Comparison Study of Gravity-Dependent Displacement of Amyloplasts in Statocytes of Cress Roots and Hypocotyls

In this study, the kinetics of gravity-dependent movement of amyloplasts (statoliths) along root statocytes and hypocotyls (endodermis cells) has been analyzed and compared in order to testify cytoskeleton involvement in the displacement of statoliths in cress (Lepidium sativum L.) seedling statocytes. After 32 h of growth at 1 g or under a fast clinorotation (50 rpm), the seedlings were treated for 24 min as follows: exposition to clinorotation or 180° inversion and the action of gravitational force in root tip or hypocotyl tip direction. Statolith displacement was studied by light microscopy on semi-thin longitudinal sections of hypocotyls and root caps, measuring the distance between the centre of plastids and morphological cell bottom. Considerable temporal differences have been determined between the kinetics of the longitudinal motion of amyloplasts in root and hypocotyl statocytes of 1-g seedlings upon exposition to fast clinorotation and inversion. In statocytes of both organs of seedlings grown under fast clinorotation, the gravity provoked displacement of statoliths in the direction of its action; however, the displacement was much faster in hypocotyl than in root statocytes. It has been assumed that the gravity-determined longitudinal transport of amyloplasts, both in hypocotyl endodermic cells and root statocytes of cress seedlings, is modulated by the cytoskeleton.     

Comparative studies on gravisensitive protists on ground (2D and 3D clinostats) and in microgravity

In order to prepare and support space experiments, 2D and 3D clinostats are widely applied to study the influence of simulated weightlessness on biological systems. In order to evaluate the results a comparison between the data obtained in simulation experiments and in real microgravity is necessary. We are currently analyzing the gravity-dependent behavior of the protists Paramecium biaurelia (ciliate) and Euglena gracilis (photosynthetic flagellate) on these different experimental platforms. So far, first results are presented concerning the behaviour of Euglena on a 2D fast rotating clinostat and a 3D clinostat as well as under real microgravity conditions (TEXUS sounding rocket flight), of Paramecium on a 2D clinostat and in microgravity. Our data show similar results during 2D and 3D clinorotation compared to real microgravity with respect to loss of orientation (gravitaxis) of Paramecium and Euglena and a decrease of linearity of the cell tracks of Euglena. However, the increase of the mean swimming velocities, especially during 3D clinorotation (Euglena) and 2D clinorotation of Paramecium might indicate a persisting mechanostimulation of the cells. Further studies including long-term 2D and 3D clinostat exposition will enable us to demonstrate the qualification of the applied simulation methods.     

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.     

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.     

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.     

The stress protein level under clinorotation in context of the seedling developmental program and the stress response

Heat-shock proteins (HSP70 and HSP90) are present in plant cells under the normal growth conditions. At the same time, a variety of environmental disruptions results in their rapid synthesis as a substantial part of adaptation. HSP amounts can be indicative of a cellular stress level. Altered gravity (clinorotation) is unnatural for plants, so it may be a kind of stress. The aim of this study was to analyze the influence of horizontal clinorotation on the HSP70 and HSP90 level during seedling development. Pea (Pisum sativum L.) seedlings grown for 3 days from seed imbibitions in stationary control and under slow clinorotation (2 rpm) are used for this investigation. Western blot analysis indicated that HSP70 and HSP90 were abundant in the embryos of dry seeds and their amount decreased significantly during seed germination. But under horizontal clinorotation, their level in seedlings remained higher compared to the control. Furthermore, a comparison of the influence of horizontal and vertical clinorotation on the HSP level was carried out. On the ELISA data, HSP70 and HSP90 amounts in the 3-day old seedlings were higher after horizontal clinorotation than after vertical. The obtained data show an increased HSP70 and HSP90 level in pea seedlings under clinorotation. Both, rotation and change in the cell position relatively to a gravity vector affect the HSP level.     

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.     

Chlorophyll,Carotenoid and Anthocyanin Accumulation in Mung Bean Seedling Under Clinorotation

The accumulation of plant pigments in mung bean (Vigna radiata L.) seedlings was measured after clinorotation (2 rpm for 2-4 days), and compared to a stationary control. The pigments measured included chlorophyll and carotenoid in primary leaves, and the anthocyanin in seedlings. While significant changes in chlorophyll and carotenoid accumulation were not observed during the initial 2 to 4 days of cultivation, by day 4 the seedlings grown on the clinostat had lower levels of anthocyanin, compared to those in the control seedlings. To further detail the cause for the observed reduction in anthocyanin accumulation under altered gravity conditions, seedlings were grown in the presence of silver nitrate, a known ethylene inhibitor, for 4 days, since it is known ethylene has a negative impact on anthocyanin accumulation. Silver nitrate promoted anthocyanin accumulation in the clinostat seedlings, and as a result there was no significant difference between the control and clinostat seedlings in anthocyanin accumulation. The results suggest that slow clinorotation negatively impacts anthocyanin pigmentation in mung bean seedlings, with endogenous ethylene suspected to be involved in this.     

Actin Microfilament Organization in the Transition Zone of Arabidopsis-ABD2-GFP Roots Under Clinorotation

Seedlings of Arabidopsis thaliana-ABD2-GFP were grown under slow clinorotation (2?rpm) and treated with actin and tubulin disrupting drugs in order to characterize the role of actin microfilaments in cell growth and gravisensing. Changes in microfilament organization and cell parameters have shown that the transition root zone (TZ) is rather sensitive to microfilament disruption in control plants. It is assumed that under clinorotation, organization of actin cytoskeleton in the TZ is coordinated in a different way than in the control. Organization of microfilaments depends upon organization of microtubules and clinorotation does not influence this interrelation significantly.     

Physiological Responses of Synechocystis sp. PCC 6803 under Clinorotation

Photosystem efficiency and the characteristic on oxidative stress were examined to elucidate the metabolic responses of Synechocystis sp. PCC 6803 to short-term clinorotation. Results compiled when using clinostat to simulate microgravity for 60?h, showed that clinorotation clearly prohibited the photochemical quantum yield, but promoted the synthesis of chlorophyll and total protein. This may be a compensatory mechanism for the algal cell to maintain its normal metabolism. An increased malondialdehyde (MDA) content of algal cell upon clinorotation, together with an enhanced catalase (CAT) activity was observed during the whole period of clinorotation. One conclusion is that short-term clinorotation acts as a kind of stress, and that these physiological responses may be a special way for an algal cell to adapt itself to a different environment other than earth gravity.     

Experimental and numerical studies on the performance and surface streamlines on the blades of a horizontal-axis wind turbine

Investigating laminar separation over the turbine blade of a horizontal-axis wind turbine (HAWT) has been considered an important task to improve the aerodynamic performance of a wind turbine. To better understand the laminar separation phenomena, in this study, the aerodynamic forces of a SD8000 airfoil (representing the sectional blade shape) in the steady-state conditions were first predicted using an incompressible Reynolds-averaged Navier–Stokes solver with the γ–Re _θt and k–k _L–ω transition models. By comparing simulation and experimental results, the k–k _L–ω transition model was chosen to simulate the laminar separation on three-dimensional (3D) turbine blade. Experimentally, a HAWT with three blades was then tested in a close-circuit wind tunnel between the tip speed ratios (TSRs) of 2 and 7 at the wind speed of 10 m/s. In addition, through computational fluid dynamics, the turbine performance and flow characteristics on the blade as blade is rotating were investigated. It is shown that 3D simulations agreed well with the experimental results with regard to the mechanical power of the HAWT at the testing TSRs. Moreover, the separation and reattachment lines on the suction surface of the turbine blade were also observed through the skin friction line, indicating that laminar separation moved toward the trailing edge with the increasing TSR at the blade tip region.     

Effects of Wall Vessel Rotation on the Growth of Larval Zebrafish Inner Ear Otoliths

Stimulus dependence is a general feature of developing sensory systems. It has been shown earlier that the growth of otoliths of late-stage Cichlid fish (Oreochromis mossambicus) and Zebrafish (Danio rerio) was slowed down by hypergravity, whereas microgravity during spaceflight yielded an opposite effect, i.e., larger than 1 g otoliths, in Swordtail (Xiphophorus helleri) late-stage embryos. Using ground-based techniques to apply simulated weightlessness, long-term clinorotation (exposure on a fast-rotating clinostat with one axis of rotation for 7 days) led to larger than 1 g otoliths in late-stage Cichlid fish, which is fully in line with the results obtained on Swordtails from spaceflight. Hitherto, early-staged fish have not yet been subjected to (simulated or real) long-term (i.e., more than 3 or 4 days) weightlessness to investigate otolith growth. The present study was carried out in order to fill this gap. Therefore, we subjected Zebrafish at a somite-stage to Wall Vessel Rotation (WVR; a method regarded to provide simulated weightlessness), when the anlage of the inner ear already is present (10 h post fertilisation, hpf). Siblings were maintained under WVR for 3, 6, 9 and 12 days. Further short-term experiments (3 days) were carried out on 10 hpf animals as well as on very early larvae (1 K cell stage, 3 hpf) at two different rotation speeds. WVR (both rotation speeds) had no effect on otolith biogenesis in both stages as all otoliths were present after the experiments. In comparison with 1 g controls, WVR had significantly increased otolith growth (normalised by fish length) after 3 and 6 days of exposure, but significant differences of otolith growth between experimental animals and controls were not found after 9 and 12 days. In conclusion, WVR (at least within a time-span of exposure of up to 6 days) brings, comparable to the situation in real microgravity, a kind of feedback mechanism into action, resulting in larger otoliths. Later, possible effects of WVR might be overruled by normal allometric growth since the action of the feedback mechanism may be discontinued in the course of an adaptation.     

In-field Conductivity Fluctuations in Ba<Subscript>0.72</Subscript>K<Subscript>0.28</Subscript>Fe<Subscript>2</Subscript>As<Subscript>2</Subscript> Single Crystals

Fluctuations in the conductivity of Ba_0.72K_0.28Fe₂As₂ single crystal are studied systematically by resistance measurements as a function of temperature and magnetic field. A clear Maki?Thompson and Aslamakov?Larkin (MT–AL) two- to three-dimensional (2D–3D) crossover is found on the excess conductivity (Δσ) curves as the temperature approaches the superconducting critical temperature, T _c. 3D fluctuations in superconductivity are realized near T _c that are well fitted to experimental data by the 3D Aslamazov–Larkin theory. The Maki–Thompson model shows a 2D conductivity fluctuation above the 2D-3D temperature transition, T ₀, which depends on magnetic field. Results show that the 2D-3D dimensional crossover moves to lower temperature with increasing magnetic field. The values of the transition temperature and the crossover in the reduced temperature, ln(ε ₀), as functions of magnetic field were used to determine the coherence length and the lifetime, τ _φ , of the fluctuational pairs at the temperature of 35 K. Analysis of the Ba_0.72K_0.28Fe₂As₂ single crystal gives a value of 3.76 × 10^??12 s for the τ _φ in the absence of magnetic field and it decreases to 2.4 × 10^??12 s in magnetic field of 13 T.     

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.     

Analysis,design and implementation of sensorless <Emphasis Type="Italic">V/f</Emphasis> control in a surface-mounted PMSM without damper winding

This paper presents a novel, reliable and efficient V/f control implementation on a 8-pole, 750 rpm, 5 kW surface-mounted permanent magnet synchronous motor (PMSM) without damper winding. In the absence of a damper winding, open loop V/f control of SM is inherently unstable, particularly at high speeds. Stabilisation can be done with proper stator frequency modulation in accordance with the change in rotor speed to provide for effect of damping. This has been implemented here without use of any shaft-mounted encoder. The change in rotor speed is observed from power perturbation, thereby eliminating the need for using a speed sensor in the drive. The efficiency of the drive is further increased with appropriate control of the power factor, irrespective of load and frequency variations. Simulated and experimental results are presented for both open-loop and the proposed V/f control. These results establish the accuracy of the design of the proposed V/f control strategy and the precision of hardware implementation. A comparative study between the proposed V/f control method and standard vector control method, as implemented on this PMSM, has also been presented here to establish the advantages of the proposed scheme. The PMSM itself was designed and fabricated in the laboratory.     

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) Hemocytes

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.