Differential effects of hypergravity on immune dysfunctions induced by simulated microgravity.

Microgravity (µg) is among the major stressors in space causing immune cell dysregulations. These are frequently expressed as increased pro-inflammatory states of monocytes and reduced activation capacities in T cells. Hypergravity (as artificial gravity) has shown to have beneficial effects on the musculoskeletal and cardiovascular system both as a countermeasure option for µg-related deconditioning and as "gravitational therapy" on Earth. Since the impact of hypergravity on immune cells is sparsely explored, we investigated if an application of "mild" mechanical loading of 2.8 g is able to avoid or treat µg-mediated immune dysregulations. For this, T cell and monocyte activation states and cytokine pattern were first analyzed after whole blood antigen incubation in simulated µg (s-µg) by using the principle of fast clinorotation or in hypergravity. Subsequent hypergravity countermeasure approaches were run at three different sequences: one preconditioning setting, where 2.8 g was applied before s-µg exposure and two therapeutic approaches in which 2.8 g was set either intermediately or at the end of s-µg. In single g-grade exposure experiments, monocyte pro-inflammatory state was enhanced in s-µg and reduced in hypergravity, whereas T cells displayed reduced activation when antigen incubation was performed in s-µg. Hypergravity application in all three sequences did not alleviate the increased pro-inflammatory potential of monocytes. However, in T cells the preconditioning approach restored antigen-induced CD69 expression and IFNγ secretion to 1 g control values and beyond. This in vitro study demonstrates a proof of concept that mild hypergravity is a gravitational preconditioning option to avoid adaptive immune cell dysfunctions induced by (s-)µg and that it may act as a booster of immune cell functions.

Omics Studies of Specialized Cells and Stem Cells under Microgravity Conditions.

The primary objective of omics in space with focus on the human organism is to characterize and quantify biological factors that alter structure, morphology, function, and dynamics of human cells exposed to microgravity. This review discusses exciting data regarding genomics, transcriptomics, epigenomics, metabolomics, and proteomics of human cells and individuals in space, as well as cells cultured under simulated microgravity. The NASA Twins Study significantly heightened interest in applying omics technologies and bioinformatics in space and terrestrial environments. Here, we present the available publications in this field with a focus on specialized cells and stem cells exposed to real and simulated microgravity conditions. We summarize current knowledge of the following topics: (i) omics studies on stem cells, (ii) omics studies on benign specialized different cell types of the human organism, (iii) discussing the advantages of this knowledge for space commercialization and exploration, and (iv) summarizing the emerging opportunities for translational regenerative medicine for space travelers and human patients on Earth.

Low-Speed Clinorotation of Brachypodium distachyon and Arabidopsis thaliana Seedlings Triggers Root Tip Curvatures That Are Reminiscent of Gravitropism.

Clinostats are instruments that continuously rotate biological specimens along an axis, thereby averaging their orientation relative to gravity over time. Our previous experiments indicated that low-speed clinorotation may itself trigger directional root tip curvature. In this project, we have investigated the root curvature response to low-speed clinorotation using Arabidopsis thaliana and Brachypodium distachyon seedlings as models. We show that low-speed clinorotation triggers root tip curvature in which direction is dictated by gravitropism during the first half-turn of clinorotation. We also show that the angle of root tip curvature is modulated by the speed of clinorotation. Arabidopsis mutations affecting gravity susception (pgm) or gravity signal transduction (arg1, toc132) are shown to affect the root tip curvature response to low-speed clinorotation. Furthermore, low-speed vertical clinorotation triggers relocalization of the PIN3 auxin efflux facilitator to the lateral membrane of Arabidopsis root cap statocytes, and creates a lateral gradient of auxin across the root tip. Together, these observations support a role for gravitropism in modulating root curvature responses to clinorotation. Interestingly, distinct Brachypodium distachyon accessions display different abilities to develop root tip curvature responses to low-speed vertical clinorotation, suggesting the possibility of using genome-wide association studies to further investigate this process.

The Fight against Cancer by Microgravity: The Multicellular Spheroid as a Metastasis Model.

Cancer is a disease exhibiting uncontrollable cell growth and spreading to other parts of the organism. It is a heavy, worldwide burden for mankind with high morbidity and mortality. Therefore, groundbreaking research and innovations are necessary. Research in space under microgravity (µg) conditions is a novel approach with the potential to fight cancer and develop future cancer therapies. Space travel is accompanied by adverse effects on our health, and there is a need to counteract these health problems. On the cellular level, studies have shown that real (r-) and simulated (s-) µg impact survival, apoptosis, proliferation, migration, and adhesion as well as the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors in cancer cells. Moreover, the µg-environment induces in vitro 3D tumor models (multicellular spheroids and organoids) with a high potential for preclinical drug targeting, cancer drug development, and studying the processes of cancer progression and metastasis on a molecular level. This review focuses on the effects of r- and s-µg on different types of cells deriving from thyroid, breast, lung, skin, and prostate cancer, as well as tumors of the gastrointestinal tract. In addition, we summarize the current knowledge of the impact of µg on cancerous stem cells. The information demonstrates that µg has become an important new technology for increasing current knowledge of cancer biology.

Rapid Cellular Perception of Gravitational Forces in Human Jurkat T Cells and Transduction into Gene Expression Regulation.

Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.

Guanylyl Cyclase-cGMP Signaling Pathway in Melanocytes: Differential Effects of Altered Gravity in Non-Metastatic and Metastatic Cells.

Human epidermal melanocytes as melanin producing skin cells represent a crucial barrier against UV-radiation and oxidative stress. It was shown that the intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), generated by the guanylyl cyclases (GCs), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) and the natriuretic peptide-activated particulate GC (GC-A/GC-B), plays a role in the melanocyte response to environmental stress. Importantly, cGMP is involved in NO-induced perturbation of melanocyte-extracellular matrix interactions and in addition, increased NO production during inflammation may lead to loss of melanocytes and support melanoma metastasis. Further, the NO-sensitive sGC is expressed predominantly in human melanocytes and non-metastatic melanoma cells, whereas absence of functional sGC but up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are detected in metastatic cells. Thus, suppression of sGC expression as well as up-regulated expression of GC-A/GC-B/iNOS appears to correlate with tumor aggressiveness. As the cGMP pathway plays important roles in melanocyte (patho)physiology, we present an overview on the differential effects of altered gravity (hypergravity/simulated microgravity) on the cGMP signaling pathway in melanocytes and melanoma cells with different metastatic potential. We believe that future experiments in real microgravity may benefit from considering cGMP signaling as a possible factor for melanocyte transformation and in medication.

Time-averaged simulated microgravity (taSMG) inhibits proliferation of lymphoma cells, L-540 and HDLM-2, using a 3D clinostat.

BACKGROUND: Gravity is omnipresent on Earth; however, humans in space, such as astronauts at the International Space Station, experience microgravity. Long-term exposure to microgravity is considered to elicit physiological changes, such as muscle atrophy, in the human body. In addition, certain types of cancer cells demonstrate inhibited proliferation under condition of time-averaged simulated microgravity (taSMG). However, the response of human Hodgkin's lymphoma cancer cells to reduced gravity, and the associated physiological changes in these cells, have not been elucidated. METHODS: In this study, the proliferation of human Hodgkin's lymphoma cancer cells (L-540 and HDLM-2) under taSMG condition (<10-3 G, 1 G is defined as 9.8 m/s2) was studied using a 3D clinostat. Normal human dermal fibroblast (HDF) was proliferated in the same condition as a control group. For the development of 3D clinostat, two motors were used to actuate the frames. Electrical wires for power supply and communication were connected via slip ring. For symmetrical path of gravitational vector, optimal angular velocities of the motors were found using simulation results. Under the condition of taSMG implemented by the 3D clinostat, proliferation of the cells was observed for 3 days. RESULTS: The results indicated that proliferation of these cancer cells was significantly (p < 0.0005) inhibited under taSMG, whereas proliferation of normal HDF cells was not affected. CONCLUSIONS: Findings in this study could be significantly valuable in developing novel strategies for selective killing of cancer cells such as lymphoma.

Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.

Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

The impact of microgravity-based proteomics research.

Proteomics is performed in microgravity research in order to determine protein alterations occurring qualitatively and quantitatively, when single cells or whole organisms are exposed to real or simulated microgravity. To this purpose, antibody-dependent (Western blotting, flow cytometry, Luminex(®) technology) and antibody-independent (mass spectrometry, gene array) techniques are applied. The anticipated findings will help to understand microgravity-specific behavior, which has been observed in bacteria, as well as in plant, animal and human cells. To date, the analyses revealed that cell cultures are more sensitive to microgravity than cells embedded in organisms and that proteins changing under microgravity are highly interactive. Furthermore, one has to distinguish between primary gravity-induced and subsequent interaction-dependent changes of proteins, as well as between direct microgravity-related effects and indirect stress responses. Progress in this field will impact on tissue engineering and medicine and will uncover possibilities of counteracting alterations of protein expression at lowered gravity.

Combining clinostating and proton irradiation for modeling the space environment: a case study with a Chernobyl accession of Arabidopsis thaliana.

PURPOSE: The study of mechanisms of plant responses to extreme conditions, particularly, microgravity and ionizing radiation, is crucial for space exploration. Modern space biology of plants focuses on increasing plant tolerance to harsh conditions of space environment. Given the limited access to the International Space Station, we designed and assembled the 3D clinostat for mimicking microgravity, which, in combination with proton irradiation, allows simulating space conditions. As a case study for testing the device, we studied the effect of clinostating on Arabidopsis thaliana accession originating from the Chernobyl exclusion zone. MATERIALS AND METHODS: Using the combined clinostating and proton irradiation, we simulated the conditions of long-term space flight for Arabidopsis thaliana plants of the Chernobyl accession - progeny of chronically irradiated plants, grown from field-collected (Masa-0) and laboratory-cultivated (Masa-0-1) seeds, and for wild-type Col-8. The clinostating and irradiation of plants were also carried out separately. Plant responses were studied as photosynthetic and phenotypic endpoints of seedlings. RESULTS AND CONCLUSIONS: Parameters of chlorophyll fluorescence estimated immediately after exposure showed that Masa-0-1 plants were resistant to the simulated space conditions, while Masa-0 demonstrated modulation of non-photochemical fluorescence quenching. Proton irradiation generally inhibited photosynthesis of Masa-0, Masa-0-1, and Col-8 seedlings. The combined effect of irradiation and clinostating modulated the photosynthetic activity of Col-8 seedlings. The leaf area of seedlings did not change after exposure to simulated conditions. The 3D clinostat model and software are published along with this article for researchers interested in the field of space biology.

Use of a microgravity analog to explore the effects of simulated microgravity on the development of Escherichia coli K12 biofilms.

The rapid development of space technologies and the increase of human presence in space has brought the discussion of the effects of microgravity on cells into the undergraduate classroom. This paper proposes an idea to simulate microgravity on a bacterial culture, suitable for an introductory microbiology laboratory. For this purpose, we show the use of a 2D clinostat designed for microbial studies, along with traditional microbiology techniques such as optical density, plate counts, and biofilm biomass measurement to test the effect of simulated microgravity on the growth of Escherichia coli K12. This exercise aims to facilitate further discussions on the effects of microgravity on bacteria growth and communication, as well as the use of technology to simulate space and predict physiological changes in cells.

Preparation for mice spaceflight: Indications for training C57BL/6J mice to adapt to microgravity effect with three-dimensional clinostat on the ground.

Like astronauts, animals need to undergo training and screening before entering space. At present, pre-launch training for mice mainly focuses on adaptation to habitat system. Training for the weightless environment of space in mice has not received much attention. Three-dimensional (3D) clinostat is a method to simulate the effects of microgravity on Earth. However, few studies have used a 3D clinostat apparatus to simulate the effects of microgravity on animal models. Therefore, we conducted a study to evaluate the feasibility and effects of long-term treatment with three-dimensional clinostat in C57BL/6 J mice. Thirty 8-week-old male C57BL/6 J mice were randomly assigned to three groups: mice in individually ventilated cages (MC group, n = 6), mice in survival boxes (SB group, n = 12), and mice in survival boxes receiving 3D clinostat treatment (CS group, n = 12). The mice showed good tolerance after 12 weeks of alternate day training. To evaluate the biological effects of simulated microgravity, the changes in serum metabolites were monitored using untargeted metabolomics, whereas bone loss was assessed using microcomputed tomography of the left femur. Compared with the metabolome of the SB group, the metabolome of the CS group showed significant differences during the first three weeks and the last three weeks. The KEGG pathways in the late stages were mainly related to the nervous system, indicating the influence of long-term microgravity on the central nervous system. Besides, a marked reduction in the trabecular number (P < 0.05) and an increasing trend of trabecular spacing (P < 0.1) were observed to occur in a time-dependent manner in the CS group compared with the SB group. These results showed that mice tolerated well in a 3D clinostat and may provide a new strategy in pre-launch training for mice and conducting relevant ground-based modeling experiments.

Transcriptome profiles of rice roots under simulated microgravity conditions and following gravistimulation.

Root system architecture affects the efficient uptake of water and nutrients in plants. The root growth angle, which is a critical component in determining root system architecture, is affected by root gravitropism; however, the mechanism of root gravitropism in rice remains largely unknown. In this study, we conducted a time-course transcriptome analysis of rice roots under conditions of simulated microgravity using a three-dimensional clinostat and following gravistimulation to detect candidate genes associated with the gravitropic response. We found that HEAT SHOCK PROTEIN (HSP) genes, which are involved in the regulation of auxin transport, were preferentially up-regulated during simulated microgravity conditions and rapidly down-regulated by gravistimulation. We also found that the transcription factor HEAT STRESS TRANSCRIPTION FACTOR A2s (HSFA2s) and HSFB2s, showed the similar expression patterns with the HSPs. A co-expression network analysis and an in silico motif search within the upstream regions of the co-expressed genes revealed possible transcriptional control of HSPs by HSFs. Because HSFA2s are transcriptional activators, whereas HSFB2s are transcriptional repressors, the results suggest that the gene regulatory networks governed by HSFs modulate the gravitropic response through transcriptional control of HSPs in rice roots.

Clinorotation inhibits myotube formation by fluid motion, not by simulated microgravity.

To study processes related to weightlessness in ground-based cell biological research, a theoretically assumed microgravity environment is typically simulated using a clinostat - a small laboratory device that rotates cell culture vessels with the aim of averaging out the vector of gravitational forces. Here, we report that the rotational movement during fast clinorotation induces complex fluid motions in the cell culture vessel, which can trigger unintended cellular responses. Specifically, we demonstrate that suppression of myotube formation by 2D-clinorotation at 60 rpm is not an effect of the assumed microgravity but instead is a consequence of fluid motion. Therefore, cell biological results from fast clinorotation cannot be attributed to microgravity unless alternative explanations have been rigorously tested and ruled out. We consider two control experiments mandatory, i) a static, non-rotating control, and ii) a control for fluid motion. These control experiments are also highly recommended for other rotation speed settings and experimental conditions. Finally, we discuss strategies to minimize fluid motion in clinorotation experiments.

The Analysis of Gravitropic Setpoint Angle Control in Plants.

The history of research on gravitropism has been largely confined to the primary root-shoot axis and to understanding how the typically vertical orientation observed there is maintained. Many lateral organs are gravitropic too and are often held at specific non-vertical angles relative to gravity. These so-called gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that root and shoot lateral organs are able to effect tropic growth both with and against the gravity vector. This chapter describes methods and considerations relevant to the investigation of mechanisms underlying GSA control.

Blueprints for Constructing Microgravity Analogs.

The desire to understand gravitational effects on living things requires the removal of the very factor that determines life on Earth. Unfortunately, the required free-fall conditions that provide such conditions are limited to a few seconds unless earth-orbiting platforms are available. Therefore, attempts have been made to create conditions that simulate reduced gravity or gravity-free conditions ever since the gravity effects have been studied. Such conditions depend mostly on rotating devices (aka clinostats) that alter the gravity vector faster than the biological response time or create conditions that compensate sedimentation by fluid dynamics. Although several sophisticated, commercial instruments are available, they are unaffordable to most individual investigators. This article describes important considerations for the design and construction of low cost but versatile instruments that are sturdy, fully programmable, and affordable. The chapter focuses on detailed construction, programming of microcontrollers, versatility, and reliability of the instrument.

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.

CAMDLES: CFD-DEM Simulation of Microbial Communities in Spaceflight and Artificial Microgravity.

We present CAMDLES (CFD-DEM Artificial Microgravity Developments for Living Ecosystem Simulation), an extension of CFDEM®Coupling to model biological flows, growth, and mass transfer in artificial microgravity devices. For microbes that accompany humans into space, microgravity-induced alterations in the fluid environment are likely to be a major factor in the microbial experience of spaceflight. Computational modeling is needed to investigate how well ground-based microgravity simulation methods replicate that experience. CAMDLES incorporates agent-based modeling to study inter-species metabolite transport within microbial communities in rotating wall vessel bioreactors (RWVs). Preexisting CFD modeling of RWVs has not yet incorporated growth; CAMDLES employs the simultaneous modeling of biological, chemical, and mechanical processes in a micro-scale rotating reference frame environment. Simulation mass transfer calculations were correlated with Monod dynamic parameters to predict relative growth rates between artificial microgravity, spaceflight microgravity, and 1 g conditions. By simulating a microbial model community of metabolically cooperative strains of Escherichia coli and Salmonella enterica, we found that the greatest difference between microgravity and an RWV or 1 g gravity was when species colocalized in dense aggregates. We also investigated the influence of other features of the system on growth, such as spatial distribution, product yields, and diffusivity. Our simulation provides a basis for future laboratory experiments using this community for investigation in artificial microgravity and spaceflight microgravity. More broadly, our development of these models creates a framework for novel hypothesis generation and design of biological experiments with RWVs, coupling the effects of RWV size, rotation rate, and mass transport directly to bacterial growth in microbial communities.

Simulated Micro-, Lunar, and Martian Gravities on Earth-Effects on Escherichia coli Growth, Phenotype, and Sensitivity to Antibiotics.

Bacterial behavior has been studied under microgravity conditions, but very little is known about it under lunar and Martian gravitational regimes. An Earth-based approach was designed and implemented using inclined clinostats and an in-house-developed code to determine the optimal clinorotation angular speed for bacterial liquid cultures of 5 RPM. With this setup, growth dynamics, phenotypic changes, and sensitivity to antibiotics (minimum inhibitory concentration (MIC) of two different classes of antibiotics) for three Escherichia coli strains (including uropathogenic) were examined under simulated micro-, lunar, and Martian gravities. The results included increased growth under simulated micro- and lunar gravities for some strains, and higher concentrations of antibiotics needed under simulated lunar gravity with respect to simulated micro- and Martian gravities. Clinostat-produced results can be considered suggestive but not determinative of what might be expected in altered gravity, as there is still a need to systematically verify these simulation devices' ability to accurately replicate phenomena observed in space. Nevertheless, this approach serves as a baseline to start interrogating key cellular and molecular aspects relevant to microbial processes on the lunar and Martian surfaces.

Analysis of Graviresponse and Biological Effects of Vertical and Horizontal Clinorotation in Arabidopsis thaliana Root Tip.

Clinorotation was the first method designed to simulate microgravity on ground and it remains the most common and accessible simulation procedure. However, different experimental settings, namely angular velocity, sample orientation, and distance to the rotation center produce different responses in seedlings. Here, we compare A. thaliana root responses to the two most commonly used velocities, as examples of slow and fast clinorotation, and to vertical and horizontal clinorotation. We investigate their impact on the three stages of gravitropism: statolith sedimentation, asymmetrical auxin distribution, and differential elongation. We also investigate the statocyte ultrastructure by electron microscopy. Horizontal slow clinorotation induces changes in the statocyte ultrastructure related to a stress response and internalization of the PIN-FORMED 2 (PIN2) auxin transporter in the lower endodermis, probably due to enhanced mechano-stimulation. Additionally, fast clinorotation, as predicted, is only suitable within a very limited radius from the clinorotation center and triggers directional root growth according to the direction of the centrifugal force. Our study provides a full morphological picture of the stages of graviresponse in the root tip, and it is a valuable contribution to the field of microgravity simulation by clarifying the limitations of 2D-clinostats and proposing a proper use.