Gene-environmental influence of space and microgravity on red blood cells with sickle cell disease.

A fundamental question in human biology and for hematological disease is how do complex gene-environment interactions lead to individual disease outcome? This is no less the case for sickle cell disease (SCD), a monogenic disorder of Mendelian inheritance, both clinical course, severity, and treatment response, is variable amongst affected individuals. New insight and discovery often lie between the intersection of seemingly disparate disciplines. Recently, opportunities for space medicine have flourished and have offered a new paradigm for study. Two recent Nature papers have shown that hemolysis and oxidative stress play key mechanistic roles in erythrocyte pathogenesis during spaceflight. This paper reviews existing genetic and environmental modifiers of the sickle cell disease phenotype. It reviews evidence for erythrocyte pathology in microgravity environments and demonstrates why this may be relevant for the unique gene-environment interaction of the SCD phenotype. It also introduces the hematology and scientific community to methodological tools for evaluation in space and microgravity research. The increasing understanding of space biology may yield insight into gene-environment influences and new treatment paradigms in SCD and other hematological disease phenotypes.

Growth and mineralization of fetal mouse long bones under microgravity and daily 1 g gravity exposure.

In a previous Space Shuttle/Spacelab experiment (STS-42), we observed direct responses of isolated fetal mouse long bones to near weightlessness. This paper aimed to verify those results and study the effects of daily 1×g exposure during microgravity on the growth and mineralization of these bones. Two experiments were conducted: one on an American Space Shuttle mission (IML-2 on STS-65) and another on a Russian Bio-Cosmos flight (Bion-10 on Cosmos-2229). Despite differences in hardware, both used 17-day-old fetal mouse metatarsals cultured for 4 days. Results showed reduced proteoglycan content under microgravity compared to 1×g conditions, with no main differences in other cellular structures. While the overall metatarsal length was unaffected, the length increase of the mineralized diaphysis was significantly reduced under microgravity. Daily 1×g exposure for at least 6 h abolished the microgravity-induced reduction in cartilage mineralization, indicating the need for long-duration exposure to 1×g as an in-flight countermeasure using artificial gravity.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

Effects of altered gravity on growth and morphology in Wolffia globosa implications for bioregenerative life support systems and space-based agriculture.

Understanding the response of plants to varied gravitational conditions is vital for developing effective food production in space bioregenerative life support systems. This study examines the impact of altered gravity conditions on the growth and morphological responses of Wolffia globosa (commonly known as "water lentils" or "duckweed"), assessing its potential as a space crop. Although an experiment testing the effect of simulated microgravity on Wolffia globosa has been previously conducted, for the first time, we investigated the effect of multiple gravity levels on the growth and morphological traits of Wolffia globosa plants. The plant responses to simulated microgravity, simulated partial gravity (Moon), and hypergravity environments were evaluated using random positioning machines and the large-diameter centrifuge. As hypothesized, we observed a slight reaction to different gravitational levels in the growth and morphological traits of Wolffia globosa. The relative growth rates (RGR) of plants subjected to simulated microgravity and partial gravity were reduced when compared to those in other gravity levels. The morphological analysis revealed differences in plant dimensions and frond length-to-width ratios under diverse gravity conditions. Our findings showed that Wolffia globosa is responsive to gravitational changes, with its growth and morphological adaptations being slightly influenced by varying gravitational environments. As for other crop species, growth was reduced by the microgravity conditions; however, RGR remained substantial at 0.33 a day. In conclusion, this study underscores the potential of Wolffia globosa as a space crop and its adaptability to diverse gravitational conditions, contributing to the development of sustainable food production and bioregenerative life support systems for future space exploration missions.

The importance of gravity vector on adult mammalian organisms: Effects of hypergravity on mouse testis.

In the age of space exploration, the effect of hypergravity on human physiology is a relatively neglected topic. However, astronauts have several experiences of hypergravity during their missions. The main disturbance of altered gravity can be imputed to cell cytoskeleton alteration and physiologic homeostasis of the body. Testis has proved to be a particularly sensible organ, subject to environmental alteration and physiological disturbance. This makes testis an organ eligible for investigating the alteration following exposure to altered gravity. In our study, mice were exposed to hypergravity (3g for 14 days) in the Large Diameter Centrifuge machine (ESA, Netherland). We have observed a morphological alteration of the regular architecture of the seminiferous tubules of testis as well as an altered expression of factors involved in the junctional complexes of Sertoli cells, responsible for ensuring the morpho-functional integrity of the organ. The expression of key receptors in physiological performance, such as Androgen Receptors and Interstitial Cells Stimulating Hormone receptors, was found lower expressed. All these findings indicate the occurrence of altered physiological organ performance such as the reduction of the spermatozoa number and altered endocrine parameters following hypergravity exposure.

Future space experiment platforms for astrobiology and astrochemistry research.

Space experiments are a technically challenging but a scientifically important part of astrobiology and astrochemistry research. The International Space Station (ISS) is an excellent example of a highly successful and long-lasting research platform for experiments in space, that has provided a wealth of scientific data over the last two decades. However, future space platforms present new opportunities to conduct experiments with the potential to address key topics in astrobiology and astrochemistry. In this perspective, the European Space Agency (ESA) Topical Team Astrobiology and Astrochemistry (with feedback from the wider scientific community) identifies a number of key topics and summarizes the 2021 "ESA SciSpacE Science Community White Paper" for astrobiology and astrochemistry. We highlight recommendations for the development and implementation of future experiments, discuss types of in situ measurements, experimental parameters, exposure scenarios and orbits, and identify knowledge gaps and how to advance scientific utilization of future space-exposure platforms that are either currently under development or in an advanced planning stage. In addition to the ISS, these platforms include CubeSats and SmallSats, as well as larger platforms such as the Lunar Orbital Gateway. We also provide an outlook for in situ experiments on the Moon and Mars, and welcome new possibilities to support the search for exoplanets and potential biosignatures within and beyond our solar system.

Gravitational effects on fibroblasts' function in relation to wound healing.

The spaceflight environment imposes risks for maintaining a healthy skin function as the observed delayed wound healing can contribute to increased risks of infection. To counteract delayed wound healing in space, a better understanding of the fibroblasts' reaction to altered gravity levels is needed. In this paper, we describe experiments that were carried out at the Large Diameter Centrifuge located in ESA-ESTEC as part of the ESA Academy 2021 Spin Your Thesis! Campaign. We exposed dermal fibroblasts to a set of altered gravity levels, including transitions between simulated microgravity and hypergravity. The addition of the stress hormone cortisol to the cell culture medium was done to account for possible interaction effects of gravity and cortisol exposure. Results show a main impact of cortisol on the secretion of pro-inflammatory cytokines as well as extracellular matrix proteins. Altered gravity mostly induced a delay in cellular migration and changes in mechanosensitive cell structures. Furthermore, 20 × g hypergravity transitions induced changes in nuclear morphology. These findings provide insights into the effect of gravity transitions on the fibroblasts' function related to wound healing, which may be useful for the development of countermeasures.

The application of artificial gravity in medicine and space.

Gravity plays a crucial role in physiology. The lack of gravity, like in long duration spaceflight missions, cause pathologies in e.g., the musculoskeletal system, cardiovascular deconditioning, immune system deprivation or brain abnormalities, to just mention a few. The application of artificial gravity through short-arm human centrifugation (SAHC) has been studied as a possible countermeasure to treat spaceflight deconditioning. However, hypergravity protocols applied by using SAHC have also been used to treat different, ground-based pathologies. Such gravitational therapies have been applied in Uruguay for more than four decades now. The aim of this overview is to summarize the most important findings about the effects of gravitational therapy in different, mainly vascular based pathologies according to the experience in the Gravitational Therapy Center and to discuss the current research in the field of hypergravity applications in medicine but also as multisystem countermeasure for near weightlessness pathologies. New insight is needed on the use of hypergravity in medicine and space research and application.

Real-time quantification of osteoclastic resorptive activity by electric cell-substrate impedance sensing.

In several diseases, bone resorption by osteoclasts is dysregulated. Thus far, no simple technique for real-time measurement of resorption is available. Here, we introduce an impedimetric bioassay for real-time monitoring of resorption by making use of the electrical insulating properties of the resorbable substrate calcium phosphate. Different chemical stimuli were applied to (pre)osteoclasts cultured on a layer of calcium phosphate in multi-well plates containing electrodes. By this, osteoclast activity can be measured continuously over days, and the effects of stimulating or inhibiting factors can be quantified. When cells were cultured in the presence of an inflammatory factor such as IL-1ß, the resorptive activity started earlier. The measured decline in resistance was higher at culture day 5 than at cultures with M-CSF or M-CSF + RANKL (M-CSF norm. Resistance = 1, M-CSF + RANKL = 0.7, M-CSF + RANKL + IL-1ß = 0.5). However, at day 11, this difference had nearly disappeared. Likewise, bisphosphonates were shown to inhibit osteoclastic activity. Our findings illustrate the importance of real-time monitoring; wherefore, this method has high potential not only for the study of osteoclast resorptive activity in the context of osteoclast function and diseases but also could find application in high-throughput drug-testing studies.

Stability Studies of UV Laser Irradiated Promethazine and Thioridazine after Exposure to Hypergravity Conditions.

Pharmaceuticals carried into space are subjected to different gravitational conditions. Hypergravity is encountered in the first stage, during spacecraft launching. The stability of medicines represents a critical element of space missions, especially long-duration ones. Therefore, stability studies should be envisaged before the implementation of drugs for future deep space travel, where the available pharmaceuticals would be limited and restocking from Earth would be impossible. Multipurpose drugs should be proposed for this reason, such as phenothiazine derivatives that can be transformed by optical methods into antimicrobial agents. Within this preliminary study, promethazine and thioridazine aqueous solutions were exposed to UV laser radiation that modified their structures and generated a mixture of photoproducts efficient against particular bacteria. Subsequently, they were subjected to 20 g in the European Space Agency's Large Diameter Centrifuge. The aim was to evaluate the impact of hypergravity on the physico-chemical and spectral properties of unirradiated and laser-irradiated medicine solutions through pH assay, UV-Vis/FTIR absorption spectroscopy, and thin-layer chromatography. The results revealed no substantial alterations in centrifuged samples when compared to uncentrifuged ones. Due to their stability after high-g episodes, laser-exposed phenothiazines could be considered for future space missions.

A novel device to study altered gravity and light interactions in seedling tropisms.

Long-duration space missions will need to rely on the use of plants in bio-regenerative life support systems (BLSSs) because these systems can produce fresh food and oxygen, reduce carbon dioxide levels, recycle metabolic waste, and purify water. In this scenario, the need for new experiments on the effects of altered gravity conditions on plant biological processes is increasing, and significant efforts should be devoted to new ideas aimed at increasing the scientific output and lowering the experimental costs. Here, we report the design of an easy-to-produce and inexpensive device conceived to analyze the effect of interaction between gravity and light on root tropisms. Each unit consisted of a polystyrene multi-slot rack with light-emitting diodes (LEDs), capable of holding Petri dishes and assembled with a particular filter-paper folding. The device was successfully used for the ROOTROPS (for root tropisms) experiment performed in the Large Diameter Centrifuge (LDC) and Random Positioning Machine (RPM) at ESA's European Space Research and Technology centre (ESTEC). During the experiments, four light treatments and six gravity conditions were factorially combined to study their effects on root orientation of Brassica oleracea seedlings. Light treatments (red, blue, and white) and a dark condition were tested under four hypergravity levels (20 g, 15 g, 10 g, 5 g), a 1 g control, and a simulated microgravity (RPM) condition. Results of validation tests showed that after 24 h, the assembled system remained unaltered, no slipping or displacement of seedlings occurred at any hypergravity treatment or on the RPM, and seedlings exhibited robust growth. Overall, the device was effective and reliable in achieving scientific goals, suggesting that it can be used for ground-based research on phototropism-gravitropism interactions. Moreover, the concepts developed can be further expanded for use in future spaceflight experiments with plants.

Use of Reduced Gravity Simulators for Plant Biological Studies.

Simulated microgravity and partial gravity research on Earth is a necessary complement to space research in real microgravity due to limitations of access to spaceflight. However, the use of ground-based facilities for reduced gravity simulation is far from simple. Microgravity simulation usually results in the need to consider secondary effects that appear in the generation of altered gravity. These secondary effects may interfere with gravity alteration in the changes observed in the biological processes under study. In addition to microgravity simulation, ground-based facilities are also capable of generating hypergravity or fractional gravity conditions whose effects on biological systems are worth being tested and compared with the results of microgravity exposure. Multiple technologies (2D clinorotation, random positioning machines, magnetic levitators, or centrifuges) and experimental hardware (different containers and substrates for seedlings or cell cultures) are available for these studies. Experimental requirements should be collectively and carefully considered in defining the optimal experimental design, taking into account that some environmental parameters, or life-support conditions, could be difficult to be provided in certain facilities. Using simulation facilities will allow us to anticipate, modify, or redefine the findings provided by the scarce available spaceflight opportunities.

Artificially altered gravity elicits cell homeostasis imbalance in planarian worms, and cerium oxide nanoparticles counteract this effect.

Gravity alterations elicit complex and mostly detrimental effects on biological systems. Among these, a prominent role is occupied by oxidative stress, with consequences for tissue homeostasis and development. Studies in altered gravity are relevant for both Earth and space biomedicine, but their implementation using whole organisms is often troublesome. Here we utilize planarians, simple worm model for stem cell and regeneration biology, to characterize the pathogenic mechanisms brought by artificial gravity alterations. In particular, we provide a comprehensive evaluation of molecular responses in intact and regenerating specimens, and demonstrate a protective action from the space-apt for nanotechnological antioxidant cerium oxide nanoparticles.

The effect of hypergravity in intestinal permeability of nanoformulations and molecules.

The oral administration of drugs remains a challenge due to rapid enzymatic degradation and minimal absorption in the gastrointestinal tract. Mechanical forces, namely hypergravity, can interfere with cellular integrity and drug absorption, and there is no study describing its influence in the intestinal permeability. In this work, it was studied the effect of hypergravity on intestinal Caco-2 cells and its influence in the intestinal permeability of different nanoformulations and molecules. It was shown that the cellular metabolic activity and integrity were maintained after exposure to different gravity-levels (g-levels). Expression of important drug transporters and tight junctions' proteins was evaluated and, most proteins demonstrated a switch of behavior in their expression. Furthermore, paracellular transport of FITC-Dextran showed to significantly increase with hypergravity, which agrees with the decrease of transepithelial electrical resistance and the increase of claudin-2 at higher g-levels. The diffusion of camptothecin released from polymeric micelles revealed a significant decrease, which agrees with the increased expression of the P-gp observed with the increase in g-levels, responsible for pumping this drug out. The neonatal Fc receptor-mediated transport of albumin-functionalized nanoparticles loaded with insulin showed no significant changes when increasing the g-levels. Thus, this study supports the effect of hypergravity on intestinal permeability is dependent on the molecule studied and the mechanism by which it is absorbed in the intestine.

Hypergravity affects cell traction forces of fibroblasts.

Cells sense and react on changes of the mechanical properties of their environment and, likewise, respond to external mechanical stress applied to them. However, whether the gravitational field as overall body force modulates cellular behavior is unclear. Different studies demonstrated that micro- and hypergravity influences the shape and elasticity of cells, initiate cytoskeleton reorganization, and influence cell motility. All these cellular properties are interconnected and contribute to forces that cells apply on their surrounding microenvironment. Yet, studies that investigated changes of cell traction forces under hypergravity conditions are scarce. Here, we performed hypergravity experiments on 3T3 fibroblast cells using the large-diameter centrifuge at the European Space Agency - European Space Research and Technology Centre. Cells were exposed to hypergravity of up to 19.5 g for 16 h in both the upright and the inverted orientation with respect to the g-force vector. We observed a decrease in cellular traction forces when the gravitational field was increased up to 5.4 g, followed by an increase of traction forces for higher gravity fields up to 19.5 g independent of the orientation of the gravity vector. We attribute the switch in cellular response to shear thinning at low g-forces, followed by significant rearrangement and enforcement of the cytoskeleton at high g-forces.

Molecular impact of launch related dynamic vibrations and static hypergravity in planarians.

Although many examples of simulated and real microgravity demonstrating their profound effect on biological systems are described in literature, few reports deal with hypergravity and vibration effects, the levels of which are severely increased during the launch preceding the desired microgravity period. Here, we used planarians, flatworms that can regenerate any body part in a few days. Planarians are an ideal model to study the impact of launch-related hypergravity and vibration during a regenerative process in a "whole animal" context. Therefore, planarians were subjected to 8.5 minutes of 4 g hypergravity (i.e. a human-rated launch level) in the Large Diameter Centrifuge (LDC) and/or to vibrations (20-2000 Hz, 11.3 Grms) simulating the conditions of a standard rocket launch. The transcriptional levels of genes (erg-1, runt-1, fos, jnk, and yki) related with the early stress response were quantified through qPCR. The results show that early response genes are severely deregulated after static and dynamic loads but more so after a combined exposure of dynamic (vibration) and static (hypergravity) loads, more closely simulating real launch exposure profiles. Importantly, at least four days after the exposure, the transcriptional levels of those genes are still deregulated. Our results highlight the deep impact that short exposures to hypergravity and vibration have in organisms, and thus the implications that space flight launch could have. These phenomena should be taken into account when planning for well-controlled microgravity studies.

Collaboration Around Rare Bone Diseases Leads to the Unique Organizational Incentive of the Amsterdam Bone Center.

In the field of rare bone diseases in particular, a broad care team of specialists embedded in multidisciplinary clinical and research environment is essential to generate new therapeutic solutions and approaches to care. Collaboration among clinical and research departments within a University Medical Center is often difficult to establish, and may be hindered by competition and non-equivalent cooperation inherent in a hierarchical structure. Here we describe the "collaborative organizational model" of the Amsterdam Bone Center (ABC), which emerged from and benefited the rare bone disease team. This team is often confronted with pathologically complex and under-investigated diseases. We describe the benefits of this model that still guarantees the autonomy of each team member, but combines and focuses our collective expertise on a clear shared goal, enabling us to capture synergistic and innovative opportunities for the patient, while avoiding self-interest and possible harmful competition.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus.

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.