bed rest results in the exercise group of subjects confirm that resistance and aerobic exercises performed during spaceflight can play an integral role in maintaining neuromuscular and cardiovascular functions, which can help in reducing decrements in functional performance. These results indicate that a countermeasure to mitigate postflight postural control dysfunction is required to maintain functional performance.
Spaceflight Study	International Space Station (ISS)	SLID-408	Dietary acid load and bone turnover during long-duration spaceflight and bed rest	160±20 Days	Dietary acid load and bone turnover changes	Background: Bed rest studies document that a lower dietary acid load is associated with lower bone resorption.Objective: We tested the effect of dietary acid load on bone metabolism during spaceflight.Design: Controlled 4-d diets with a high or low animal protein-to-potassium (APro:K) ratio (High and Low diets, respectively) were given to 17 astronauts before and during spaceflight. Each astronaut had 1 High and 1 Low diet session before flight and 2 High and 2 Low sessions during flight, in addition to a 4-d session around flight day 30 (FD30), when crew members were to consume their typical in-flight intake. At the end of each session, blood and urine samples were collected. Calcium, total protein, energy, and sodium were maintained in each crew member's preflight and in-flight controlled diets.Results: Relative to preflight values, N-telopeptide (NTX) and urinary calcium were higher during flight, and bone-specific alkaline phosphatase (BSAP) was higher toward the end of flight. The High and Low diets did not affect NTX, BSAP, or urinary calcium. Dietary sulfur and age were significantly associated with changes in NTX. Dietary sodium and flight day were significantly associated with urinary calcium during flight. The net endogenous acid production (NEAP) estimated from the typical dietary intake at FD30 was associated with loss of bone mineral content in the lumbar spine after the mission. The results were compared with data from a 70-d bed rest study, in which control (but not exercising) subjects' APro:K was associated with higher NTX during bed rest. Conclusions: Long-term lowering of NEAP by increasing vegetable and fruit intake may protect against changes in loss of bone mineral content during spaceflight when adequate calcium is consumed, particularly if resistive exercise is not being performed. This trial was registered at clinicaltrials.gov as NCT01713634.
Spaceflight Study	International Space Station (ISS)	SLID-409	Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station	6 months	Gene Expression	Adaptation to the space environment can sometimes pose physiological problems to International Space Station (ISS) astronauts after their return to earth. Therefore, it is important to develop healthcare technologies for astronauts. In this study, we examined the feasibility of using hair follicles, a readily obtained sample, to assess gene expression changes in response to spaceflight adaptation. In order to investigate the gene expression changes in human hair follicles during spaceflight, hair follicles of 10 astronauts were analyzed by microarray and real time qPCR analyses. We found that spaceflight alters human hair follicle gene expression. The degree of changes in gene expression was found to vary among individuals. In some astronauts, genes related to hair growth such as FGF18, ANGPTL7 and COMP were upregulated during flight, suggesting that spaceflight inhibits cell proliferation in hair follicles.
Spaceflight Study	International Space Station (ISS)	SLID-410	Fusarium oxysporum as an Opportunistic Fungal Pathogen on Zinnia hybrida Plants Grown on board the International Space Station	n.a.	Fungi	A plant production system called Veggie was launched to the International Space Station (ISS) in 2014. In late 2015, during the growth of Zinnia hybrida cv. ‘Profusion’ in the Veggie hardware, plants developed chlorosis, leaf curling, fungal growth that damaged leaves and stems, and eventually necrosis. The development of symptoms was correlated to reduced air flow leading to a significant buildup of water enveloping the leaves and stems in microgravity. Symptomatic tissues were returned to Earth on 18 May 2016 and were immediately processed to determine the primary causal agent of the disease. The presumptive pathogen was identified as Fusarium oxysporum by morphological features of microconidia and conidiophores on symptomatic tissues; that is, by epifluorescent microscopy (EFM), scanning electron microscopy (SEM), metabolic microarrays, and ITS sequencing. Both EFM and SEM imaging of infected tissues showed that germinating conidia were capable of stomatal penetration and thus acted as the primary method for infecting host tissues. A series of ground-based pathogenicity assays were conducted with healthy Z. hybrida plants that were exposed to reduced-airflow and high-water stress (i.e., encased in sealed bags) or were kept in an unstressed configuration. Koch's postulates were successfully completed with Z. hybrida plants in the lab, but symptoms only matched ISS-flown symptomatic tissues when the plants were stressed with high-water exposure. Unstressed plants grown under similar lab conditions failed to develop the symptoms observed with plants on board the ISS. The overall results of the pathogenicity tests imply that F. oxysporum acted as an opportunistic pathogen on severely high-water stressed plants. The source of the opportunistic pathogen is not known, but virulent strains of F. oxysporum were not recovered from unused materials in the Veggie plant pillow growth units assayed after the flight.
Spaceflight Study	International Space Station (ISS)	SLID-411	Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station	n.a.	Bacteria	Several evolutionarily distinct, near full-length draft metagenome-resolved genomes (MRG), were assembled from sequences recovered from the International Space Station (ISS) environments. The retrieval of MRGs facilitated the exploration of a large collection of archived strains (~ 500 isolates) and assisted in isolating seven related strains. The whole genome sequences (WGS) of seven ISS strains exhibited 100% identity to the 4.85 × 106 bp of four MRGs. The “metagenome to phenome” approach led to the description of a novel bacterial genus from the ISS samples. The phylogenomics and traditional taxonomic approaches suggested that these seven ISS strains and four MRGs were not phylogenetically affiliated to any validly described genera of the family Erwiniaceae, but belong to a novel genus with the proposed name Kalamiella. Comparative genomic analyses of Kalamiella piersonii strains and MRGs showed genes associated with carbohydrate (348 genes), amino acid (384), RNA (59), and protein (214) metabolisms; membrane transport systems (108), pathways for biosynthesis of cofactors, vitamins, prosthetic groups, and pigments (179); as well as mechanisms for virulence, disease, and defense (50). Even though Kalamiella genome annotation and disc diffusion tests revealed multidrug resistance, the PathogenFinder algorithm predicted that K. piersonii strains are not human pathogens. This approach to isolating microbes allows for the characterization of functional pathways and their potential virulence properties that can directly affect human health. The isolation of novel strains from the ISS has broad applications in microbiology, not only because of concern for astronaut health but it might have a great potential for biotechnological relevance. The metagenome to phenome approach will help to improve our understanding of complex metabolic networks that control fundamental life processes under microgravity and in deep space.
Spaceflight Study	International Space Station (ISS)	SLID-411	Metagenome to phenome approach enables isolation and genomics characterization of Kalamiella piersonii gen. nov., sp. nov. from the International Space Station	n.a.	Bacteria	Several evolutionarily distinct, near full-length draft metagenome-resolved genomes (MRG), were assembled from sequences recovered from the International Space Station (ISS) environments. The retrieval of MRGs facilitated the exploration of a large collection of archived strains (~ 500 isolates) and assisted in isolating seven related strains. The whole genome sequences (WGS) of seven ISS strains exhibited 100% identity to the 4.85 × 106 bp of four MRGs. The “metagenome to phenome” approach led to the description of a novel bacterial genus from the ISS samples. The phylogenomics and traditional taxonomic approaches suggested that these seven ISS strains and four MRGs were not phylogenetically affiliated to any validly described genera of the family Erwiniaceae, but belong to a novel genus with the proposed name Kalamiella. Comparative genomic analyses of Kalamiella piersonii strains and MRGs showed genes associated with carbohydrate (348 genes), amino acid (384), RNA (59), and protein (214) metabolisms; membrane transport systems (108), pathways for biosynthesis of cofactors, vitamins, prosthetic groups, and pigments (179); as well as mechanisms for virulence, disease, and defense (50). Even though Kalamiella genome annotation and disc diffusion tests revealed multidrug resistance, the PathogenFinder algorithm predicted that K. piersonii strains are not human pathogens. This approach to isolating microbes allows for the characterization of functional pathways and their potential virulence properties that can directly affect human health. The isolation of novel strains from the ISS has broad applications in microbiology, not only because of concern for astronaut health but it might have a great potential for biotechnological relevance. The metagenome to phenome approach will help to improve our understanding of complex metabolic networks that control fundamental life processes under microgravity and in deep space.
Spaceflight Study	Space Environment Survivability of Living Organisms (SESLO)	SLID-412	Nanosatellites for Biology in Space: In Situ Measurement of Bacillus subtilis Spore Germination and Growth after 6 Months in Low Earth Orbit on the O/OREOS Mission	181 Days	Bacteria	We report here complete 6-month results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment. The world’s first and only long-duration live-biology cubesat experiment, SESLO was executed by one of two 10-cm cube-format payloads aboard the 5.5-kg O/OREOS (Organism/Organic Exposure to Orbital Stresses) free-flying nanosatellite, which launched to a 72°-inclination, 650-km Earth orbit in 2010. The SESLO experiment measured the long-term survival, germination, metabolic, and growth responses of Bacillus subtilis spores exposed to microgravity and ionizing radiation including heavy-ion bombardment. A pair of radiation dosimeters (RadFETs, i.e., radiation-sensitive field-effect transistors) within the SESLO payload provided an in-situ dose rate estimate of 6–7.6 mGy/day throughout the mission. Microwells containing samples of dried spores of a wild-type B. subtilis strain and a radiation-sensitive mutant deficient in Non-Homologoous End Joining (NHEJ) were rehydrated after 14, 91, and 181 days in space with nutrient medium containing with the redox dye alamarBlue (aB), which changes color upon reaction with cellular metabolites. Three-color transmitted light intensity measurements of all microwells were telemetered to Earth within days of each 24-hour growth experiment. At 14 and 91 days, spaceflight samples germinated, grew, and metabolized significantly more slowly than matching ground-control samples, as measured both by aB reduction and optical density changes; these rate differences notwithstanding, the final optical density attained was the same in both flight and ground samples. After 181 days in space, spore germination and growth appeared hindered and abnormal. We attribute the differences not to an effect of the space environment per se, as both spaceflight and ground-control samples exhibited the same behavior, but to a pair of ~15-day thermal excursions, after the 91-day measurement and before the 181-day experiment, that peaked above 46 °C in the SESLO payload. Because the payload hardware operated nominally at 181 days, the growth issues point to heat damage, most likely to component(s) of the growth medium (RPMI 1640 containing aB) or to biocompatibility issues caused by heat-accelerated outgassing or leaching of harmful compounds from components of the SESLO hardware and electronics.
Spaceflight Study	NASA Space Shuttle Atlantis Mission STS-115	SLID-413	Spaceflight enhances cell aggregation and random budding in Candida albicans	12 Days	Fungi	This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans–induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.
Spaceflight Study	International Space Station (ISS)	SLID-414	Improvements in the re-flight of spaceflight experiments on plant tropisms	2 months	Seed	In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.
Spaceflight Study	Shuttle Endeavour,ISS	SLID-415	Microgravity does not alter plant stand gas exchange of wheat at moderate light levels and saturating CO2 concentration	73 Days	Grain	Plant stand gas exchange was measured nondestructively in microgravity during the Photosynthesis Experiment Subsystem Testing and Operations experiment conducted onboard the International Space Station. Rates of evapotranspiration and photosynthesis measured in space were compared with ground controls to determine if microgravity directly affects whole-stand gas exchange of Triticum aestivum. During six 21-day experiment cycles, evapotranspiration was determined continuously from water addition rates to the nutrient delivery system, and photosynthesis was determined from the amount of CO2 added to maintain the chamber CO2 concentration setpoint. Plant stand evapotranspiration, net photosynthesis, and water use efficiency were not altered by microgravity. Although leaf area was significantly reduced in microgravity-grown plants compared to ground control plants, leaf area distribution was not affected enough to cause significant differences in the amounts of light absorbed by the flight and ground control plant stands. Microgravity also did not affect the response of evapotranspiration to changes in chamber vapor pressure difference of 12-day-old wheat plant stands. These results suggest that gravity naïve plants grown at moderate light levels (300 μmol m−2 s−1) behave the same as ground control plants. This implies that future plant-based regenerative life support systems can be sized using 1 g data because water purification and food production rates operate at nearly the same rates as in 1 g at moderate light levels. However, it remains to be verified whether the present results are reproducible in plants grown under stronger light levels.
Spaceflight Study	International Space Station (ISS)	SLID-416	Organ-specific remodeling of the Arabidopsis transcriptome in response to spaceflight	n.a.	Plant	Background:Spaceflight presents a novel environment that is outside the evolutionary experience of terrestrial organisms. Full activation of the International Space Station as a science platform complete with sophisticated plant growth chambers, laboratory benches, and procedures for effective sample return, has enabled a new level of research capability and hypothesis testing in this unique environment. The opportunity to examine the strategies of environmental sensing in spaceflight, which includes the absence of unit gravity, provides a unique insight into the balance of influence among abiotic cues directing plant growth and development: including gravity, light, and touch. The data presented here correlate morphological and transcriptome data from replicated spaceflight experiments. Results:The transcriptome of Arabidopsis thaliana demonstrated organ-specific changes in response to spaceflight, with 480 genes showing significant changes in expression in spaceflight plants compared with ground controls by at least 1.9-fold, and 58 by more than 7-fold. Leaves, hypocotyls, and roots each displayed unique patterns of response, yet many gene functions within the responses are related. Particularly represented across the dataset were genes associated with cell architecture and growth hormone signaling; processes that would not be anticipated to be altered in microgravity yet may correlate with morphological changes observed in spaceflight plants. As examples, differential expression of genes involved with touch, cell wall remodeling, root hairs, and cell expansion may correlate with spaceflight-associated root skewing, while differential expression of auxin-related and other gravity-signaling genes seemingly correlates with the microgravity of spaceflight. Although functionally related genes were differentially represented in leaves, hypocotyls, and roots, the expression of individual genes varied substantially across organ types, indicating that there is no single response to spaceflight. Rather, each organ employed its own response tactics within a shared strategy, largely involving cell wall architecture. Conclusions:Spaceflight appears to initiate cellular remodeling throughout the plant, yet specific strategies of the response are distinct among specific organs of the plant. Further, these data illustrate that in the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development, and that the basis for that engagement lies in the differential expression of genes in an organ-specific manner that maximizes the utilization of these signals – such as the up-regulation of genes associated with light-sensing in roots.
Spaceflight Study	International Space Station (ISS)	SLID-417	RNA-seq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity	n.a.	Plant	Premise:Plants synthesize information from multiple environmental stimuli when determining their direction of growth. Gravity, being ubiquitous on Earth, plays a major role in determining the direction of growth and overall architecture of the plant. Here, we utilized the microgravity environment on board the International Space Station (ISS) to identify genes involved influencing growth and development of phototropically stimulated seedlings of Arabidopsis thaliana. Methods:Seedlings were grown on the ISS, and RNA was extracted from 7 samples (pools of 10–15 plants) grown in microgravity (μg) or Earth gravity conditions (1-g). Transcriptomic analyses via RNA sequencing (RNA-seq) of differential gene expression was performed using the HISAT2-Stringtie-DESeq2 RNASeq pipeline. Differentially expressed genes were further characterized by using Pathway Analysis and enrichment for Gene Ontology classifications. Results:For 296 genes that were found significantly differentially expressed between plants in microgravity compared to 1-g controls, Pathway Analysis identified eight molecular pathways that were significantly affected by reduced gravity conditions. Specifically, light-associated pathways (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) were significantly downregulated in microgravity. Conclusions:Gene expression in A. thaliana seedlings grown in microgravity was significantly altered compared to that of the 1-g control. Understanding how plants grow in conditions of microgravity not only aids in our understanding of how plants grow and respond to the environment but will also help to efficiently grow plants during long-range space missions.
Spaceflight Study	Space Shuttle	SLID-418	Spaceflight transcriptomes: unique responses to a novel environment	n.a.	Plant	The spaceflight environment presents unique challenges to terrestrial biology, including but not limited to the direct effects of gravity. As we near the end of the Space Shuttle era, there remain fundamental questions about the response and adaptation of plants to orbital spaceflight conditions. We address a key baseline question of whether gene expression changes are induced by the orbital environment, and then we ask whether undifferentiated cells, cells presumably lacking the typical gravity response mechanisms, perceive spaceflight. Arabidopsis seedlings and undifferentiated cultured Arabidopsis cells were launched in April, 2010, as part of the BRIC-16 flight experiment on STS-131. Biologically replicated DNA microarray and averaged RNA digital transcript profiling revealed several hundred genes in seedlings and cell cultures that were significantly affected by launch and spaceflight. The response was moderate in seedlings; only a few genes were induced by more than 7-fold, and the overall intrinsic expression level for most differentially expressed genes was low. In contrast, cell cultures displayed a more dramatic response, with dozens of genes showing this level of differential expression, a list comprised primarily of heat shock–related and stress-related genes. This baseline transcriptome profiling of seedlings and cultured cells confirms the fundamental hypothesis that survival of the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression. The comparison of intact plants with cultures of undifferentiated cells confirms a second hypothesis: undifferentiated cells can detect spaceflight in the absence of specialized tissue or organized developmental structures known to detect gravity.
Spaceflight Study	Space Shuttle Atlantis (STS-135)	SLID-419	Biological and metabolic response in STS-135 space-flown mouse skin	13 Days	Animals	There is evidence that space flight condition-induced biological damage is associated with increased oxidative stress and extracellular matrix (ECM) remodeling. To explore possible mechanisms, changes in gene expression profiles implicated in oxidative stress and in ECM remodeling in mouse skin were examined after space flight. The metabolic effects of space flight in skin tissues were also characterized. Space Shuttle Atlantis (STS-135) was launched at the Kennedy Space Center on a 13-day mission. Female C57BL/6 mice were flown in the STS-135 using animal enclosure modules (AEMs). Within 3–5 h after landing, the mice were euthanized and skin samples were harvested for gene array analysis and metabolic biochemical assays. Many genes responsible for regulating production and metabolism of reactive oxygen species (ROS) were significantly (p < 0.05) altered in the flight group, with fold changes >1.5 compared to AEM control. For ECM profile, several genes encoding matrix and metalloproteinases involved in ECM remodeling were significantly up-/down-regulated following space flight. To characterize the metabolic effects of space flight, global biochemical profiles were evaluated. Of 332 named biochemicals, 19 differed significantly (p < 0.05) between space flight skin samples and AEM ground controls, with 12 up-regulated and 7 down-regulated including altered amino acid, carbohydrate metabolism, cell signaling, and transmethylation pathways. Collectively, the data demonstrated that space flight condition leads to a shift in biological and metabolic homeostasis as the consequence of increased regulation in cellular antioxidants, ROS production, and tissue remodeling. This indicates that astronauts may be at increased risk for pathophysiologic damage or carcinogenesis in cutaneous tissue.
Spaceflight Study	International Space Station (ISS)	SLID-420	Decreased expression of myogenic transcription factors and myosin heavy chains in Caenorhabditis elegans muscles developed during spaceflight	n.a.	Animals	The molecular mechanisms underlying muscle atrophy during spaceflight are not well understood. We have analyzed the effects of a 10-day spaceflight on Caenorhabditis elegans muscle development. DNA microarray, real-time quantitative PCR, and quantitative western blot analyses revealed that the amount of MHC in both body-wall and pharyngeal muscle decrease in response to spaceflight. Decreased transcription of the body-wall myogenic transcription factor HLH-1 (CeMyoD) and of the three pharyngeal myogenic transcription factors, PEB-1, CEH-22 and PHA-4 were also observed. Upon return to Earth animals displayed reduced rates of movement, indicating a functional defect. These results demonstrate that C. elegans muscle development is altered in response to spaceflight. This altered development occurs at the level of gene transcription and was observed in the presence of innervation,not simply in isolated cells. This important finding coupled with past observations of decreased levels of the same myogenic transcription factions in vertebrates after spaceflight raises the possibility that altered muscle development is a contributing factor to spaceflight-induced muscle atrophy in vertebrates.
Spaceflight Study	Space Shuttle Atlantis (STS-135)	SLID-421	Effects of microgravity on the mouse triceps brachii muscle	13 Days	Animals	Introduction: In this study we investigated the effects of microgravity on the fiber properties of the mouse triceps brachii, a forelimb muscle that has no antigravity function. Methods: Mice (n=7) were exposed to microgravity for 13 days on the space shuttle Atlantis (Space Transportation System- 135). The fiber cross-sectional area (CSA) and succinate dehy- drogenase (SDH) staining intensity of the triceps brachii muscle were compared with those of controls (n=7). SDH activity in this muscle was also estimated. Results: Microgravity did not affect the body weight, muscle weight, or fiber CSA, but there was reduced SDH staining intensity of all types of fibers, irre- spective of the muscle region (P < 0.05). Microgravity also reduced muscle SDH activity (P < 0.05). Conclusions: Short- term exposure to microgravity induced a decrease in oxidative capacity, but not atrophy, in the triceps brachii muscle of mice.
Spaceflight Study	Shuttle STS-108	SLID-422	Effects of Space Flight on Mouse Liver versus Kidney: Gene Pathway Analyses	12 Days	Animals	Understanding genome wide, tissue-specific, and spaceflight-induced changes in gene expression is critical to develop effective countermeasures. Transcriptome analysis has been performed on diverse tissues harvested from animals flown in space, but not the kidney. We determined the genome wide gene expression using a gene array analysis of kidney and liver tissue from mice flown in space for 12 days versus ground based control animals. By comparing the transcriptome of liver and kidney from animals flown in space versus ground control animals, we tested a unique hypothesis: Are there common gene expression pathways activated in multiple tissue types in response to spaceflight stimuli? Although there were tissue-specific changes, both liver and kidney overexpressed genes in the same four areas: (a) cellular responses to peptides, hormones, and nitrogen/organonitrogen compounds; (b) apoptosis and cell death; (c) fat cell differentiation and (d) negative regulation of protein kinase.
Spaceflight Study	STS-131,STS-135,Bion-M1	SLID-423	Effects of spaceflight on the muscles of the murine shoulder	15 Days,13 Days,30 Days	Animals	Mechanical loading is necessary for the development and maintenance of the musculoskeletal system. Removal of loading via microgravity, paralysis, or bed rest leads to rapid loss of muscle mass and function; however, the molecular mechanisms that lead to these changes are largely unknown, particularly for the spaceflight (SF) microgravity environment. Furthermore, few studies have explored these effects on the shoulder, a dynamically stabilized joint with a large range of motion; therefore, we examined the effects of microgravity on mouse shoulder muscles for the 15-d Space Transportation System (STS)-131, 13-d STS-135, and 30-d Bion-M1 missions. Mice from STS missions were euthanized within 4 h after landing, whereas mice from the Bion-M1 mission were euthanized within 14 h after landing. The motion-generating deltoid muscle was more sensitive to microgravity than the joint-stabilizing rotator cuff muscles. Mice from the STS-131 mission exhibited reduced myogenic (Myf5 and -6) and adipogenic (Pparg, Cebpa, and Lep) gene expression, whereas either no change or an increased expression of these genes was observed in mice from the Bion-M1 mission. In summary, muscle responses to microgravity were muscle-type specific, short-duration SF caused dramatic molecular changes to shoulder muscles and responses to reloading upon landing were rapid.-Shen, H., Lim, C., Schwartz, A. G., Andreev-Andrievskiy, A., Deymier, A. C., Thomopoulos, S. Effects of spaceflight on the muscles of the murine shoulder.
Spaceflight Study	Space Shuttle Endeavour (STS)-126	SLID-424	Evaluation of in vitro macrophage differentiation during space flight	14 Days	Animals	We differentiated mouse bone marrow cells in the presence of recombinant macrophage colony stimulating (rM-CSF) factor for 14 days during the flight of space shuttle Space Transportation System (STS)-126. We tested the hypothesis that the receptor expression for M-CSF, c-Fms was reduced. We used flow cytometry to assess molecules on cells that were preserved during flight to define the differentiation state of the developing bone marrow macrophages; including CD11b, CD31, CD44, Ly6C, Ly6G, F4/80, Mac2, c-Fos as well as c-Fms. In addition, RNA was preserved during the flight and was used to perform a gene microarray. We found that there were significant differences in the number of macrophages that developed in space compared to controls maintained on Earth. We found that there were significant changes in the distribution of cells that expressed CD11b, CD31, F4/80, Mac2, Ly6C and c-Fos. However, there were no changes in c-Fms expression and no consistent pattern of advanced or retarded differentiation during space flight. We also found a pattern of transcript levels that would be consistent with a relatively normal differentiation outcome but increased proliferation by the bone marrow macrophages that were assayed after 14 days of space flight. There also was a surprising pattern of space flight influence on genes of the coagulation pathway. These data confirm that a space flight can have an impact on the in vitro development of macrophages from mouse bone marrow cells.
Spaceflight Study	Bion M1	SLID-425	Exposure to microgravity for 30 days onboard Bion M1 caused muscle atrophy and impaired regeneration in murine femoral Quadriceps	30 Days	Animals	Mechanical unloading in microgravity during spaceflight is known to cause muscular atrophy, changes in muscle fiber composition, gene expression, and reduction in regenerative muscle growth. Although some limited data exists for long-term effects of microgravity in human muscle, these processes have mostly been studied in rodents for short periods of time. Here we report on how long-term (30-day long) mechanical unloading in microgravity affects murine muscles of the femoral Quadriceps group. To conduct these studies we used muscle tissue from 6 microgravity mice, in comparison to habitat (7), and vivarium (14) ground control mice from the NASA Biospecimen Sharing Program conducted in collaboration with the Institute for Biomedical Problems of the Russian Academy of Sciences, during the Russian Bion M1 biosatellite mission in 2013. Muscle histomorphology from microgravity specimens showed signs of extensive atrophy and regenerative hypoplasia relative to ground controls. Specifically, we observed a two-fold decrease in the number of myonuclei, compared to vivarium and ground controls, and central location of myonuclei, low density of myofibers in the tissue, and of myofibrils within a fiber, as well as fragmentation and swelling of myofibers. Despite obvious atrophy, muscle regeneration nevertheless appeared to have continued after 30 days in microgravity as evidenced by thin and short newly formed myofibers. Many of them, however, showed evidence of apoptotic cells and myofibril degradation, suggesting that long-term unloading in microgravity may affect late stages of myofiber differentiation. Ground asynchronous and vivarium control animals demonstrated normal, well-developed tissue structure with sufficient blood and nerve supply and evidence of regenerative formation of new myofibers free of apoptotic nuclei. Regenerative activity of satellite cells in muscles was observed both in microgravity and ground control groups, using Pax7 and Myogenin immunolocalization, as well as Myogenin expression analysis. In addition, we have detected positive nuclear immunolocalization of c-Jun and c-Myc proteins indicating their sensitivity to changes in gravitational loading in a given model. In summary, long-term spaceflight in microgravity caused significant atrophy and degeneration of the femoral Quadriceps muscle group, and it may interfere with muscle regenerative processes by inducing apoptosis in newly-formed myofibrils during their differentiation phase.
Spaceflight Study	STS-131	SLID-426	Fifteen days of microgravity causes growth in calvaria of mice	15 Days	Animals	Bone growth may occur in spaceflight as a response to skeletal unloading and head-ward fluid shifts. While unloading causes significant loss of bone mass and density in legs of animals exposed to microgravity, increased blood and interstitial fluid flows accompanying microgravity-induced fluid redistribution may elicit an opposite effect in the head. Seven 23-week-old, adult female wild-type C57BL/6 mice were randomly chosen for exposure to 15 days of microgravity on the STS-131 mission, while eight female littermates served as ground controls. Upon mission completion, all 15 murine calvariae were imaged on a micro-computed tomography scanner. A standardized rectangular volume was placed on the parietal bones of each calvaria for analyses, and three parameters were determined to measure increased parietal bone volume: bone volume (BV), average cortical thickness (Ct.Th), and tissue mineral density (TMD). Microgravity exposure caused a statistically significant increase in BV of the spaceflight (SF) group compared to that of the ground control (GC) group, the mean BV±SD for the SF group was 1.904±0.842 mm3, compared to 1.758±0.122 mm3 for the GC group (p<0.05). Ct.Th demonstrated a trend of increase from 0.099±0.006 mm in the GC group to 0.104±0.005 mm in the SF group (p=0.12). TMD was similar between the two groups with 0.878±0.029 g/cm3 for the GC group and 0.893±0.028 g/cm3 for the SF group (p=0.31). Our results indicate that microgravity causes responsive changes in calvarial bones that do not normally bear weight. These findings suggest that fluid shifts alone accompanying microgravity may initiate bone adaptation independent of skeletal loading by tissue.
Spaceflight Study	Russian spacecrafts Foton M-2 and M-3	SLID-427	Functional changes in the snail statocyst system elicited by microgravity	n.a.	Animals	Background: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. Methodology/principal findings: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. Conclusions/significance: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space.
Spaceflight Study	Space Transport System-108/UF-1	SLID-428	Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets	12 Days	Animals	This portion of the study quantified the effects of a 12-day space shuttle mission (Space Transport System-108/UF-1) on body and lymphoid organ masses, activation marker expression, cytokine secretion, and erythrocyte and thrombocyte characteristics in C57BL/6 mice. Animals in flight (Flt group) had 10-12% lower body mass compared with ground controls housed either in animal enclosure modules or under standard vivarium conditions (P < 0.001) and the smallest thymus and spleen masses. Percentages of CD25(+) lymphocytes, CD3(+)/CD25(+) T cells, and NK1.1(+)/CD25(+) natural killer cells from Flt mice were higher compared with both controls (P < 0.05). In contrast, CD71 expression was depressed in the Flt and animal enclosure module control mice compared with vivarium control animals (P < 0.001). Secretion of interferon-gamma, IL-2, and IL-4, but not tumor necrosis factor-alpha and IL-5, by splenocytes from Flt mice was decreased relative to either one or both ground controls (P < 0.05). Flt mice also had high red blood cell and thrombocyte counts compared with both sets of controls; low red blood cell volume and distribution width, percentage of reticulocytes, and platelet volume were also noted (P < 0.05) and were consistent with dehydration. These data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become significant during long-term space missions.
Spaceflight Study	Space Shuttle Atlantis (STS-135)	SLID-429	Is spaceflight-induced immune dysfunction linked to systemic changes in metabolism?	13 Days	Animals	The Space Shuttle Atlantis launched on its final mission (STS-135) on July 8, 2011. After just under 13 days, the shuttle landed safely at Kennedy Space Center (KSC) for the last time. Female C57BL/6J mice flew as part of the Commercial Biomedical Testing Module-3 (CBTM-3) payload. Ground controls were maintained at the KSC facility. Subsets of these mice were made available to investigators as part of NASA's Bio-specimen Sharing Program (BSP). Our group characterized cell phenotype distributions and phagocytic function in the spleen, catecholamine and corticosterone levels in the adrenal glands, and transcriptomics/metabolomics in the liver. Despite decreases in most splenic leukocyte subsets, there were increases in reactive oxygen species (ROS)-related activity. Although there were increases noted in corticosterone levels in both the adrenals and liver, there were no significant changes in catecholamine levels. Furthermore, functional analysis of gene expression and metabolomic profiles suggest that the functional changes are not due to oxidative or psychological stress. Despite changes in gene expression patterns indicative of increases in phagocytic activity (e.g. endocytosis and formation of peroxisomes), there was no corresponding increase in genes related to ROS metabolism. In contrast, there were increases in expression profiles related to fatty acid oxidation with decreases in glycolysis-related profiles. Given the clear link between immune function and metabolism in many ground-based diseases, we propose a similar link may be involved in spaceflight-induced decrements in immune and metabolic function.
Spaceflight Study	Space Shuttle Atlantis (STS-135)	SLID-430	Masticatory muscles of mouse do not undergo atrophy in space	13 Days	Animals	Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System-135. Age- and sex-matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 ± 3% decreased muscle mass, 2-fold decreased phosphorylated (P)-Akt, and 4- to 12-fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3-fold increase in P-focal adhesion kinase, 1.5-fold increase in P-Akt, and 50-90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3-fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril-specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.
Spaceflight Study	STS-118	SLID-431	Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors	13 Days	Animals	The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, 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 < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). 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.
Ground Study	n.a.	SLID-432	Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments	n.a.	Animals	Background: Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved. Objectives: The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C. Methods: Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH). Results: Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6-7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months. Conclusion: These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.
Spaceflight Study	SpaceX CRS-3,SpaceX CRS-11	SLID-433	Prolonged Exposure to Microgravity Reduces Cardiac Contractility and Initiates Remodeling in Drosophila	30 Days	Animals	Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which we found dramatically elevated proteasome aggregates co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.
Spaceflight Study	Shuttle	SLID-434	Reduction of anabolic signals and alteration of osteoblast nuclear morphology in microgravity	n.a.	Animals	Bone loss has been repeatedly documented in astronauts after flight, yet little is known about the mechanism of bone loss in space flight. Osteoblasts were activated during space flight in microgravity (microg) with and without a 1 gravity (1 g) field and 24 genes were analyzed for early induction. Induction of proliferating cell nuclear antigen (PCNA), transforming growth factor beta (TGFbeta), cyclo-oxygenase-2 (cox-2), cpla2, osteocalcin (OC), c-myc, fibroblast growth factor-2 (fgf-2), bcl2, bax, and fgf-2 message as well as FGF-2 protein were significantly depressed in microg when compared to ground (gr). Artificial onboard gravity normalized the induction of c-myc, cox-2, TGFbeta, bax, bcl2, and fgf-2 message as well as FGF-2 protein synthesis in spaceflight samples. In normal gravity, FGF-2 induces bcl2 expression; we found that bcl2 expression was significantly reduced in microgravity conditions. Since nuclear shape is known to elongate in the absence of mitogens like FGF-2, we used high-resolution image-based morphometry to characterize changes in osteoblast nuclear architecture under microgravity, 1 g flight, and ground conditions. Besides changes in cell shape (roundish/elliptic), other high-resolution analyses show clear influences of gravity on the inner nuclear structure. These changes occur in the texture, arrangement, and contrast of nuclear particles and mathematical modeling defines the single cell classification of the osteoblasts. Changes in nuclear structure were evident as early as 24 h after exposure to microgravity. This documented alteration in nuclear architecture may be a direct result of decreased expression of autocrine and cell cycle genes, suggesting an inhibition of anabolic response in microg. Life on this planet has evolved in a normal gravity field and these data suggest that gravity plays a significant role in regulation of osteoblast transcription.
Spaceflight Study	STS-131,STS-135	SLID-435	Response of the mouse sublingual gland to spaceflight	15 Days,13 Days	Animals	The ultrastructure and immunohistochemistry of secretory proteins of sublingual glands were studied in mice flown on the US space shuttles Discovery [Space Transportation System (STS)-131] and Atlantis (STS-135). No differences in mucous acinar or serous demilune cell structure were observed between sublingual glands of ground (control) and flight mice. In contrast, previous studies showed autophagy and apoptosis of parotid serous acinar cells in flight mice. The expression of parotid secretory protein (PSP) in sublingual demilune cells of STS-131 flight mice was significantly increased compared with ground (control) mice but decreased in STS-135 flight mice. Similarly, expression of mucin (MUC-19) in acinar cells and expression of the type II regulatory subunit of protein kinase A (PKA-RII) in demilune cells were increased in STS-131 flight mice and decreased in STS-135 flight mice, but not significantly. Demilune cell and parotid protein (DCPP) was slightly decreased in mice from both flights, and nuclear PKA-RII was slightly increased. These results indicate that the response of salivary glands to spaceflight conditions varies among the different glands, cell types, and secretory proteins. Additionally, the spaceflight environment, including the effects of microgravity, modifies protein expression. Determining changes in salivary proteins may lead to development of non-invasive methods to assess the physiological status of astronauts.
Spaceflight Study	STS-133,STS-135	SLID-436	Retinal non-visual photoreception in space	12 Days,13 Days	Animals	Background: Circadian rhythm disruption occurs during spaceflight, leading to crew health and performance decrements. Spaceflight-related retinal changes, including oxidative stress and neuronal loss, have been previously reported in mice. Methods: Animal tissue from experiments aboard shuttle missions STS-133 (BALB/cJ mice, albino strain) and STS-135 (C57BL mice, pigmented strain), along with ground controls, was examined to determine survival of intrinsically photosensitive retinal ganglion cells (ipRGC) and melanopsin expression in retinas of mice exposed to the spaceflight environment. Real-time qPCR (RTqPCT) and microarray approaches were used to analyze Opn4 (melanopsin) gene expression, while immunohistologic studies were conducted to detect melanopsin localization in the retina. Results: Opn4 expression was decreased in albino BALB/cJ mice exposed to spaceflight, as measured by RTqPCR, but not in C57BL mice samples as analyzed by microarray. Opn4 expression returned to control levels at 7 d postreturn in the BALB/cJ samples. Melanopsin positive RGCs were found in the expected proportion in all samples, except for the BALB/cJ samples at 1 d after flight, where virtually no immunoreactive cells were found. Discussion: Spaceflight environmental factors may affect the nonvisual function of the retina, mediated by a reduction in melanopsin expression and ipRGC survival, contributing to circadian disruption.
Spaceflight Study	Bion-11,Foton-M3	SLID-437	Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity	14 Days,12 Days	Animals	The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.
Spaceflight Study	Bion-11,Foton-M3	SLID-437	Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity	14 Days,12 Days	Animals	The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.
Ground Study	n.a.	SLID-438	Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation	n.a.	Animals	Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (μg) conditions. Scientists on the ground use two models of simulated μg (sμg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true μg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true μg and sμg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sμg models provide an excellent test bed for scientists to develop baseline studies and augment true μg in spaceflight experiments. Ultimately, sμg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.
Spaceflight Study	STS-131	SLID-439	Spaceflight reduces vasoconstrictor responsiveness of skeletal muscle resistance arteries in mice	15 Days	Animals	Cardiovascular adaptations to microgravity undermine the physiological capacity to respond to orthostatic challenges upon return to terrestrial gravity. The purpose of the present study was to investigate the influence of spaceflight on vasoconstrictor and myogenic contractile properties of mouse gastrocnemius muscle resistance arteries. We hypothesized that vasoconstrictor responses acting through adrenergic receptors [norepinephrine (NE)], voltage-gated Ca(2+) channels (KCl), and stretch-activated (myogenic) mechanisms would be diminished following spaceflight. Feed arteries were isolated from gastrocnemius muscles, cannulated on glass micropipettes, and physiologically pressurized for in vitro experimentation. Vasoconstrictor responses to intraluminal pressure changes (0-140 cmH(2)O), KCl (10-100 mM), and NE (10(-9)-10(-4) M) were measured in spaceflown (SF; n = 11) and ground control (GC; n = 11) female C57BL/6 mice. Spaceflight reduced vasoconstrictor responses to KCl and NE; myogenic vasoconstriction was unaffected. The diminished vasoconstrictor responses were associated with lower ryanodine receptor-2 (RyR-2) and ryanodine receptor-3 (RyR-3) mRNA expression, with no difference in sarcoplasmic/endoplasmic Ca(2+) ATPase 2 mRNA expression. Vessel wall thickness and maximal intraluminal diameter were unaffected by spaceflight. The data indicate a deficit in intracellular calcium release via RyR-2 and RyR-3 in smooth muscle cells as the mechanism of reduced contractile activity in skeletal muscle after spaceflight. Furthermore, the results suggest that impaired end-organ vasoconstrictor responsiveness of skeletal muscle resistance arteries contributes to lower peripheral vascular resistance and less tolerance of orthostatic stress in humans after spaceflight.
Spaceflight Study	STS-131	SLID-440	Spaceflight-induced synaptic modifications within hair cells of the mammalian utricle	15 Days	Animals	Exposure to the microgravity conditions of spaceflight alleviates the load normally imposed by the Earth's gravitational field on the inner ear utricular epithelia. Previous ultrastructural investigations have shown that spaceflight induces an increase in synapse density within hair cells of the rat utricle. However, the utricle exhibits broad physiological heterogeneity across different epithelial regions, and it is unknown whether capabilities for synaptic plasticity generalize to hair cells across its topography. To achieve systematic and broader sampling of the epithelium than was previously conducted, we used immunohistochemistry and volumetric image analyses to quantify synapse distributions across representative utricular regions in specimens from mice exposed to spaceflight (a 15-day mission of the space shuttle Discovery). These measures were compared with similarly sampled Earth-bound controls. Following paraformaldehyde fixation and microdissection, immunohistochemistry was performed on intact specimens to label presynaptic ribbons (anti-CtBP2) and postsynaptic receptor complexes (anti-Shank1A). Synapses were identified as closely apposed pre- and postsynaptic puncta. Epithelia from horizontal semicircular canal cristae served as "within-specimen" controls, whereas utricles and cristae from Earth-bound cohorts served as experimental controls. We found that synapse densities decreased in the medial extrastriolae of microgravity specimens compared with experimental controls, whereas they were unchanged in the striolae and horizontal cristae from the two conditions. These data demonstrate that structural plasticity was topographically localized to the utricular region that encodes very low frequency and static changes in linear acceleration, and illuminates the remarkable capabilities of utricular hair cells for synaptic plasticity in adapting to novel gravitational environments.NEW & NOTEWORTHY Spaceflight imposes a radically different sensory environment from that in which the inner ear utricle normally operates. We investigated synaptic modifications in utricles from mice flown aboard a space shuttle mission. Structural synaptic plasticity was detected in the medial extrastriola, a region associated with encoding static head position, as decreased synapse density. These results are remarkably congruent with a recent report of decreased utricular function in astronauts immediately after returning from the International Space Station.
Spaceflight Study	Shuttle	SLID-441	Toll mediated infection response is altered by gravity and spaceflight in Drosophila	n.a.	Animals	Space travel presents unlimited opportunities for exploration and discovery, but requires better understanding of the biological consequences of long-term exposure to spaceflight. Immune function in particular is relevant for space travel. Human immune responses are weakened in space, with increased vulnerability to opportunistic infections and immune-related conditions. In addition, microorganisms can become more virulent in space, causing further challenges to health. To understand these issues better and to contribute to design of effective countermeasures, we used the Drosophila model of innate immunity to study immune responses in both hypergravity and spaceflight. Focusing on infections mediated through the conserved Toll and Imd signaling pathways, we found that hypergravity improves resistance to Toll-mediated fungal infections except in a known gravitaxis mutant of the yuri gagarin gene. These results led to the first spaceflight project on Drosophila immunity, in which flies that developed to adulthood in microgravity were assessed for immune responses by transcription profiling on return to Earth. Spaceflight alone altered transcription, producing activation of the heat shock stress system. Space flies subsequently infected by fungus failed to activate the Toll pathway. In contrast, bacterial infection produced normal activation of the Imd pathway. We speculate on possible linkage between functional Toll signaling and the heat shock chaperone system. Our major findings are that hypergravity and spaceflight have opposing effects, and that spaceflight produces stress-related transcriptional responses and results in a specific inability to mount a Toll-mediated infection response.
Spaceflight Study	n.a.	SLID-442	Effects of Space Flight on the Chemical Constituents and Anti-Inflammatory Activity of Licorice (Glycyrrhiza uralensis Fisch)	18 Days	Seed	Licorice, the oldest Chinese traditional medicine, is widely used in the treatment of human diseases. Due to the deficiency of wild resource, selecting and breeding becomes a key issue to expanding the supply of licorice. Spaceflight technology will become a new method for medicinal plants. The aim of this study was to investigate the effect of spaceflight on the components and anti-inflammatory activity in licorice. After flowing on a recoverable satellite for 18 days, licorice seeds were germinated and grown to maturity and the parallel ground-based seeds were also planted under the same conditions. The main components in licorice root were analyzed through HPLC. The contents of two components in spaceflight groups were higher than that of the ground control ones. Three acute inflammatory models including xylene-induced auricular edema, carrageenan-induced paw edema and acetic acid-induced vascular permeability were utilized to compare the anti-inflammatory activity of licorice pre and post spaceflight. The licorice extract showed the significant anti-inflammation activity. After the spaceflight, the pharmacological activity of licorice got higher than that of the ground control one. All of the models gained the tendency that the spaceflight group of species Hangjinqi had the strongest activity than other groups. The research provided the scientific data for a new breeding of medicinal plant through the spaceflight and indicated that the technology of space flight may be a new effective method for the breeding and cultivation of licorice.
Spaceflight Study	International Space Station (ISS)	SLID-443	Effect of Spaceflight on Tomato Seed Quality and Biochemical Characteristics of Mature Plants	6 months	Fruit	Intensive space exploration includes profound investigations on the effect of weightlessness and cosmic radiation on plant growth and development. Tomato seeds are often used in such experiments though up to date the results have given rather vague information about biochemical changes in mature plants grown from seeds subjected to spaceflight. The effect of half a year of storage in the International Space Station (ISS) on tomato seeds (cultivar Podmoskovny ranny) was studied by analyzing the biochemical characteristics and mineral content of mature plants grown from these seeds both in greenhouse and field conditions. A significant increase was recorded in ascorbic acid, polyphenol and carotenoid contents, and total antioxidant activity (AOA), with higher changes in the field conditions compared to greenhouse. Contrary to control plants, the ones derived from space-stored seeds demonstrated a significant decrease in root AOA. The latter plants also showed a higher yield, but lower content of fruit dry matter, sugars, total dissolved solids and organic acids. The fruits of plants derived from space-stored seeds demonstrated decreased levels of Fe, Cu and taste index. The described results reflect the existence of oxidative stress in mature tomato plants as a long-term consequence of the effect of spaceflight on seed quality, whereas the higher yield may be attributed to genetic modifications.
Spaceflight Study	Tiangong-2 Space Flight	SLID-444	Research on lettuce growth technology onboard Chinese Tiangong II Spacelab	27 Days	Vegetable	Lettuce was grown in a space vegetable cultivation facility onboard the Tiangong Ⅱ Spacelab during October 18 to November 15, 2016, in order to testify the key cultivating technology in CELSS under spaceflight microgravity condition. Potable water was used for irrigation of rooting substrate and the SRF (slowly released fertilizer) offered mineral nutrition for plant growth. Water content and electric conductivity in rooting substrate were measured based on FDR(frequency domain reflectometry) principle applied first in spaceflight. Lettuce germinated with comparative growth vigor as the ground control, showing that the plants appeared to be not stressed by the spaceflight environment. Under microgravity, lettuce grew taller and showed deeper green color than the ground control. In addition, the phototropism of the on-orbit plants was more remarkable. The nearly 30-d spaceflight test verified the seed fixation technology and water& nutrition management technology, which manifests the feasibility of FDR being used for measuring moisture content and electric conductivity in rooting zone under microgravity. Furthermore, the edibility of the space-grown vegetable was proved, providing theoretical support for astronaut to consume the space vegetable in future manned spaceflight.
Spaceflight Study	International Space Station (ISS)	SLID-445	International Space Station environmental microbiome-microbial inventories of ISS filter debris	n.a.	Bacteria	Despite an expanding array of molecular approaches for detecting microorganisms in a given sample, rapid and robust means of assessing the differential viability of the microbial cells, as a function of phylogenetic lineage, remain elusive. A propidium monoazide (PMA) treatment coupled with downstream quantitative polymerase chain reaction (qPCR) and pyrosequencing analyses was carried out to better understand the frequency, diversity, and distribution of viable microorganisms associated with debris collected from the crew quarters of the International Space Station (ISS). The cultured bacterial counts were more in the ISS samples than cultured fungal population. The rapid molecular analyses targeted to estimate viable population exhibited 5-fold increase in bacterial (qPCR-PMA assay) and 25-fold increase in microbial (adenosine triphosphate assay) burden than the cultured bacterial population. The ribosomal nucleic acid-based identification of cultivated strains revealed the presence of only four to eight bacterial species in the ISS samples, however, the viable bacterial diversity detected by the PMA-pyrosequencing method was far more diverse (12 to 23 bacterial taxa) with the majority consisting of members of actinobacterial genera (Propionibacterium, Corynebacterium) and Staphylococcus. Sample fractions not treated with PMA (inclusive of both live and dead cells) yielded a great abundance of highly diverse bacterial (94 to 118 taxa) and fungal lineages (41 taxa). Even though deep sequencing capability of the molecular analysis widened the understanding about the microbial diversity, the cultivation assay also proved to be essential since some of the spore-forming microorganisms were detected only by the culture-based method. Presented here are the findings of the first comprehensive effort to assess the viability of microbial cells associated with ISS surfaces, and correlate differential viability with phylogenetic affiliation.
Spaceflight Study	International Space Station (ISS)	SLID-446	Four-year bacterial monitoring in the International Space Station-Japanese Experiment Module "Kibo" with culture-independent approach	4 Years	Bacteria	Studies on the relationships between humans and microbes in space habitation environments are critical for success in long-duration space missions, to reduce potential hazards to the crew and the spacecraft infrastructure. We performed microbial monitoring in the Japanese Experiment Module “Kibo”, a part of the International Space Station, for 4 years after its completion, and analyzed samples with modern molecular microbiological techniques. Sampling was performed in September 2009, February 2011, and October 2012. The surface of the incubator, inside the door of the incubator, an air intake, air diffuser, and handrail were selected as sampling sites. Sampling was performed using the optimized swabbing method. Abundance and phylogenetic affiliation of bacteria on the interior surfaces of Kibo were determined by quantitative PCR and pyrosequencing, respectively. Bacteria in the phyla Proteobacteria (γ-subclass) and Firmicutes were frequently detected on the interior surfaces in Kibo. Families Staphylococcaceae and Enterobacteriaceae were dominant. Most bacteria detected belonged to the human microbiota; thus, we suggest that bacterial cells are transferred to the surfaces in Kibo from the astronauts. Environmental bacteria such as Legionella spp. were also detected. From the data on bacterial abundance and phylogenetic affiliation, Kibo has been microbiologically well maintained; however, the microbial community structure in Kibo may change with prolonged stay of astronauts. Continuous monitoring is required to obtain information on changes in the microbial community structure in Kibo.
Spaceflight Study	International Space Station (ISS)	SLID-447	Survey of environmental biocontamination on board the International Space Station	6 Years	Bacteria	The International Space Station (ISS) is an orbital living and working environment extending from the original Zarya control module built in 1998. The expected life span of the completed station is around 10 years and during this period it will be constantly manned. It is inevitable that the ISS will also be home to an unknown number of microorganisms. This survey reports on microbiological contamination in potable water, air, and on surfaces inside the ISS. The viable counts in potable water did not exceed 1.0 x 10(2) CFU/ml. Sphingomonas sp. and Methylobacterium sp. were identified as the dominant genera. Molecular analysis demonstrated the presence of nucleic acids belonging to various pathogens, but no viable pathogens were recovered. More than 500 samples were collected at different locations over a period of 6 years to characterize air and surface contamination in the ISS. Concentrations of airborne bacteria and fungi were lower than 7.1 x 10(2) and 4.4 x 10(1) CFU/m3, respectively. Staphylococcus sp. was by far the most dominant airborne bacterial genus, whereas Aspergillus sp. and Penicillium sp. dominated the fungal population. The bacterial concentrations in surface samples fluctuated from 2.5 x 10(1) to 4.3 x 10(4) CFU/100 cm2. Staphylococcus sp. dominated in all of these samples. The number of fungi varied between 2.5 x 10(1) and 3.0 x 10(5) CFU/100 cm2, with Aspergillus sp. and Cladosporium sp. as the most dominant genera. Furthermore, the investigations identified the presence of several (opportunistic) pathogens and strains involved in the biodegradation of structural materials.
Spaceflight Study	International Space Station (ISS)	SLID-448	Bacterial monitoring with adhesive sheet in the international space station-"Kibo", the Japanese experiment module	n.a.	Bacteria	Microbiological monitoring is important to assure microbiological safety, especially in long-duration space habitation. We have been continuously monitoring the abundance and diversity of bacteria in the International Space Station (ISS)-“Kibo” module to accumulate knowledge on microbes in the ISS. In this study, we used a new sampling device, a microbe-collecting adhesive sheet developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. We first validated the adhesive sheet as a sampling device to be used in a space habitat with regard to the stability of the bacterial number on the sheet during prolonged storage of up to 12 months. Bacterial abundance on the surfaces in Kibo was then determined and was lower than on the surfaces in our laboratory (105 cells [cm2]-1), except for the return air grill, and the bacteria detected in Kibo were human skin microflora. From these aspects of microbial abundance and their phylogenetic affiliation, we concluded that Kibo has been microbiologically well maintained; however, microbial abundance may increase with the prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in Kibo is required.
Spaceflight Study	STS-87	SLID-449	Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings	16 Days	Grain	In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.
Spaceflight Study	STS-73,STS-63,STS-87	SLID-450	Composition and physical properties of starch in microgravity-grown plants	16 Days,5 Days,6 Days	Grain	The effect of spaceflight on starch development in soybean (Glycine max L., BRIC-03) and potato (Solanum tuberosum, Astroculture-05) was compared with ground controls by biophysical and biochemical measurements. Starch grains from plants from both flights were on average 20-50% smaller in diameter than ground controls. The ratio delta X/delta rho (delta X --difference of magnetic susceptibilities, delta rho--difference of densities between starch and water) of starch grains was ca. 15% and 4% higher for space-grown soybean cotyledons and potato tubers, respectively, than in corresponding ground controls. Since the densities of particles were similar for all samples (1.36 to 1.38 g/cm3), the observed difference in delta X/delta rho was due to different magnetic susceptibilities and indicates modified composition of starch grains. In starch preparations from soybean cotyledons (BRIC-03) subjected to controlled enzymatic degradation with alpha-amylase for 24 hours, 77 +/- 6% of the starch from the flight cotyledons was degraded compared to 58 +/- 12% in ground controls. The amylose content in starch was also higher in space-grown tissues. The good correlation between the amylose content and delta X/delta rho suggests, that the magnetic susceptibility of starch grains is related to their amylose content. Since the seedlings from the BRIC-03 experiment showed elevated post-flight ethylene levels, material from another flight experiment (GENEX) which had normal levels of ethylene was examined and showed no difference to ground controls in size distribution, density, delta X/delta rho and amylose content. Therefore the role of ethylene appears to be more important for changes in starch metabolism than microgravity.
Spaceflight Study	STS-73,STS-63,STS-87	SLID-450	Composition and physical properties of starch in microgravity-grown plants	16 Days,5 Days,6 Days	Grain	The effect of spaceflight on starch development in soybean (Glycine max L., BRIC-03) and potato (Solanum tuberosum, Astroculture-05) was compared with ground controls by biophysical and biochemical measurements. Starch grains from plants from both flights were on average 20-50% smaller in diameter than ground controls. The ratio delta X/delta rho (delta X --difference of magnetic susceptibilities, delta rho--difference of densities between starch and water) of starch grains was ca. 15% and 4% higher for space-grown soybean cotyledons and potato tubers, respectively, than in corresponding ground controls. Since the densities of particles were similar for all samples (1.36 to 1.38 g/cm3), the observed difference in delta X/delta rho was due to different magnetic susceptibilities and indicates modified composition of starch grains. In starch preparations from soybean cotyledons (BRIC-03) subjected to controlled enzymatic degradation with alpha-amylase for 24 hours, 77 +/- 6% of the starch from the flight cotyledons was degraded compared to 58 +/- 12% in ground controls. The amylose content in starch was also higher in space-grown tissues. The good correlation between the amylose content and delta X/delta rho suggests, that the magnetic susceptibility of starch grains is related to their amylose content. Since the seedlings from the BRIC-03 experiment showed elevated post-flight ethylene levels, material from another flight experiment (GENEX) which had normal levels of ethylene was examined and showed no difference to ground controls in size distribution, density, delta X/delta rho and amylose content. Therefore the role of ethylene appears to be more important for changes in starch metabolism than microgravity.
Spaceflight Study	n.a.	SLID-451	Influence of Microgravity Environment on Root Growth, Soluble Sugars, and Starch Concentration of Sweetpotato Stem Cuttings	5 Days	Grain	Because sweetpotato [Ipomoea batatas (L.) Lam.] stem cuttings regenerate very easily and quickly, a study of their early growth and development in microgravity could be useful to an understanding of morphological changes that might occur under such conditions for crops that are propagated vegetatively. An experiment was conducted aboard a U.S. Space Shuttle to investigate the impact of microgravity on root growth, distribution of amyloplasts in the root cells, and on the concentration of soluble sugars and starch in the stems of sweetpotatoes. Twelve stem cuttings of ‘Whatley/Loretan’ sweetpotato (5 cm long) with three to four nodes were grown in each of two plant growth units filled with a nutrient agarose medium impregnated with a half-strength Hoagland solution. One plant growth unit was flown on Space Shuttle Colombia for 5 days, whereas the other remained on the ground as a control. The cuttings were received within 2 h postflight and, along with ground controls, processed in ≈45 min. Adventitious roots were counted, measured, and fixed for electron microscopy and stems frozen for starch and sugar assays. Air samples were collected from the headspace of each plant growth unit for postflight determination of carbon dioxide, oxygen, and ethylene levels. All stem cuttings produced adventitious roots and growth was quite vigorous in both ground-based and flight samples and, except for a slight browning of some root tips in the flight samples, all stem cuttings appeared normal. The roots on the flight cuttings tended to grow in random directions. Also, stem cuttings grown in microgravity had more roots and greater total root length than ground-based controls. Amyloplasts in root cap cells of ground-based controls were evenly sedimented toward one end compared with a more random distribution in the flight samples. The concentration of soluble sugars, glucose, fructose, and sucrose and total starch concentration were all substantially greater in the stems of flight samples than those found in the ground-based samples. Carbon dioxide levels were 50% greater and oxygen marginally lower in the flight plants, whereas ethylene levels were similar and averaged less than 10 nL·L −1. Despite the greater accumulation of carbohydrates in the stems, and greater root growth in the flight cuttings, overall results showed minimal differences in cell development between space flight and ground-based tissues. This suggests that the space flight environment did not adversely impact sweetpotato metabolism and that vegetative cuttings should be an acceptable approach for propagating sweetpotato plants for space applications.
Spaceflight Study	Mir Space Station	SLID-452	From seed-to-seed experiment with wheat plants under space-flight conditions	167 Days	Grain	An important goal with plant experiments in microgravity is to achieve a complete life cycle, the "seed-to-seed experiment." Some Soviet attempts to reach this goal are described, notably an experiment with the tiny mustard, Arabidopsis thaliana, in the Phyton 3 device on Salyut 7. Normal seeds were produced although yields were reduced and development was delayed. Several other experiments have shown abnormalities in plants grown in space. In recent work, plants of wheat (Triticum aestivum) were studied on the ground and then in a preliminary experiment in space. Biometric indices of vegetative space plants were 2 to 2.5 times lower than those of controls, levels of chlorophyll a and b were reduced (no change in the ratio of the two pigments), carotenoids were reduced, there was a serious imbalance in major minerals, and membrane lipids were reduced (no obvious change in lipid patterns). Following the preliminary studies, an attempt was made with the Svetoblock-M growth unit to grow a super-dwarf wheat cultivar through a life cycle. The experiment lasted 167 d on Mir. Growth halted from about day 40 to day 100, when new shoots appeared. Three heads had appeared in the boot (surrounded by leaves) when plants were returned to earth. One head was sterile, but 28 seeds matured on earth, and most of these have since produced normal plants and seeds. In principle, a seed-to-seed experiment with wheat should be successful in microgravity.
Spaceflight Study	SJ-10 Satellite	SLID-453	Metabolomics Analysis in Different Development Stages on SP0 Generation of Rice Seeds After Spaceflight	12 Days	Seed	Spaceflight is a special abiotic stress condition. In recent years, it has been confirmed that the spaceflight caused the stress response of rice seeds, and the protein level, transcription level, and methylation level will change during the planting process after returning to the ground. However, the changes at the metabolome level are not very clear. In this study, two kinds of rice seeds, Dongnong423 (DN3) and Dongnong416 (DN6), were carried on the ShiJian-10 retractable satellite (SJ-10) for 12.5 days in orbit, returned to the ground and planted in the field until the three-leaf (TLP) and tillering stage (TS). The results of antioxidant enzyme activity, soluble sugar, and electron leakage rate revealed that the spaceflight caused the stress response of rice. The TLP and TS of DN3 identified 110 and 57 different metabolites, respectively, while the TLP and TS of DN6 identified 104 and 74 different metabolites, respectively. These metabolites included amino acids, sugars, fatty acids, organic acids and secondary metabolites. We used qRT-PCR technology to explore the changes of enzyme genes in the tricarboxylic acid cycle (TCA) and amino acid metabolism pathway. Combined with the results of metabolomics, we determined that during the TLP, the TCA cycle rate of DN3 was inhibited and amino acid metabolism was activated, while the TCA cycle rate of DN6 was activated and amino acid metabolism was inhibited. In TS, the TCA cycle rate of DN3 was inhibited, and amino acid metabolism was not significantly changed, while the TCA cycle rate of DN6 was activated and amino acid metabolism was inhibited. These results suggested that the response mechanisms of the two different rice strains to spaceflight stress are different, and these differences may be reflected in energy consumption and compound biosynthesis of rice in different growth and development stages. This study provided new insights for further exploring the effects of spaceflight.
Spaceflight Study	SJ-10 Satellite	SLID-454	The Memory of Rice Response to Spaceflight Stress: From the Perspective of Metabolomics and Proteomics	12 Days	Seed	The stress response of plants to spaceflight has been confirmed in contemporary plants, and plants retained the memory of spaceflight through methylation reaction. However, how the progeny plants adapt to this cross-generational stress memory was rarely reported. Here, we used the ShiJian-10 retractable satellite carrying Dongnong416 rice seeds for a 12.5-day on-orbit flight and planted the F2 generation after returning to the ground. We evaluated the agronomic traits of the F2 generation plants and found that the F2 generation plants had no significant differences in plant height and number of tillers. Next, the redox state in F2 plants was evaluated, and it was found that the spaceflight broke the redox state of the F2 generation rice. In order to further illustrate the stress response caused by this redox state imbalance, we conducted proteomics and metabolomics analysis. Proteomics results showed that the redox process in F2 rice interacts with signal transduction, stress response, and other pathways, causing genome instability in the plant, leading to transcription, post-transcriptional modification, protein synthesis, protein modification, and degradation processes were suppressed. The metabolomics results showed that the metabolism of the F2 generation plants was reshaped. These metabolic pathways mainly included amino acid metabolism, sugar metabolism, cofactor and vitamin metabolism, purine metabolism, phenylpropane biosynthesis, and flavonoid metabolism. These metabolic pathways constituted a new metabolic network. This study confirmed that spaceflight affected the metabolic changes in offspring rice, which would help better understand the adaptation mechanism of plants to the space environment.
Spaceflight Study	SJ-10 Satellite	SLID-455	Combining Proteomics and Metabolomics to Analyze the Effects of Spaceflight on Rice Progeny	12 Days	Seed	Spaceflight is a special abiotic stress, the biological effect mechanism of which on contemporary rice has been clarified, However, its effect on offspring rice was still unclear. In order to understand the response mechanism of F2 generation plants to space flight, this study used SJ-10 recoverable satellite to carry DN423 rice seeds for 12.5 days in orbit flight. After returning to the ground, the plants were then planted to F2 generation to explore the biological effect mechanism. Our research showed that in the F2 generation of TLS, the rice plant height of the space flight group increased by 33.8%, the ear length and thousand-grain weight decreased by 9.7 and 4.6%, respectively, and the grain number per panicle increased by 6.5%. Moreover, related proteins that control changes in agronomic traits have been identified. The changes of MDA, H2O2, soluble sugar, electron leakage and antioxidant enzyme activity confirmed the stress response in F2 generation plants. ITRAQ and LC-MS technology were used to reveal the change pattern of protein levels and metabolite levels in F2 generation plants, 389 and 405 proteins were identified as differentially abundant proteins in TLS and TS, respectively. In addition, there were 124 and 125 metabolites that changed during these two periods. The proteome and metabolome result further confirmed that the F2 generation plants still retained the memory of space flight stress, and retained the memory of space flight stress through genome instability. Oxidative stress signals activated sugar signals to rebuild metabolic networks to adapt to space flight stress. The reconstruction of energy metabolism, amino acid metabolism, phenylalanine metabolism, and flavonoid metabolism played an important role in the process of adapting to space flight stress. The results of this study broaden the perspective of space biological effects and provide a basis for studying the effects of abiotic stress on plant progeny.
Spaceflight Study	Shenzhou 5 Space Flight,Shenzhou 6 Space Flight	SLID-456	Molecular Variation and Application from Aerospace Mutagenesis in Upland Rice Huhan 3 and Huhan 7	n.a.	Seed	To further improve upland rice varieties Huhan 3 and Huhan 7,seed samples were sent to outer space with two recoverable spaceships for approximately 1 and 5 d and were propagated for 7 and 5 generations,respectively.Phenotypic analysis revealed that the morphological traits and the protein and amylose contents of grains changed.Characterization of genomic mutations by the gene-associated simple sequence repeat (SSR) and insertion-delete (InDel) markers indicated that the mutation pattern was very complex.Most of the mutations occurred at the 3′or 5′-end of the fragments in the simple sequence repeat fragment.Reverse transcription-polymerase chain reaction (RT-PCR) assay showed that mutations in those parts of the SSR affected their gene expression,indicating that gene associated markers would be helpful to isolate functional genes.Field survey for breeding also revealed that more lines with high yield,high quality and drought-tolerance could be selected through aerospace breeding.The results indicate that aerospace mutagenesis resulted in molecular variation,as well as physiological and morphological changes for rice breeding.
Spaceflight Study	Space Shuttle Challenger	SLID-457	Germination, growth rates, and electron microscope analysis of tomato seeds flown on the LDEF	69 Months	Fruit	The purpose of the experiment was to determine cosmic rays long-term effects on living tissue. A batch of tomato seeds were flown in orbit aboard the Long Duration Exposure Facility (LDEF) for almost 6 y. During this time, the seeds received an abundant exposure to cosmic radiation. Upon the return of the LDEF to Earth, the seeds were distributed throughout the United States and 30 foreign countries for analysis. Our university analysis included germination and growth rates as well as scanning electron microscopy (SEM) and X-ray analysis of the control as well as space exposed tomato seeds. In analyzing the seeds under the electron microscope, usual observations were performed on the nutritional and epidermis layer of the seed. These layers appeared to be more porous in the space exposed seeds than on Earth-based control seeds. This unusual characteristic may explain the increases in the space seeds growth pattern. (Several test results showed that the space-exposed seeds germinated sooner than Earth-based seeds. Also, the space-exposed seeds grew at a faster rate.) The porous nutritional region may allow the seeds to receive necessary nutrients and liquids more readily, thus enabling the plant to grow at a faster rate. Roots, leaves and stems were cut into small sections and mounted. After sputter coating the specimens with argon/gold palladium plasma, they were viewed under the electron microscope. Many micrographs were taken. The X-ray analysis displayed possible identifications of calcium, potassium, chlorine, copper, aluminum, silicon, phosphate, carbon, and sometimes sulfur and iron. The highest concentrations were shown in potassium and calcium. As a result of the electron interaction and X-ray production within the open seeds, the traditional layers of the space-exposed seed gave peaks of Mg, P and S, while the Earth seed gave an iron peak, which was not detected in the space-exposed seed because of electron beam positioning difference. The space-exposed seed and the Earth-control seed specimens displayed high concentrations of copper.
Spaceflight Study	Chinese spacelab TG-2	SLID-458	Molecular Basis to Integrate Microgravity Signals into the Photoperiodic Flowering Pathway in Arabidopsis thaliana under Spaceflight Condition	n.a.	Seed to mature plant	Understanding the effects of spaceflight on plant flowering regulation is important to setup a life support system for long-term human space exploration. However, the way in which plant flowering is affected by spaceflight remains unclear. Here, we present results from our latest space experiments on the Chinese spacelab Tiangong-2, in which Arabidopsis wild-type and transgenic plants pFT::GFP germinated and grew as normally as their controls on the ground, but the floral initiation under the long-day condition in space was about 20 days later than their controls on the ground. Time-course series of digital images of pFT::GFP plants showed that the expression rhythm of FT in space did not change, but the peak appeared later in comparison with those of their controls on the ground. Whole-genome microarray analysis revealed that approximately 16% of Arabidopsis genes at the flowering stage changed their transcript levels under spaceflight conditions in comparison with their controls on the ground. The GO terms were enriched in DEGs with up-regulation of the response to temperature, wounding, and protein stabilization and down-regulation of the function in circadian rhythm, gibberellins, and mRNA processes. FT and SOC1 could act as hubs to integrate spaceflight stress signals into the photoperiodic flowering pathway in Arabidopsis in space.
Spaceflight Study	SJ-10 Satellite	SLID-459	Transcriptomic Analysis of the Interaction Between FLOWERING LOCUS T Induction and Photoperiodic Signaling in Response to Spaceflight	12 Days	Seed to mature plant	Spaceflight has an impact on the growth and development of higher plants at both the vegetative stage and reproductive stage. A great deal of information has been available on the vegetative stage in space, but relatively little is known about the influence of spaceflight on plants at the reproductive stage. In this study, we constructed transgenic Arabidopsis thaliana plants expressing the flowering control gene, FLOWERING LOCUS T (FT), together with the green fluorescent protein gene (GFP) under control of a heat shock-inducible promoter (HSP17.4), by which we induced FT expression inflight through remote controlling heat shock (HS) treatment. Inflight photography data showed that induction of FT expression in transgenic plants in space under non-inductive short-day conditions could promote flowering and reduce the length of the inflorescence stem in comparison with that of wild-type plants under the same conditions. Whole-genome microarray analysis of gene expression changes in leaves of wild-type and these transgenic plants grown under the long-day and short-day photoperiod conditions in space indicated that the function of the photoperiod-related spaceflight responsive genes is mainly involved in protein synthesis and post-translation protein modulation, notably protein phosphorylation. In addition, changes of the circadian component of gene expression in response to spaceflight under different photoperiods indicated that roles of the circadian oscillator could act as integrators of spaceflight response and photoperiodic signals in Arabidopsis plants grown in space.
Spaceflight Study	Mir Space Station	SLID-460	Human thermohomeostasis onboard “Mir” and in simulated microgravity studies	174 Days	Thermoregulatory response	Significant changes of thermogomeostatic parameters was obtained by thermotopometric method using the techniques simulate of microgravity effects: bed rest, pressurized isolation, suit immersion (SI). However, each of ground models made rectal temperature (T) trend downward. The autothermometric study (24 and 12 sessions, 2–13th and 6– 174th flight days) was carried out onboard “Mir” by two flight engineers who had preliminary tested at SI (1–2 days). Studies of German investigators onboard “Mir” confirmed: rectal T must be higher in space flight as compared to the normal environment (n=4). Comparative studies suggest that microgravity is a key factor for the human body surface T raise and abolishment of the external/internal T-gradient. T-homeostasis was not really changing during missions and could be regarded as acute effect of microgravity. After delineation of changes in body surface T — by Carnot's thermodynamic law — rectal T raise should have been anticipated. Facts pointing to the excess entropy of human body must not be passed over.
Spaceflight Study	Mir Space Station	SLID-461	Body temperature and thermoregulation during submaximal exercise after 115-day spaceflight	115 Days	Thermoregulatory response	Background: Altered thermoregulation has been reported following spaceflight simulations (bed rest and water immersion) but has never been examined after actual spaceflight.Hypothesis: We tested the null hypothesis that body temperatures and heat loss responses during exercise would be similar before and after spaceflight.Methods: Two male crewmembers of the 115-d Mir 18 mission performed supine submaximal cycle exercise (20 min at 40% and 20 min at 65% of preflight VO2peak) once at 145-146 d preflight and once at 5 d postflight (R + 5).Results: After flight neither crewmember could complete the exercise protocol, stopping after 28-29 min. The core temperature (Tin, ingestible telemetry pill) at test termination was similar (37.8 degrees C for both subjects) pre- and postflight despite shorter postflight test duration. The slopes of the skin blood flow (laser Doppler)/Tin relationship (subject 1: 396 vs. 214; subject 2: 704 vs. 143% change Perfusion Unit/degree C), and the sweating rate (dew point hygrometry)/Tin relationship (subject 1: 6.3 vs. 2.0; subject 2: 4.6 vs. 0.7 mg.min-1.cm-2.degree C-1), were both reduced postflight without appreciable change in the Tin thresholds for sweating or skin blood flow.Conclusion: In this preliminary report for two crewmembers, the sensitivity of the heat loss responses were reduced after long-duration spaceflight, resulting in a faster rate of rise in core temperature.

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