NOTE: End date changed to 5/31/2026 per F. Hernandez/ARC (Ed., 7/1/25)

NOTE: End date changed to 5/31/2025 per F. Hernandez/ARC (Ed., 10/22/24)

NOTE: End date changed to 9/30/2024 per F. Hernandez/ARC (Ed., 6/14/24)

NOTE: End date changed to 5/31/2024 per NSSC information (Ed., 3/17/24)

NOTE: End date changed to 5/31/2023 per NSSC information (Ed., 6/15/22)

NOTE: End date changed to 5/31/2022 per NSSC information (Ed., 9/23/21)

NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 7/17/20)

NOTE: End date changed to 5/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/14/19)

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

NOTE: End date changed to 5/31/2026 per F. Hernandez/ARC (Ed., 7/1/25)

NOTE: End date changed to 5/31/2025 per F. Hernandez/ARC (Ed., 10/22/24)

NOTE: End date changed to 9/30/2024 per F. Hernandez/ARC (Ed., 6/14/24)

NOTE: End date changed to 5/31/2024 per NSSC information (Ed., 3/17/24)

NOTE: End date changed to 5/31/2023 per NSSC information (Ed., 6/15/22)

NOTE: End date changed to 5/31/2022 per NSSC information (Ed., 9/23/21)

NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 7/17/20)

NOTE: End date changed to 5/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/14/19)

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

Metagenomic Sequencing Microbiome analysis, using STARMAPs (Similarity Test for Accordant and Reproducible Microbiome Abundance Patterns) (Jiang et al., Microbiome, 2019), focuses on the changes or differences in microbiome composition between groups, rather than the composition of the microbiome itself. This approach is extremely informative for comparisons between experiments, since the composition can differ greatly between cohorts of animals, even from the same source. Using this approach, we have identified flight vs. ground differences reproducible between Space Shuttle mission STS-135, Rodent Research-1 (RR-1), and Rodent Research-7 (RR-7).

Metagenomic sequencing of the ground control re-run (Blue-enriched vs. White light study) has been completed. Fecal samples corresponding to the same timepoints and conditions as the original study were examined. This was done under the supervision of co-PI Stefan Green (Director of Genomics and Microbiome Core Facility, Rush University) using primers and methods comparable to the original study. Overall, the results highlight a lack of any significant effect of the different light conditions on the mice gut microbiomes. Minor effects seen in the PERMANOVA model (and to some extent in the principal component analysis / PCA plots) are most likely attributable to a “founder effect”, as these effects was present even at the baseline. From this preliminary analysis, we feel confident in moving forward with analyzing the RR-7 datasets, and our concerns are allayed regarding the mismatched light spectrum affecting the microbiome.

Evaluation of Activity/Rest Behaviors of Mice. The RR-7 mission included obtaining three 48-hour continuous video recordings at approximately monthly intervals during the mission. We are developing machine-learning assisted scoring to enhance our analysis of this valuable data. However, many behavioral features that would signify sleep or rest cannot be relied upon in a microgravity environment. The obstructed view of animals in the nestbox further complicates interpretation of the video.

Serum ELISA Assays. Assays of the serum levels of the peptide hormone Leptin and the chemokine CINC-1 were completed. Previous data from our laboratory indicates that CINC-1 is elevated by sleep disruption (Bowers et al., 2021). In both strains, the CINC-1 levels increased over time in the blue-enriched light exposed animals, while differences between 25 and 75 days’ exposure were not seen in white light exposed animals. C57BL/6J mice exposed to blue-enriched light had the highest CINC-1 values on average. We will compare whether our mouse grimace score (MGS) results align with these chemokine data in indicating sleep-disturbing effects of blue-enriched light.

NOTE: End date changed to 5/31/2023 per NSSC information (Ed., 6/15/22)

NOTE: End date changed to 5/31/2022 per NSSC information (Ed., 9/23/21)

NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 7/17/20)

NOTE: End date changed to 5/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/14/19)

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

To avoid confounding effects of sequencing done at different times, RNA-seq analysis of tissue samples from the original and the new control groups will be done at the same time. Samples are being prepared for shipment to the NASA GeneLab for this analysis.

Because of pronounced cohort effects on the gut microbiome, shotgun metagenomic sequencing of the microbiome samples from the original study has already been completed. In the past year, extensive analysis of this data has been completed and a manuscript is under preparation. Key findings include: 1) pronounced strain differences in the impact of the spaceflight on both alpha and beta diversity of the gut microbiome and in the changes in community structure over time; and 2) reproduction in RR-7 of the effects of spaceflight on the gut microbiome reported previously in RR-1 and STS-135 (Jiang et al., Microbiome, 2019).

Analysis of video records in the past year has revealed strain differences in behavioral rhythms and in the use of the nestbox/hut within the habitat in flight, compared to ground habitats.

In the coming year, we expect to complete analysis of our samples now that this can be done, including the new control groups.

Reference: Jiang P, Green SJ, Chlipala GE, Turek FW, Vitaterna MH. "Reproducible changes in the gut microbiome suggest a shift in microbial and host metabolism during spaceflight." Microbiome. 2019 Aug 9;7(1):113. https://doi.org/10.1186/s40168-019-0724-4 ; PMID: 31399081; PMCID: PMC6689164

NOTE: End date changed to 5/31/2022 per NSSC information (Ed., 9/23/21)

NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 7/17/20)

NOTE: End date changed to 5/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/14/19)

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

This year, an additional 40 Ground control mice were exposed to the identical ISSES conditions and schedule, as a test of the impact of the habitat light spectrum. This provided a side-by-side comparison of the light spectra of the original Flight ("white") and Ground ("blue-enriched") lighting. All mice completed the study without significant health issues.

In addition, using shotgun metagenomic sequencing, we characterized the microbiome in fecal and environmental samples from the 2018 study animals. The sequencing data were processed using the NASA GeneLab metagenomics processing pipeline ( https://github.com/AstrobioMike/NASA-GeneLab-working/tree/master/Metagenomics/Illumina ), and the taxonomic abundance and microbial gene content data were derived from sequencing reads via both read-alignment-based and assembly-based approaches. Preliminary findings based on only the read-based taxonomic abundance data suggest mouse(host)-strain-dependent changes in the fecal microbiome during spaceflight. Using principal component analysis (PCA) followed by Permutational Multivariate ANOVA (PERMANOVA) on isometric-log-ratio-transformed species abundance data, we found significant differences in the fecal microbiome composition between B6 and C3H mice throughout the mission (P < 0.0001). By mission day 25, clear effects of the habitat (i.e., the ISS hardware and environment used by Flight and Ground groups vs. the standard caging used by Vivarium and Basal groups; P < 0.0001), spaceflight (P < 0.0001), as well as habitat × mouse strain (P = 0.015) and spaceflight × mouse strain (P < 0.0001) were observed. This pattern was persistent till the end of the study (i.e., day 75). These findings are consistent with our hypothesis that the host genetic background influences the effects of the space environment on the gut microbiome.

NOTE: End date changed to 5/31/2021 per F. Hernandez/ARC and NSSC information (Ed., 7/17/20)

NOTE: End date changed to 5/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/14/19)

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

All mice completed the study without significant health issues. Samples were obtained from a total of 80 mice.

In addition, fecal samples, food samples, and environmental swabs were obtained every 2 weeks from the Vivarium, Ground, and Flight conditions. Three 48-hour continuous video recording sessions were obtained from the six cages/habitats of these conditions as well, at approximately monthly intervals.

Advances in the past year:

1) We developed, validated, and applied a new statistical method for comparing microbiome changes, which revealed reproducible spaceflight-induced microbiome changes from RR-1 and STS-135 missions. These findings have been published in Microbiome (Jiang et al., 2019).

2) In collaboration with GeneLab, we validated our specimen processing methods for RR-7 tissues that were preserved in RNA-later.

3) We completed a first-pass analysis of the behaviors of mice inside the ground and the flight habitats.

4) We validated our specimen processing methods for RR-7 microbiome samples and DNA extractions from 381 samples are complete.

Issues encountered in the past year:

1) We learned that the light spectrum of the flight and the ground habitats did not match ; 2) A re-run of the ground control animals including both blue-enriched and white light habitats is planned ; 3) To avoid batch effects, RNA-seq analysis will not be done until samples from these additional control animals can be included ; 4) Metagenomic sequencing for microbiome analysis was delayed by laboratory closures due to COVID-19.

NOTE: End date changed to 5/31/2019 per NSSC information (Ed., 6/7/18)

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

In the past year, we successfully launched and completed data and sample collection from all 80 experimental animals for RR-7. This included sampling both prior to launch and during the duration of the flight. Samples from the ISS were returned to Earth in January 2019, and dissections completed with the Biospecimen Sharing Program in February 2019. Work is now underway to process samples and analyze data.

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

1. Evaluation of fecal samples from the spaceflight reference mission RR-1 has been done. To examine the gastrointestinal bacterial populations, fecal samples from C57BL/6J mice housed onboard International Space Station (ISS) for one month, as well as fecal samples from mice of three control groups (basal = preflight; vivarium = standard housing; ground = habitat and environment matched to flight) were studied. DNA was extracted, and regions of the 16S ribosomal RNA gene were copied and sequenced in order to identify the bacteria types present and their relative abundances. Findings: Substantial differences between housing type were found, i.e., differences between the (basal and vivarium) and (ground and flight) conditions were evident at all taxonomic levels. However, significant differences between the ground control and flight subjects were found, with the flight subjects having higher ratios of the phyla Firmicutes: Bacteroidetes. Firmicutes and Bacteroidetes are the two bacterial phyla that account for the greatest proportion of the gut microbiome. This preliminary study confirms that gut microbiome changes do occur within 1 month of spaceflight, and thus our planned evaluation of the timecourse of these changes (and the effects of host genotype) will be informative.

2. Using the spaceflight analog Hindlimb Unloading (HU) four different inbred strains of mice were evaluated in parallel. This was done to insure that a) strains that were tolerant of this analogous stressor were chosen for flight and b) two strains with contrasting responses to this analogous stressor were chosen to improve the likelihood that our side-by-side strain comparison would be informative. Findings: Four widely-used inbred strains of mice were examined: C57BL/6J, C3H/HeJ, BALB/cJ, and A/J mice. A/J mice were judged to be least tolerant of the analog stress and were removed from consideration. C57BL/6J and BALB/cJ mice exhibited similar patterns of sleep changes in response to HU, while C3H/HeJ mice did not have increased sleep fragmentation as did the other two strains. In addition, C3H/HeJ mice have pineal glands that secrete melatonin, while the other strains have genetic deficiency in melatonin production. For these and other reasons, C57BL/6J and C3H/HeJ were selected for flight.

3. In an effort to optimize the habitat configuration for flight, we considered the balance between providing enrichment huts and providing space for running/climbing/activity. Specifically, we tested the utilization of enrichment huts as a function of the number of mice housed together. Findings: No significant differences between 10 mice provided one hut vs. 10 mice provided two huts were detected in hut occupancy (number of mice in the hut) over time. When two huts were provided, mice preferred to stay in one hut; the second hut was not used for sleeping.

4. To develop an improved method of sample preservation, we tested and validated a minimal dissection and RNA-later stabilization for spaceflight samples. This allows for high RNA Integrity Numbers even from internal structures while reducing crew time demands in dissection. Findings: Three large segments kept at 4 deg for 48 hr in RNA-later before transferring to -80 deg freeze was selected as the method of choice for RR-7.

Two manuscripts are under preparation.

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.

Working with NASA scientists and engineers, our Experiment Requirements Document has been drafted and our experiment is scheduled for flight—RR7—in June 2018. Two inbred strains of mice will be compared at two time points in flight. Additional details have been modified to increase the feasibility of the study.

In consultation with NASA scientists and flight engineers, we have modified our plans for dissection and preservation of tissues for flight. This fixative/stabilization solution enables histology to be done as well as RNA and proteins to be extracted for gene expression analysis or western blots. We currently are working on optimizing our post-storage RNA extraction/processing methods.

However, the impact of the proposed studies extends beyond understanding the mechanisms at play in the unique stresses of spaceflight. The proposed studies will elucidate mechanisms underlying interactions between GI, immune, metabolic, and sleep functions and specifically the role of the microbiota in these interactions. This knowledge has tremendous potential for guiding development of dysbiosis-targeted interventions for disorders in all of these systems.