Simulating Parabolic Flight like <Emphasis Type="Italic">g</Emphasis>-Profiles on Ground - A Combination of Centrifuge and Clinostat

Clinostats and centrifuges are widely used to create simulated microgravity or hypergravity, respectively, in order to study the impact of gravity on biosystems. Here, we used a clinostat and a centrifuge in alternating modes of operation in order to create a simulated parabolic flight like g-profile. To our knowledge, it is the first time that both devices were run in connection. In order to test the method, we investigated the production of reactive oxygen species of immune cells (macrophages) during oxidative burst in an on-line kinetic approach, which has been extensively studied under real (parabolic flight) and simulated microgravity (clinostat) as well as under hypergravity conditions (centrifuge). Our results indicate that clinostat and centrifuge can be operated in an alternating way to simulate the repetitive changes of gravity during parabolic flight. Although the switch from one gravity level to the other could not be carried out as quickly as it takes place during actual parabolic flight due to technical and operational reasons, it can be concluded that running experiments in a clinostat aboard a centrifuge on ground are suitable for studying gravity-related phenomena.     

The Impact of Hypergravity and Vibration on Gene and Protein Expression of Thyroid Cells

Experiments in space either on orbital missions on-board the ISS, or in suborbital missions using sounding rockets, like TEXUS as well as parabolic flight campaigns are still the gold standard to achieve real microgravity conditions in the field of gravitational biology and medicine. However, during launch, and in flight, hypergravity and vibrations occur which might interfere with the effects of microgravity. It is therefore important to know these effects and discriminate them from the microgravity effects. This can be achieved by ground-based facilities like centrifuges or vibration platforms. Recently, we have conducted several experiments with different thyroid cancer cell lines. This study, as part of the ESA-CORA-GBF 2010-203 project, focused on the influence of vibration and hypergravity on benign human thyroid follicular epithelial cells (Nthy-ori 3-1 cell line). Gene and in part protein expression regulation under both conditions were analyzed for VCAN, ITGA10, ITGB1, OPN, ADAM19, ANXA1, TNFA, ABL2, ACTB, PFN2, TLN1, EZR, RDX, MSN, CTGF, PRKCA, and PRKAA1 using quantitative real-time PCR and Western Blot. We found that hypergravity and vibration affected genes and proteins involved in the extracellular matrix, the cytoskeleton, apoptosis, cell growth and signaling. Vibration always led to a down-regulation, whereas hypergravity resulted in a more heterogeneous expression pattern. Overall we conclude that both conditions can influence gene regulation and production of various genes and proteins. As a consequence, it is important to perform control experiments on hypergravity and vibration facilities in parallel to flight experiments.     

Hypergravity of 10<Emphasis Type="Italic">g</Emphasis> Changes Plant Growth,Anatomy, Chloroplast Size,and Photosynthesis in the Moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

The photosynthetic and anatomical responses of bryophytes to changes in gravity will provide crucial information for estimating how these plant traits evolved to adapt to changes in gravity in land plant history. We performed long-term hypergravity experiments at 10g for 4 and 8 weeks using the moss Physcomitrella patens with two centrifuges equipped with lighting systems that enable long-term plant growth under hypergravity with irradiance. The aims of this study are (1) to quantify changes in the anatomy and morphology of P. patens, and (2) to analyze the post-effects of hypergravity on photosynthesis by P. patens in relation to these changes. We measured photosynthesis by P. patens for a population of gametophores (e.g., canopy) in Petri dishes and plant culture boxes. Gametophore numbers increased by 9% for a canopy of P. patens, with 24–27% increases in chloroplast sizes (diameter and thickness) in leaf cells. In a canopy of P. patens, the area-based photosynthesis rate (A _canopy) was increased by 57% at 10g. The increase observed in A _canopy was associated with greater plant numbers and chloroplast sizes, both of which involved enhanced CO₂ diffusion from the atmosphere to chloroplasts in the canopies of P. patens. These results suggest that changes in gravity are important environmental stimuli to induce changes in plant growth and photosynthesis by P. patens, in which an alteration in chloroplast size is one of the key traits. We are now planning an ISS experiment to investigate the responses of P. patens to microgravity.     

Intracellular Calcium Decreases Upon Hyper Gravity-Treatment of <Emphasis Type="Italic">Arabidopsis Thaliana</Emphasis> Cell Cultures

Cell cultures of Arabidopsis thaliana (A.t.) respond to changes in the gravitational field strength with fluctuations of the amount of cytosolic calcium (Ca²⁺). In parabolic flight experiments, where hyper- and μg phases follow each other, μg clearly increased Ca²⁺, while hyper-g caused a slight reduction. Since the latter observation had not been reported before, we studied this effect in more detail. Using a special centrifuge for heavy items (ZARM, Bremen, Germany), we determined the hyper-g-dependent intracellular Ca²⁺ level with transgenic cell lines expressing the Ca²⁺ sensor, cameleon. This sensor exhibits a shift in fluorescence from 480 to 530 nm in response to Ca²⁺ binding. The data show a drop in the intracellular Ca²⁺ concentration with a threshold gravity of around 3 g. This is above hypergravity levels achieved during parabolic flights (1.8 g). The use of mutants with different sub-cellular targets of cameleon expression (nucleus, tonoplast, plasma membrane) gave the same results, i.e. Ca²⁺ is obviously exported from several intracellular compartments.     

The First European Parabolic Flight Campaign with the Airbus A310 ZERO-G

Aircraft parabolic flights repetitively provide up to 23 seconds of reduced gravity during ballistic flight manoeuvres. Parabolic flights are used to conduct short microgravity investigations in Physical and Life Sciences and in Technology, to test instrumentation prior to space flights and to train astronauts before a space mission. The use of parabolic flights is complementary to other microgravity carriers (drop towers, sounding rockets), and preparatory to manned space missions on board the International Space Station and other manned spacecraft, such as Shenzhou and the future Chinese Space Station. After 17 years of using the Airbus A300 ZERO-G, the French company Novespace, a subsidiary of the ’Centre National d’Etudes Spatiales’ (CNES, French Space Agency), based in Bordeaux, France, purchased a new aircraft, an Airbus A310, to perform parabolic flights for microgravity research in Europe. Since April 2015, the European Space Agency (ESA), CNES and the ‘Deutsches Zentrum für Luft- und Raumfahrt e.V.’ (DLR, the German Aerospace Center) use this new aircraft, the Airbus A310 ZERO-G, for research experiments in microgravity. The first campaign was a Cooperative campaign shared by the three agencies, followed by respectively a CNES, an ESA and a DLR campaign. This paper presents the new Airbus A310 ZERO-G and its main characteristics and interfaces for scientific experiments. The experiments conducted during the first European campaign are presented.     

The Impact of Simulated Microgravity on the Growth of Different Genotypes of the Model Legume Plant <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Simulated microgravity has been a useful tool to help understand plant development in altered gravity conditions. Thirty-one genotypes of the legume plant Medicago truncatula were grown in either simulated microgravity on a rotating clinostat, or in a static, vertical environment. Twenty morphological features were measured and compared between these two gravity treatments. Within-species genotypic variation was a significant predictor of the phenotypic response to gravity treatment in 100% of the measured morphological and growth features. In addition, there was a genotype–environment interaction (G × E) for 45% of the response variables, including shoot relative growth rate (p <?0.0005), median number of roots (p ～ 0.02), and root dry mass (p <?0.005). Our studies demonstrate that genotype does play a significant role in M. truncatula morphology and affects the response of plants to the gravity treatment, influencing both the magnitude and direction of the gravity response. These findings are discussed in the context of improving future studies in plant space biology by controlling for genotypic differences. Thus, manipulation of genotype effects, in combination with M. truncatula’s symbiotic relationships with bacteria and fungi, will be important for optimizing legumes for cultivation on long-term space missions.     

Effects of Short-Term Hypergravity Exposure are Reversible in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. Caryopses

Short-term hypergravity exposure is shown to retard seed germination, growth and photosynthesis in wheat caryopses. This study investigates the reversibility of effects of short-term hypergravity on imbibed wheat (Triticum aestivum var L.) caryopses. After hypergravity exposure (500 × g ? 2500 × g for 10 min) on a centrifuge, exposed caryopses were kept under normal gravity (1 × g) up to six days and then sown on agar. Results of the present study showed that percentage germination and growth were completely restored for DAY 6 compared to DAY 0. Restoration of germination and growth was accompanied by increased α-amylase activity. The specific activity of antioxidative enzyme viz. catalase and guaiacol peroxidase was lowered on DAY 6 compared to DAY 0 suggesting an alleviation of oxidative cellular damage against hypergravity stress. Chlorophyll pigment recovery along with chlorophyll fluorescence (PI and Fv/Fm) on DAY 6 indicates a transient rather than permanent damage of the photosynthetic apparatus. Thus, our findings demonstrate that short-term hypergravity effects are reversible in wheat caryopses. The metabolic cause of restoration of seed germination and growth upon transferring the caryopses to normal gravity is performed by a reactivation of carbohydrate- metabolizing enzymes, α-amylase and alleviation of oxidative stress damage with subsequent recovery of chlorophyll biosynthesis and photosynthetic activity.     

Responses of Microcrustaceans to Simulated Microgravity (2D-Clinorotation) - Preliminary Assessments for the Development of Bioregenerative Life Support Systems (BLSS)

Bioregenerative Life Support Systems (BLSS) are an endeavor to create environments able to maintain human life e.g. on future long-duration space missions like flights to Mars. Based on cyclic biological processes, these systems will be independent from material resupply (such as food, water and oxygen). Due to their central role in limnic ecosystems, herbivorous microcrustaceans could act as key player in aquatic BLSS as they link oxygen liberating, autotrophic producers like algae to higher trophic levels, such as fish. However, before such BLSS can be utilized in space, organisms inhabiting these systems have to be studied thoroughly to disclose the gravitational impact on the biological processes. This is possible in real microgravity, but requires high financial resources, is opportunity-limited or periods of microgravity are very short. Yet, cost-effective and almost permanently accessible tools for gravitational research are ground-based facilities (GBFs), providing simulated microgravity. Among those GBFs is the so called 2D-clinostat. In the present study we demonstrate, that rotation of clinostat tubes does not generate acceleration in form of (predator resembling) small scale turbulence, which can be perceived by Daphnia cucullata. Additionally, embryonal development is not disturbed in subitaneous eggs of Daphnia magna and resting eggs of the ostracod Heterocypris incongruens (besides through restrictions in space within the narrow clinostat tubes), just as subsequent hatching from the respective eggs. Hence, our results indicate that clinorotation is a suitable method to simulate microgravity for microcrustaceans.     

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.     

Life History Responses and Feeding Behavior of Microcrustacea in Altered Gravity – Applicability in Bioregenerative Life Support Systems (BLSS)

Manned space missions, as for example to the planet Mars, are a current objective in space exploration. During such long-lasting missions, aquatic bioregenerative life support systems (BLSS) could facilitate independence of resupply from Earth by regenerating the atmosphere, purifying water, producing food and processing waste. In such BLSS, microcrustaceans could, according to their natural role in aquatic ecosystems, link oxygen liberating, autotrophic algae and higher trophic levels, such as fish. However, organisms employed in BLSS will be exposed to high acceleration (hyper- g) during launch of spacecrafts as well as to microgravity (μg) during space travel. It is thus essential that these organisms survive, perform and reproduce under altered gravity conditions. In this study we present the first data in this regard for the microcrustaceas Daphnia magna and Heterocypris incongruens. We found that after hyper-g exposure (centrifugation) approximately one third of the D. magna population died within one week (generally indicating that possible belated effects have to be considered when conducting and interpreting experiments during which hyper-g occurs). However, suchlike and even higher losses could be countervailed by the surviving daphnids’ unaltered high reproductive capacity. Furthermore, we can show that foraging and feeding behavior of D. magna (drop tower) and H. incongruens (parabolic flights) are rarely altered in μg. Our results thus indicate that both species are suitable candidates for BLSS utilized in space.     

Effects of fast clinostat treatment and microgravity on Vicia faba L. mesophyll cell protoplast ubiquitin pools and actin isoforms

With parabolic rocket flights and fast clinostat treatments, the effect of microgravity on ubiquitin, ubiquitin-protein conjugates, and actin isoforms of Vicia faba mesophyll protoplasts was studied. Western immunoblotting with ubiquitin antibodies revealed that simulated and particularly, real microgravity influenced the amount of free ubiquitin and of 18, 19, and 40 kD ubiquitin conjugates by inducing strong oscillations in the proteins concentrations over time. Simulated microgravity and microgravity-phase during parabolic rocket flights resulted in a decrease of actin isoforms. Results obtained support the assumption, that microgravity and fast clinostat treatment have a direct effect on Vicia faba mesophyll protoplast metabolic activities.     

Microgravity performance of micro pulsating heat pipes

A micro pulsating heat pipe made of a thin clear Teflon tube of 1.6 mm ID was used to observe the pulsating flow inside a heat pipe under different gravity levels using parabolic flights. More vigorous pulsating flow was observed under microgravity, compared to the depressed movements under hypergravity. Two metallic micro pulsating heat pipes made of an aluminum plate with small internal channels were also tested to investigate the effect of gravity on their heat transfer characteristics. Reduced gravity experiments were performed aboard Falcon 20 aircraft flying parabolic trajectories. Under normal and hypergravity conditions, both the orientation of the pulsating heat pipe and locations of the heated and cooled sections affected the heat transfer performance. Under reduced gravity, however, the heat pipes showed better operating and heat transfer performance than that under normal and hypergravity. These experiments have for the first time confirmed that pulsating heat pipes are capable of operating under reduced gravity and thus are suitable for deployment in space applications such as satellites.     

Response of Haloalkaliphilic Archaeon <Emphasis Type="Italic">Natronococcus Jeotgali</Emphasis> RR17 to Hypergravity

The survival of archaeabacteria in extreme inhabitable environments on earth that challenge organismic survival is ubiquitously known. However, the studies related to the effect of hypergravity on the growth and proliferation of archaea are unprecedented. The survival of organisms in hypergravity and rocks in addition to resistance to cosmic radiations, pressure and other extremities is imperative to study the possibilities of microbial travel between planets and endurance in hyperaccelerative forces faced during ejection of rocks from planets. The current investigation highlights the growth of an extremophilic archaeon isolated from a rocky substrate in hypergravity environment. The haloalkaliphilic archaeon, Natronococcus jeotgali RR17 was isolated from an Indian laterite rock, submerged in the Arabian sea lining Coastal Maharashtra, India. The endolithic haloarchaeon was subjected to hypergravity from 56 – 893 X gusing acceleration generated by centrifugal rotation. The cells of N. jeotgali RR17 proliferated and demonstrated good growth in hypergravity (223 X g). This is the first report on isolation of endolithic haloarchaeon N. jeotgali RR17 from an Indian laterite rock and its ability to proliferate in hypergravity. The present study demonstrates the ability of microbial life to survive and proliferate in hypergravity. Thus the inability of organismic growth in hypergravity may no longer be a limitation for astrobiology studies related to habitability of substellar objects, brown dwarfs and other planetary bodies in the universe besides planet earth.     

Fish Inner Ear Otolith Growth Under Real Microgravity (Spaceflight) and Clinorotation

Using late larval stages of cichlid fish (Oreochromis mossambicus) we have shown earlier that the biomineralization of otoliths is adjusted towards gravity by means of a neurally guided feedback loop. Centrifuge experiments, e.g., revealed that increased gravity slows down otolith growth. Microgravity thus should yield an opposite effect, i.e., larger than normal otoliths. Consequently, late larval cichlids (stage 14, vestibular system operational) were subjected to real microgravity during the 12 days FOTON-M3 spaceflight mission (OMEGAHAB-hardware). Controls were kept at 1g on ground within an identical hardware. Animals of another batch were subsequently clinorotated within a submersed fast-rotating clinostat with one axis of rotation (2d-clinostat), a device regarded to simulate microgravity. Temperature and light conditions were provided in analogy to the spaceflight experiment. Controls were maintained at 1g within the same aquarium. After all experiments, animals had reached late stage 21 (fish can swim freely). Maintenance under real microgravity during spaceflight resulted in significantly larger than normal otoliths (both lapilli and sagittae, involved in sensing gravity and the hearing process, respectively). This result is fully in line with an earlier spaceflight study in the course of which otoliths from late-staged swordtails Xiphophorus helleri were analyzed. Clinorotation resulted in larger than 1g sagittae. However, no effect on lapilli was obtained. Possibly, an effect was present but too light to be measurable. Overall, spaceflight obviously induces an adaptation of otolith growth, whereas clinorotation does not fully mimic conditions of microgravity regarding late larval cichlids.     

Gravity effect on spray impact and spray cooling

An experimental study of the film produced by the spray impact on a heated target and of the spray cooling has been performed in normal gravity and in microgravity conditions during parabolic flights. A convex shape of the target allowed visualization of the film evolution and determination of the film characteristics using the image processing. The effects of the spray parameters and of the gravity level on heat transfer have been investigated. It has been found that the spray cooling efficiency depends on the water flow rate in a non-monotonous way. A range of spray parameters at which the effect of gravity level on heat transfer is significant has been determined. It has been found that the spray cooling is less effective in microgravity conditions in comparison with normal gravity and hypergravity.     

Real-Time Video-Microscopy of Migrating Immune Cells in Altered Gravity During Parabolic Flights

In our project we developed a technical equipment which allows to visualize migration of cells in real-time video-microscopy during altered gravity conditions of NOVESPACE Airbus A300 ZERO-G parabolic flights. For validation of the experimental device we have used fast moving human neutrophils as example, because their migration is fundamental to keep the organism under immunological surveillance. Their migration is indispensable for immune effector function, where the cells leave the blood vessels and navigate to places of infection to fulfill their main task of phagocytosis. Thereby, we have analyzed if their migration is affected during altered gravity conditions and if pharmacological modification of cytoskeletal dynamics influences neutrophil migratory activity. Whereas we detected no change in neutrophil locomotory behaviour in microgravity, we found a significant inhibitory influence of hypergravity, irrespective of the chemical stimulus used. Our results suggest that hypergravity, following a microgravity environment, could represent a hazard to the human immune system function. Thus, our cell migration assay offers an optimum experimental device for studying the migratory activity and underlying signal transduction mechanisms of neutrophils to assess the immunological fitness of humans in space to fight infection, but also for investigating the locomotion of other cell types or unicellular organisms such as ciliates.     

Analysis of Statoliths Displacement in <Emphasis Type="Italic">Chara</Emphasis> Rhizoids for Validating the Microgravity-Simulation Quality of Clinorotation Modes

In single-celled rhizoids of the green algae Chara, positively gravitropic growth is governed by statoliths kept in a dynamically stable position 10–25 μ m above the cell tip by a complex interaction of gravity and actomyosin forces. Any deviation of the tube-like cells from the tip-downward orientation causes statoliths to sediment onto the gravisensitive subapical cell flank which initiates a gravitropic curvature response. Microgravity experiments have shown that abolishing the net tip-directed gravity force results in an actomyosin-mediated axial displacement of statoliths away from the cell tip. The present study was performed to critically assess the quality of microgravity simulation provided by different operational modes of a Random Positioning Machine (RPM) running with one axis (2D mode) or two axes (3D mode) and different rotational speeds (2D), speed ranges and directions (3D). The effects of 2D and 3D rotation were compared with data from experiments in real microgravity conditions (MAXUS sounding rocket missions). Rotational speeds in the range of 60–85 rpm in 2D and 3D modes resulted in a similar kinetics of statolith displacement as compared to real microgravity data, while slower clinorotation (2–11 rpm) caused a reduced axial displacement and a more dispersed arrangement of statoliths closer to the cell tip. Increasing the complexity of rotation by adding a second rotation axis in case of 3D clinorotation did not increase the quality of microgravity simulation, however, increased side effects such as the level of vibrations resulting in a more dispersed arrangement of statoliths. In conclusion, fast 2D clinorotation provides the most appropriate microgravity simulation for investigating the graviperception mechanism in Chara rhizoids, whereas slower clinorotation speeds and rotating samples around two axes do not improve the quality of microgravity simulation.