Background. Microgravity, a Space stressor, is associated with cellular and molecular alterations of the immunological profile that impact on the human body homeostasis, gastro-intestinal (GI) tract included[1]. Noteworthy, the onset, progression, and outcome of inflammatory processes are regulated by specific endogenous bioactive lipids, that can be synthesized by specific fatty acid precursors like arachidonic acid (ARA) and docosahexaenoic acid (DHA). Experiments performed in recent years on primary human immune cells onboard the International Space Station (ISS) – like ROALD [2], RESLEM [3] and SERiSM [4] – and under simulated microgravity conditions[5], demonstrated that metabolism of bioactive lipids and signal transduction thereof are dysregulated in microgravity. Hence, we sought to interrogate whether bioactive lipids may influence human intestinal cells’ response to authentic or simulated microgravity, thus contributing to maintain GI homeostasis. Among the bioactive lipids, endocannabinoids (eCBs) represent strong pro-homeostatic signals [6]. Accordingly, nutraceutical or pharmacological modulators of the receptors that bind such lipids, as well as of their metabolic enzymes, were employed. The goal was to develop potential countermeasures against Space-related human diseases. Methods. Human Caco-2 cells were chosen as a model of intestinal epithelial cells. During ground control studies (1xg) Caco-2 cells were treated with ARA, DHA, and the short chain fatty acids (SCFAs) butyrate (But) and propionate (Pro) [7,8]. In addition, Caco-2 cells were subjected to simulated microgravity (10-5xg) by means of the Rotary Cell Culture System (RCCS) developed by NASA. Western blotting and qPCR were performed to interrogate possible effects on the eCB system (ECS) at a protein and genic level. Results. In the 1xg experiments, the 50% critical micelle concentration (½CMC) of ARA and DHA (30 μM), as well as of But and Pro (2.5 mM) was chosen to treat Caco-2 cells. Briefly, ARA induced a decrease in the expression of the eCB-binding cannabinoid (CB1 and CB2) receptors, whereas DHA downregulated the expression of CB2 only. The SCFAs did not modulate any component of the ECS. Regarding the microgravity studies, Cytodex3 microcarriers were necessary for Caco-2 survival [9,10]. Of note, simulated microgravity downregulated PPARγ gene expression and increased MAGL gene expression. At the protein level, CB1 and CB2 were downregulated. Conclusions. Most of the treatments induced downregulation of eCB-binding receptors at the protein level, with CB1 and CB2 being involved in intestinal motility [11]. These effects, along with the gene and protein expression of the main eCBs metabolic enzymes, will be better elucidated in ongoing experiments.

Introduction/Background: Moon expeditions pose unique challenges due to the lunar environment's characteristics, including lunar dust and reduced gravity. Understanding the impact of lunar dust exposure and lunar gravity on human health is crucial for future missions. This study encapsulates the ongoing research endeavor utilizing an Earth-based Differential Gravity Analog Random Positioning Machine (RPM) to investigate these hazards in simulated lunar conditions. Lunar dust, composed of abrasive and reactive particles, poses considerable health threats, including respiratory issues and potential long-term health implications from inhalation. Moreover, lunar dust's propensity to adhere to surfaces raises concerns about spacecraft contamination and life support system integrity. Additionally, adaptation to lunar gravity, approximately one-sixth of Earth's gravity, presents physiological challenges such as muscle atrophy, bone density loss, and cardiovascular deconditioning.
Method/Experiment: We explored two experimental scenarios: a. In the first set of experiments, we exposed human lung epithelial cells and dermal fibroblasts to lunar dust simulant (JSC-A1). to evaluate their responses. The second experiment was performed with human B cells and to analog lunar gravity using the new RPM 2.0
Results: Analysis of the two sets of experiments revealed several significant findings. Firstly, exposure to lunar dust simulant under reduced gravity conditions led to significant functional deficits in skin fibroblasts, lung epithelial cells and lymphocytes. Secondly, the cells exhibited altered behavior and gene expression patterns when exposed to lunar dust. Separate experiments were conducted with a new apparatus called the RPM 2.0 which has specific algorithms to simulate microgravity, moon, and mars gravity for cell culture. We exposed human B lymphocytes to lunar gravity on the RPM over 72 hours. We harvested the cells and supernatants. We then examined the cytokine expression in lunar gravity exposed cells by Luminex multianalyte profiling. Inflammatory cytokines such as RANTES, VEGF-alpha and TGF-beta were found to be significantly upregulated in lunar gravity cultured B cells. These results indicate that lunar gravity by itself might produce immune function decline in human cells.
Conclusion: Our study highlights the importance of considering lunar dust exposure and reduced gravity impacts in the planning and execution of moon expeditions especially for humans. The findings suggest that lunar dust, and reduced gravity, can affect the function of biological systems. Understanding these effects is critical for designing effective countermeasures and ensuring the success and safety of future lunar missions. Further research is warranted to explore potential mitigation strategies and refine our understanding of lunar environment interactions.

Background
Exposure of the human body to prolonged microgravity causes a significant reduction in skeletal muscle and bone mass, SMB, loss. This negatively affects the physical performance of astronauts during space flights and the process of their adaptation back to life on Earth. Apart from the loss of gravity, the other main factor responsible for SMB loss in microgravity, is space anaemia, when the loss of erythrocytes is combined with a decrease in the volume of blood plasma, which is responsible for the transfer of O2 through the capillary wall to tissue.
This reduction in peripheral tissue oxygenation would down-regulate mitochondria growth/biogenesis and energy production, hence SMB mass and performance.

Terrestrial Studies
At the Symposium we would like to present the results of our studies on the range of carotenoid-based nutraceutical products, developed by our team, and which can increase peripheral tissue oxygenation including in skeletal muscle. The efficacy of the products has been validated in a number of clinical trials on elderly people and / or patients with moderate muscle atrophy.

Different people age at different rates and respond differently to the same health challenges, including to microgravity. To personalise intervention with our products, hence improve their efficacy, we have developed for self-diagnostics a point-of-care express test to measure tissue oxygen supply levels. This test can be performed by a medically untrained person and does not require any equipment other than a smartphone camera. The results can be ready in 2 minutes.

Proposal for Microgravity Study
In addition, we would also present a proposal for a study on personalised administration of one of our leading nutraceuticals to support peripheral tissue oxygenation, and skeletal muscle mass and performance in volunteer astronauts flying in microgravity conditions at the International Space Station.

A potential benefit of this study would not only be helpful for astronauts to improve their quality of life and work at the ISS, but could perhaps also help to extend their living and working time beyond the current average duration of their space missions of six months.

Countermeasure exercise is a ubiquitous feature of long duration spaceflight as prolonged exposure to microgravity (μG) on the International Space Station (ISS) results in a reduction in physical fitness, bone mineral density, and muscle strength. Approximately 25% of each working day on ISS is spent on countermeasure exercise but the effectiveness of the current program has not been optimized and some astronauts still experience significant de-conditioning.

Jumping and hopping exercises are potentially highly effective forms of countermeasure exercise and their efficacy has been demonstrated by the European Space Agency (ESA) during long-term bed-rest. The ‘High Frequency Impulse for Microgravity’ (HIFIm) is a novel supine jumping sled which is designed to overcome the current limitations of traditional CM exercise by facilitating repeated horizontal jumping in microgravity. A key feature of HIFIm is that it is designed to reduce the forces produced during exercise to levels within the capabilities of standard aircraft vibration isolation systems. In addition, HIFIm is a multi-exercise system that provides a repertoire of over 100 exercises that can work all parts of the body.

A microgravity environment can be simulated terrestrially during parabolic flight. Despite this, the current resistance training device used on ISS was not tested in parabolic flight due to the technical challenges and safety limitations involved in using this platform. Similarly, it has not been possible to test E4D, one of the main candidates for the Gateway countermeasure in parabolic flight. In contrast, HIFIm has been tested in two different parabolic flight campaigns, and has been tested for almost 70 minutes in microgravity. Firstly, during the 77th ESA Parabolic Flight Campaign (PFC) we showed that repeated jumping in microgravity is possible using HIFIm, and that the loading provided by this exercise is similar to that experienced terrestrially. Secondly, during the 64th CNES PFC we validated HIFIm's engineering approach and showed that HIFIm isolates the force and vibration to levels that are within the operational window for Gateway, without the need for additional vibration isolation systems. The combination of these results mean that HIFIm is at a TRL of 6, and that repeated jumping is at a countermeasure exercise readiness level of 7.

In an earlier experiment in the European facility for biological research onboard the Space Shuttle / Spacelab, Biorack (IML-1 on STS-42), we reported for the first time, direct responses of near weightlessness isolated fetal mouse long bones. The aim of the experiments addressed in this study was to verify the earlier results and to study, during microgravity, the effects of varying periods of daily 1×g exposure on growth and mineralization in isolated fetal mouse long bones. The paper describes the results of two experiments with mouse metatarsals, one performed on an American Space Shuttle mission (IML-2 on STS–65) and another on a Russian Bio-Cosmos flight (Bion-10 on Cosmos-2229). Experiments differed in hardware and experimental conditions, but were comparable in the biological material: 17-day-old fetal mouse metatarsal long bones cultured for 4 days. In both experiments, cultures under microgravity were compared with cultures in an on-board 1×g centrifuge. From ultrastructural analyses it appeared that proteoglycan numbers are reduced in the groups exposed to microgravity compared to the groups exposed to 1×g conditions. No differences were found in the nuclear / cytoplasmic ratio, cell divisions, glycogen granules and the size and orientation of collagen fibrils. The increase in overall metatarsal length appeared not to be affected by microgravity. In contrast, the increase in length of the mineralized diaphysis was significantly reduced under microgravity. These results are fully comparable with a previous microgravity experiment (IML-1 on STS-42) using 16-day-old mouse fetal metatarsals. We have also demonstrated, for the first time, that the microgravity-induced reduction of cartilage mineralization is completely abolished by exposing long bones daily for periods of at least 6 hours to 1×g while no effects were seen after 3 hours 1×g exposure. No effects of intermittent 1×g exposure were found on overall growth. These result indicate that rather long duration exposure might be required when on-board centrifugation is foreseen as, multisystem, countermeasure for spaceflight near weightlessness pathologies. Future research using in vivo systems, like rodents, should be explored to see of pars of a day exposure to 1g could prevent microgravity related diseases.

Background
Important alterations occurring in living organisms during space flight concern the trabecular compartment of load-bearing bones, and result in significant bone mineral loss and decay of mechanical properties [1, 2].
Composition, mineral content, and the complex micro-scale trabecular microarchitecture contribute together to the macro-scale functional strength of bone as a whole [3]. Bone alterations, including those due to reduced gravitational load conditions, are mainly assessed by measuring bone density, even though, alone, it cannot comprehensively assess skeletal integrity [4]. Micro-tomographic techniques [5], not suitable for monitoring, allow for a pre- and post-mission examination of the trabecular bone component, which undergoes the fastest and most important alterations, placing astronauts at serious risk of fracture upon re-entry [5]. Mesoscale studies in modeled microgravity conditions combined with numerical simulations, show that degradation of apparent mechanical properties must be considered to achieve an accurate description of bone performance [6, 7].
To quantify the pathological alterations in the bone micro-architecture in a clinical setting, a patented, CE marked, software medical device, the Bone Elastic Structure Test, BES TEST, has been developed. Results are uncorrelated to BMD and independent of load [8, 9]. BES TEST has a diagnostic accuracy of 78% as a 3-year fracture risk estimator [10] and can be used to complete the densitometry picture and as monitoring tool for bone follow-up in in rheumatology [9], oncology [11], nephrology [12] and rare bone diseases [13].
Its prospective application for bone alteration monitoring during spaceflights is discussed.
Method
BES TEST simulates the application of forces on an X-ray functional biopsy of the patient’s hand [14-19]. Results are combined in an index, BSI, and its T-score and Z-score (Fig.1). Characteristics: X-ray dose < 0.0005 µSV; CV intra-operator=0.06; 95%CI±8 BSI; CV inter-operator =0.11; 95% CI=±10.8 BSI [20, 21], in line with the diagnostic gold standard.
Requirements for investigation of BES TEST space application:
- Acquisition: x-ray scanner, small detector. Several possible arrangements are possible, tests in simulated space flight will clarify the best configuration.
- Calibration: the acquisition set-up will likely differ from the clinical one.
- Analysis: radiograms upload to automatic service.
Results
BES TEST monitors trabecular bone, which changes more rapidly than cortical bone and BMD in response to physio-pathological alterations, like those occurring during spaceflight.
Conclusion
BESTEST is fast, easy to perform, cost-effective and can be significantly repeated within just weeks, showing potential for monitoring the changes in bone functionality during long-duration space missions.

The present experiment investigates the behavior of marine actinobacteria in the International Space Station (ISS). More specifically, the aim of the experiment is to examine the growth rate and antibiotic production of the actinobacteria and as a result the correlation between the growth rate and the viscosity of the liquid (mix of actinobacteria and nutrient agar). The
experiment is performed in cooperation with Nanoracks and launched via Falcon 9, Space – X and remained in the ISS for a 90-day time period under constant temperature (4 degree Celsius), being stirred by the astronauts on a weekly basis. There is a medical and pharmacological
interest since marine actinobacteria are a source of bioactive natural and antibiotic products, beneficial for the human organism, producing a variety of secondary metabolites. The experiment in the ISS indicates growth similar to the experiment on Earth, with slightly higher values showing that the bacteria survived the microgravity conditions. The viscosity is slightly
greater in the ISS, potentially due to the change in the density of the liquid, following the growth of the bacteria.