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 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 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.     

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.     

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.     

Studies of the Local Structure and the g Factors of the Cu<Superscript>2+</Superscript> Site in Y<Subscript>2</Subscript>BaCuO<Subscript>5</Subscript>

The local structure and the g factors (g _x , g _y , and g _z ) of the Cu²⁺ site in Y₂BaCuO₅ are theoretically studied using the perturbation formulas of the g factors for a 3d⁹ ion in orthorhombically elongated octahedra. The orthorhombic field parameters in these formulas are determined from the superposition model and the local geometry of the system. From the calculations, the oxygen octahedron is found to undergo the local elongation ΔZ (≈0.05 Å) along c-axis and the relative bond length variation ΔX (≈0.1 Å) along a- and b-axes, respectively. The calculated g factors based on the above local structure are in good agreement with the experimental data. The relationships between the anisotropies of the g factors and the low symmetrical (orthorhombic) distortions of the Cu²⁺ site in Y₂BaCuO₅ are discussed.     

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.     

Effect of γ-irradiation on the frequency-independent parameters of an equivalent circuit for two-dimensional island platinum films

Four sets (A, B, C and D) of two-dimensional island platinum films [2D-I(Pt)Fs] were prepared by the thermal evaporation technique. The mass thicknesses for the films of such sets are 1, 5, 10 and 20 Å, respectively. The frequency-independent parameters of one of the equivalent circuit for island metal films in general are R _b, R _g and C _g where R _b is the resistance of the islands themselves while R _g is the resistance that accompanies the transfer of electrons across the gaps among islands and C _g is the capacitance that results from the separated metallic islands. The least-square values of R _b, R _g and C _g for our films were deduced via a computerized analysis. The stabilized films were exposed to γ-rays at doses 100, 200, 300, 500 and 700 Gy using ¹³⁷Cs (0.662 MeV) radiation source of dose rate 0.5 Gy/min. It was found that (1) For any particular mass thickness: (a) R _b and R _g decreases with the increase in the dose of γ-rays while C _g increases (b) the absolute value of the fractional change in R _b, R _g and C _g increases with the increase in dose. (2) For any particular dose the increase in the fractional change in R _b, R _g and C _g becomes more pronounced when the mass thickness increases. Qualitative interpretation for our results was given on the ground that γ-rays changed the island’s shape from spherical to prolate spheroid one and this has been checked experimentally via imaging the films by Atomic Force Microscope before and after γ-irradiations.     

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.     

Synthesis and thermoelectric characterization of bulk-type tellurium nanowire/polymer nanocomposites

A simple method to fabricate three-dimensionally (3-D) aligned thermoelectric nanowires attached polymer particle was demonstrated by combination of solution casting of thermoelectric nanostructures (e.g., tellurium nanowires (Te NWs)) on the surface of thermoplastic polymer (e.g., poly(methyl methacrylate (PMMA)) microbeads followed by hot compaction of thermoplastic matrix. The percolation threshold of composite with 3-D assembled Te NWs (i.e., 3.45 vol%) significantly was lower than that of a randomly dispersed Te NWs (i.e., 5.26 vol%), which resulted in an order of magnitude greater thermoelectric figure of merit (ZT of 2.8 × 10^?3) compared to randomly dispersed Te NWs in PMMA matrix (ZT of 6.4 × 10^?4) at room temperature by enhancing the electrical conductivity without increasing thermal conductivity.     

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.     

Development of new ionic gelation strategy: Towards the preparation of new monodisperse and stable hyaluronic acid/β-cyclodextrin-grafted chitosan nanoparticles as drug delivery carriers for doxorubicin

In the present study, β-cyclodextrin-grafted chitosan nanoparticles (β-CD-g-CS NPs) were prepared using a new ionic gelation strategy involving a synergistic effect of NaCl (150 mmol/L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mmol/L), and water bath sonication. This new strategy afforded smaller and more monodisperse β-CD-g-CS NPs vs. the classical ionic gelation method. New HA/β-CD-g-CS NPs were also prepared using the above-mentioned strategy by adding hyaluronic acid (HA) to the β-CD-g-CS copolymer at different weight ratios until the ZP values conversion. The best result was obtained with the weight ratio of w(HA):w(β-CD-g-CS) = 2:1 and furnished new spherical and smooth HA/β-CD-g-CS NPs. Furthermore, the stability of β- CD-g-CS NPs and HA/β-CD-g-CS NPs at 4°C in physiological medium (pH 7.4) was compared for 3 weeks period and showed that HA/β-CD-g-CS NPs were more stable all maintaining their monodispersity and high negative ZP values compared to β-CD-g-CS NPs. Finally, preliminary study of HA/β-CD-g-CS NPs as carrier for the controlled release of the anticancer drug doxorubicin was investigated. These new HA/β-CD-g-CS NPs can potentially be used as drug delivery and targeting systems for cancer treatment.     

The size and impact of the elite set of publications in scientometric assessments

Several scientometric impact indicators [total citations, h, g, and π-index, percentage rank position (PRP), weighted citation share (WCS)] of 190 elite papers of 15 members of the Hungarian Academy of Sciences active in three different fields were calculated. From the indices the PRP indicator proved to be independent of the citation practices in the fields. The PRP index of a journal paper can be calculated in per cent as unity minus (the rank number of the paper by citation frequency within the publishing journal minus one divided with the total number of papers in the journal) times hundred. The sum of the PRP index of the elite papers of a scientist may characterize his or her total publication performance. The size of the elite set of journal papers within the total was calculated by different methods. The h-index and g-index corresponds to the size of the elite, i.e. number of the elite papers according to the h-statistics and g-statistics, respectively. The number of papers in the π-set is equal to the square root of total papers. The π-index equals to one hundredth of citations to the π-set papers. In the present paper the size of the elite set is determined as the number of papers in the h-set, g-set, or π-set, and as 10 % of total papers, or number of papers cited 2, 3, or 5 times the mean citation rate (MCR) of the publishing journal. The π-citation threshold model is presented for demonstrating how MCR and the distribution of citations over the papers may influence the size of the elite set and the corresponding π-index. It was found that the scientific performances concluded from the π-index obtained from elite sets of different size are in good agreement.     

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.     

Visible-light-induced hydrogen evolution reaction with WS<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript>Se<Subscript>2−<Emphasis Type="Italic">x</Emphasis></Subscript>

WS₂ is a promising catalyst for the hydrogen evolution reaction. We have explored photocatalytic properties of ternary sulphoselenides of tungsten (WS _x Se _2?x ) by the dye-sensitized hydrogen evolution. WS _x Se _2?x solid solutions are found to exhibit high activity reaching 2339 μmol h^?1 g^?1 for WSSe, which is three times higher than that of WS₂ alone (866 μmol h^?1 g^?1 ). The turnover frequency is also high (0.7 h^?1 ). Such synergistic effect of selenium substitution in WS₂ is noteworthy.