Defining the resilience of the human salivary microbiota by a 520-day longitudinal study in a confined environment: the Mars500 mission.

BACKGROUND: The human microbiota plays several roles in health and disease but is often difficult to determine which part is in intimate relationships with the host vs. the occasional presence. During the Mars500 mission, six crewmembers lived completely isolated from the outer world for 520 days following standardized diet regimes. The mission constitutes the first spaceflight simulation to Mars and was a unique experiment to determine, in a longitudinal study design, the composition and importance of the resident vs. a more variable microbiota-the fraction of the human microbiota that changes in time and according to environmental conditions-in humans. METHODS: Here, we report the characterization of the salivary microbiota from 88 samples taken during and after Mars500 mission for a total of 720 days. Amplicon sequencing of the V3-V4 regions of 16S rRNA gene was performed, and results were analyzed monitoring the diversity of the microbiota while evaluating the effect of the three main variables present in the experimental system: time, diet, and individuality of each subject. RESULTS: Results showed statistically significant effects for either time, diet, and individuality of each subject. The main contribution came from the individuality of each subject, emphasizing salivary microbiota-personalized features, and an individual-based resilience of the microbiota. CONCLUSIONS: The uniqueness of Mars500 mission, allowed to dampen the effect of environmental variables on salivary microbiota, highlighting its pronounced personalization even after sharing the same physical space for more than a year. Video abstract.

A Longitudinal Epigenetic Aging and Leukocyte Analysis of Simulated Space Travel: The Mars-500 Mission.

Astronauts undertaking long-duration space missions may be vulnerable to unique stressors that can impact human aging. Nevertheless, few studies have examined the relationship of mission duration with DNA-methylation-based biomarkers of aging in astronauts. Using data from the six participants of the Mars-500 mission, a high-fidelity 520-day ground simulation experiment, we tested relationships of mission duration with five longitudinally measured blood DNA-methylation-based metrics: DNAmGrimAge, DNAmPhenoAge, DNA-methylation-based estimator of telomere length (DNAmTL), mitotic divisions (epigenetic mitotic clock [epiTOC2]), and pace of aging (PoA). We provide evidence that, relative to baseline, mission duration was associated with significant decreases in epigenetic aging. However, only decreases in DNAmPhenoAge remained significant 7 days post-mission. We also observed significant changes in estimated proportions of plasmablasts, CD4T, CD8 naive, and natural killer (NK) cells. Only decreases in NK cells remained significant post-mission. If confirmed more broadly, these findings contribute insights to improve the understanding of the biological aging implications for individuals experiencing long-duration space travel.

Changes of brain DTI in healthy human subjects after 520 days isolation and confinement on a simulated mission to Mars.

INTRODUCTION: Long-term confinement is known to be a stressful experience with multiple psycho-physiological effects. In the MARS500 project, a real-time simulation of a space-flight to Mars conducted in a hermetically isolated habitat, effects of long-term confinement could be investigated in a unique manner. The aim of this study was to evaluate effects of long-term-confinement on brain cytoarchitecture. MATERIAL & METHODS: The participants of the MARS500 project underwent 3T-MR imaging including a dedicated DTI-sequence before the isolation, right after ending of confinement and 6 months after the experiment. Voxelwise statistical analysis of the DTI data was carried out using tract-based-spatial statistics, comparing an age-matched control group. RESULTS: At all three sessions, significant lower fractional anisotropy (FA) than in controls was found in the anterior parts of the callosal body of the participants. Furthermore, after ending of confinement a wide-spread FA reduction could be seen in the right hemisphere culminating in the temporo-parietal-junction-zone. All these areas with decreased FA predominantly showed an elevated radial diffusivity and mean diffusivity while axial diffusivity was less correlated. DISCUSSION: Long-term confinement does have measurable effects on the microstructure of the brain white matter. We assume effects of sensory deprivation to account for the regional FA reductions seen in the right TPJ. The differences in the Corpus callosum were interpreted as due to preliminary conditions, e.g. personality traits or training effects. FA and radial diffusivity were the predominant DTI parameters with significant changes, suggesting underlying processes of myelin plasticity.

Syndrome Differentiation of Chinese Medicine in Mars 500 Long-Term Closed Environment.

OBJECTIVE: To summarize and elucidate the characteristics and evolvement of Chinese medicine (CM) patterns reflecting the physical and mental conditions of participants in the Mars 500 long-term closed environment. METHODS: The DS01-T Digital TCM Four-Diagnostic Instrument and CM Inquiring Diagnostic Questionnaire were used to collect information from 6 participants in the Mars 500 International Joint Research Project, through diagnostic methods of observation, palpation and inquiry according to CM theory. During the 520 days of the experiment, data collection was performed 37 times; a total of over 400 digital images of tongues and facial complexion and over 20,000 data were collected. These data were then analyzed by a team of experts in CM, statistics, and data mining. RESULTS: The CM pattern evolvement of participants in the long-term closed environment followed some common trends. Qi deficiency was the main CM pattern observed, with individual features depending on constitutional differences [manifested in varying degrees of accompanying patterns of Gan (Liver) qi stagnation, Pi (Spleen) deficiency, dampness encumbering, or yin deficiency]. CONCLUSION: The research has verified the effectiveness of CM syndrome differentiation based on the four diagnostic methods, which should serve as a solid foundation for observation, monitoring, and intervention in regard to the health conditions of astronauts in long-term space flights in the future.

Syndrome Differentiation of Chinese Medicine in Mars 500 Long-Term Closed Environment / 中国结合医学杂志

OBJECTIVE@#To summarize and elucidate the characteristics and evolvement of Chinese medicine (CM) patterns reflecting the physical and mental conditions of participants in the Mars 500 long-term closed environment.@*METHODS@#The DS01-T Digital TCM Four-Diagnostic Instrument and CM Inquiring Diagnostic Questionnaire were used to collect information from 6 participants in the Mars 500 International Joint Research Project, through diagnostic methods of observation, palpation and inquiry according to CM theory. During the 520 days of the experiment, data collection was performed 37 times; a total of over 400 digital images of tongues and facial complexion and over 20,000 data were collected. These data were then analyzed by a team of experts in CM, statistics, and data mining.@*RESULTS@#The CM pattern evolvement of participants in the long-term closed environment followed some common trends. Qi deficiency was the main CM pattern observed, with individual features depending on constitutional differences [manifested in varying degrees of accompanying patterns of Gan (Liver) qi stagnation, Pi (Spleen) deficiency, dampness encumbering, or yin deficiency].@*CONCLUSION@#The research has verified the effectiveness of CM syndrome differentiation based on the four diagnostic methods, which should serve as a solid foundation for observation, monitoring, and intervention in regard to the health conditions of astronauts in long-term space flights in the future.

Personalized Epigenome Remodeling Under Biochemical and Psychological Changes During Long-Term Isolation Environment.

It has been reported that several aspects of human health could be disturbed during a long-term isolated environment (for instance, the Mars-500 mission), including psychiatric disorders, circadian disruption, temporal dynamics of gut microbiota, immune responses, and physical-activity-related neuromuscular performance. Nevertheless, the mechanisms underlying these disturbances and the interactions among different aspects of human adaptation to extreme environments remain to be elucidated. Epigenetic features, like DNA methylation, might be a linking mechanism that explains the involvement of environmental factors between the human genome and the outcome of health. We conducted an exploration of personalized longitudinal DNA methylation patterns of the peripheral whole blood cells, profiling six subjects across six sampling points in the Mars-500 mission. Specifically, we developed a Personalized Epigenetic-Phenotype Synchronization Analysis (PeSa) algorithm to explore glucose- and mood-state-synchronized DNA methylation sites, focusing on finding the dynamic associations between epigenetic patterns and phenotypes in each individual, and exploring the underling epigenetic connections between glucose and mood-state disturbance. Results showed that DMPs (differentially methylated-probes) were significantly enriched in pathways related to glucose metabolism (Type II diabetes mellitus pathway), mood state (Long-term depression) and circadian rhythm (Circadian entrainment pathway) during the mission. Furthermore, our data revealed individualized glucose-synchronized and mood-state-synchronized DNA methylation sites, and PTPRN2 was found to be associated with both glucose and mood state disturbances across all six subjects. Our findings suggest that personalized phenotype-synchronized epigenetic features could reflect the effects on the human body, including the disturbances of glucose and mood-states. The association analysis of DNA methylation and phenotypes, like the PeSa analysis, could provide new possibilities in understanding the intrinsic relationship between phenotypic changes of the human body adapting to long-term isolation environmental factors.

Effects of prolonged isolation in a confined space on status of oxidative stress and prothrombogenesis: In preparation for possible future manned space expedition to Mars

@#Introduction: Apart from inflammation and endothelial dysfunction, other key components in the development of atherogenesis include prothrombogenesis and oxidative stress. The effects of long-term confinement and isolation, exposure to radiation and different gravity forces during space travel could potentially increase the long-term risk of atherosclerosis. To the best of our knowledge, this is the first study determining the status of prothrombogenesis and oxidative stress in six cosmonauts subjected to the longest duration of confined isolation period of 520 days in preparation for prospective undetermined manned space travel to Mars. Materials and Methods: This collaborative research between the National Space Agency (ANGKASA), Universiti Teknologi MARA, Malaysia and Institute of Biomedical Problems (IBMP), Russia was conducted at the Russian Academy of Sciences IBMP, Moscow, Russia. Six multi-national cosmonauts were assigned to live in a ground-based confined module for 520 days. Standard exercise and diet regime were instituted throughout the isolation phase. Six age, ethnic and gender-matched healthy, free-living ground controls were recruited in parallel. Serial serum and whole blood were analysed for biomarkers of prothrombogenesis [plasminogen activator inhibitor-1 (PAI-1) and homocysteine] and oxidative stress [oxidised low-density lipoprotein (ox-LDL) and malondialdehyde (MDA)]. Results: There were significantly lower concentrations of PAI-1 and homocysteine in cosmonauts during confinement compared to the controls. There were no significant differences seen in the concentrations of biomarkers of oxidative stress during confinement but there was a significant percentage change increment for serum MDA in cosmonauts. Conclusion: Long-term confinement decreased the risk of prothrombogenesis and this could be attributed to the exercise and diet regime which includes omega-3 fatty acids supplementation given to the crew members during their confinement period. However, oxidative damage could not be excluded and may be attributed to the influence of psychological stress during this prolonged confinement.

Body composition and metabolic changes during a 520-day mission simulation to Mars.

PURPOSE: The "Mars-500 project" allowed to evaluate the changes in psychological/physiological adaptation over a prolonged confinement, in order to gather information for future missions. Here, we evaluated the impact of confinement and isolation on body composition, glucose metabolism/insulin resistance and adipokine levels. METHODS: The "Mars-500 project" consisted of 520 consecutive days of confinement from June 3, 2010 to Nov 4, 2011. The crew was composed of six male subjects (three Russians, two Europeans, and one Chinese) with a median age of 31 years (range 27-38 years). RESULTS: During the 520-day confinement, total body mass and BMI progressively decreased, reaching a significant difference at the end (417 days) of the observation period (- 9.2 and - 5.5%, respectively). Fat mass remained unchanged. A progressive and significant increase of fasting plasma glucose was observed between 249 and 417 days (+ 10/+ 17% vs baseline), with a further increase at the end of confinement (up to + 30%). Median plasma insulin showed a non-significant early increment (60 days; + 86%). Total adiponectin halved (- 47%) 60 days after hatch closure, remaining at this nadir (- 51%) level for a further 60 days. High molecular weight adiponectin remained significantly lower from 60 to 168 days. CONCLUSIONS: Based on these data, countermeasures may be envisioned to balance the potentially harmful effects of prolonged confinement, including a better exercise program, with accurate monitoring of (1) the individual activity and (2) the relationship between body composition and metabolic derangement.

The effect of long-term confinement and the efficacy of exercise countermeasures on muscle strength during a simulated mission to Mars: data from the Mars500 study.

BACKGROUND: Isolation and long duration spaceflight are associated with musculoskeletal deconditioning. Mars500 was a unique, high-fidelity analogue of the psychological challenges of a 520-day manned mission to Mars. We aimed to explore the effect of musculoskeletal deconditioning on three outcome measures: (1) if lower limb muscle strength was reduced during the 520-day isolation; (2) if type I or II muscle fibres were differentially affected; and (3) whether any 70-day exercise interventions prevented any isolation-induced loss of strength. METHODS: Six healthy male subjects (mean ± SEM) (34 ± 3 years; 1.76 ± 0.02 metres; 83.7 ± 4.8 kg) provided written, informed consent to participate. The subjects' maximal voluntary contraction (MVC) was assessed isometrically in the calf (predominantly type I fibres), and maximal voluntary isokinetic force (MVIF) was assessed in the quadriceps/hamstrings (predominantly type II fibres) at 0.2 and 0.4 ms-1 using the Multifunctional Dynamometer for Space (MDS) at 35-day intervals throughout Mars500. Exercise interventions were completed 3-7 days/week throughout the 520-day isolation in a counterbalanced design excluding 142-177 days (rest period) and 251-284 days (simulated Mars landing). Exercise interventions included motorized treadmill running, non-motorized treadmill running, cycle ergometry, elastomer-based resistance exercise, whole-body vibration (WBV), and resistance exercise using MDS. RESULTS: Calf MVC did not reduce across the 520-day isolation and MDS increased strength by 18% compared to before that of 70-day exercise intervention. In contrast, there was a significant bilateral loss of MVIF across the 520 days at both 0.2 ms-1 (R 2 = 0.53; P = 0.001) and 0.4 ms-1 (0.4 ms-1; R 2 = 0.42; P = 0.007). WBV (+ 3.7 and 8.8%) and MDS (+ 4.9 and 5.2%) afforded the best protection against isolation-induced loss of MVIF, although MDS was the only intervention to prevent bilateral loss of calf MVC and leg MVIF at 0.2 and 0.4 ms-1. CONCLUSIONS: Mars500 induced significant loss of quadriceps/hamstrings MVIF but not calf MVC. Collectively, these data suggest that muscles with predominantly type I fibres were affected less by isolation compared to type II dominant muscles. MDS and WBV afforded the best protection against isolation-induced loss of strength and thus may have virtue in exploration class missions.

Preparing for the crewed Mars journey: microbiota dynamics in the confined Mars500 habitat during simulated Mars flight and landing.

BACKGROUND: The Mars500 project was conceived as the first full duration simulation of a crewed return flight to Mars. For 520 days, six crew members lived confined in a specifically designed spacecraft mock-up. The herein described "MIcrobial ecology of Confined Habitats and humAn health" (MICHA) experiment was implemented to acquire comprehensive microbiota data from this unique, confined manned habitat, to retrieve important information on the occurring microbiota dynamics, the microbial load and diversity in the air and on various surfaces. In total, 360 samples from 20 (9 air, 11 surface) locations were taken at 18 time-points and processed by extensive cultivation, PhyloChip and next generation sequencing (NGS) of 16S rRNA gene amplicons. RESULTS: Cultivation assays revealed a Staphylococcus and Bacillus-dominated microbial community on various surfaces, with an average microbial load that did not exceed the allowed limits for ISS in-flight requirements indicating adequate maintenance of the facility. Areas with high human activity were identified as hotspots for microbial accumulation. Despite substantial fluctuation with respect to microbial diversity and abundance throughout the experiment, the location within the facility and the confinement duration were identified as factors significantly shaping the microbial diversity and composition, with the crew representing the main source for microbial dispersal. Opportunistic pathogens, stress-tolerant or potentially mobile element-bearing microorganisms were predicted to be prevalent throughout the confinement, while the overall microbial diversity dropped significantly over time. CONCLUSIONS: Our findings clearly indicate that under confined conditions, the community structure remains a highly dynamic system which adapts to the prevailing habitat and micro-conditions. Since a sterile environment is not achievable, these dynamics need to be monitored to avoid spreading of highly resistant or potentially pathogenic microorganisms and a potentially harmful decrease of microbial diversity. If necessary, countermeasures are required, to maintain a healthy, diverse balance of beneficial, neutral and opportunistic pathogenic microorganisms. Our results serve as an important data collection for (i) future risk estimations of crewed space flight, (ii) an optimized design and planning of a spacecraft mission and (iii) for the selection of appropriate microbial monitoring approaches and potential countermeasures, to ensure a microbiologically safe space-flight environment.

Temporal dynamics of the gut microbiota in people sharing a confined environment, a 520-day ground-based space simulation, MARS500.

BACKGROUND: The intestinal microbial communities and their temporal dynamics are gaining increasing interest due to the significant implications for human health. Recent studies have shown the dynamic behavior of the gut microbiota in free-living, healthy persons. To date, it is not known whether these dynamics are applicable during prolonged life sharing in a confined and controlled environment. RESULTS: The MARS500 project, the longest ground-based space simulation ever, provided us with a unique opportunity to trace the crew microbiota over 520 days of isolated confinement, such as that faced by astronauts in real long-term interplanetary space flights, and after returning to regular life, for a total of 2 years. According to our data, even under the strictly controlled conditions of an enclosed environment, the human gut microbiota is inherently dynamic, capable of shifting between different steady states, typically with rearrangements of autochthonous members. Notwithstanding a strong individuality in the overall gut microbiota trajectory, some key microbial components showed conserved temporal dynamics, with potential implications for the maintenance of a health-promoting, mutualistic microbiota configuration. CONCLUSIONS: Sharing life in a confined habitat does not affect the resilience of the individual gut microbial ecosystem, even in the long term. However, the temporal dynamics of certain microbiota components should be monitored when programming future mission simulations and real space flights, to prevent breakdowns in the metabolic and immunological homeostasis of the crewmembers.

Spaceflight induced changes in the human proteome.

INTRODUCTION: Spaceflight is one of the most extreme conditions encountered by humans: Individuals are exposed to radiation, microgravity, hypodynamia, and will experience isolation. A better understanding of the molecular processes induced by these factors may allow us to develop personalized countermeasures to minimize risks to astronauts. Areas covered: This review is a summary of literature searches from PubMed, NASA, Roskosmos and the authors' research experiences and opinions. The review covers the available proteomic data on the effects of spaceflight factors on the human body, including both real space missions and ground-based model experiments. Expert commentary: Overall, the authors believe that the present background, methodology and equipment improvements will enhance spaceflight safety and support accumulation of new knowledge on how organisms adapt to extreme conditions.

How stressful are 105 days of isolation? Sleep EEG patterns and tonic cortisol in healthy volunteers simulating manned flight to Mars.

Spaceflights "environment" negatively affects sleep and its functions. Among the different causes promoting sleep alterations, such as circadian rhythms disruption and microgravity, stress is of great interest also for earth-based sleep medicine. This study aims to evaluate the relationships between stress related to social/environmental confinement and sleep in six healthy volunteers involved in the simulation of human flight to Mars (MARS500). Volunteers were sealed in a spaceship simulator for 105 days and studied at 5 specific time-points of the simulation period. Sleep EEG, urinary cortisol (24 h preceding sleep EEG recording) and subjectively perceived stress levels were collected. Cognitive abilities and emotional state were evaluated before and after the simulation. Sleep EEG parameters in the time (latency, duration) and frequency (power and hemispheric lateralization) domains were evaluated. Neither cognitive and emotional functions alterations nor abnormal stress levels were found. Higher cortisol levels were associated to: (i) decrease of sleep duration, increase of arousals, and shortening of REM latency; (ii) reduction of delta power and enhancement of sigma and beta in NREM N3; and (iii) left lateralization of delta activity (NREM and REM) and right lateralization of beta activity (NREM). Stressful conditions, even with cortisol fluctuations in the normal range, alter sleep structure and sleep EEG spectral content, mirroring pathological conditions such as primary insomnia or insomnia associated to depression. Correlations between cortisol fluctuations and sleep changes suggest a covert risk for developing allostatic load, and thus the need to develop ad-hoc countermeasures for preventing sleep alterations in long lasting manned space missions.