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.     

The “<Emphasis Type="Italic">Daphnia</Emphasis>” Lynx Mark I Suborbital Flight Experiment: Hardware Qualification at the Drop Tower Bremen

The Drop Tower Bremen, a ground-based facility enabling research under real microgravity conditions, is an excellent platform for testing new types of experimental hardware to ensure full performance when deployed in costly and rare flight opportunities such as suborbital flights. Here we describe the “Daphnia” experiment which will fly on XCOR Aerospace Lynx Mark I and our experience from the hardware tests with the catapult system at the drop tower. The aim of the “Daphnia” experiment is to obtain data on the biological performance of daphnids and predator-prey interactions in microgravity, which are important for the development of aquatic bioregenerative life support systems (BLSS). The experiment consists of two subunits: The first unit is dedicated to predator-prey interactions, where behavioural analysis should reveal if microgravity interfere with prey (Daphnia) detection or feeding and therefore may interrupt the trophic cascade. The functioning of such an artificial food web is indispensable for a long-lasting BLSS suitable for long-duration manned space missions or Earth-based explorations to extreme habitats. The second unit is designed to investigate the impact of microgravity on gene expression and the cytoskeleton in Daphnia. Next to data collection, the real microgravity conditions at the drop tower have helped to identify the weak points of the “Daphnia” experimental hardware and lead to further improvement. Hence, the drop tower is ideal for testing new experimental hardware which is indispensable before the implementation in suborbital flights.     

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.     

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.     

Annotated Gene and Proteome Data Support Recognition of Interconnections Between the Results of Different Experiments in Space Research

In a series of studies, human thyroid and endothelial cells exposed to real or simulated microgravity were analyzed in terms of changes in gene expression patterns or protein content. Due to the limitation of available cells in many space research experiments, comparative and control experiments had to be done in a serial manner. Therefore, detected genes or proteins were annotated with gene names and SwissProt numbers, in order to allow searches for interconnections between results obtained in different experiments by different methods. A crosscheck of several studies on the behavior of cytoskeletal genes and proteins suggested that clusters of cytoskeletal components change differently under the influence of microgravity and/or vibration in different cell types. The result that LOX and ISG15 gene expression were clearly altered during the Shenzhou-8 spaceflight mission could be estimated by comparison with the results of other experiments. The more than 100-fold down-regulation of LOX supports our hypothesis that the amount and stability of extracellular matrix have a great influence on the formation of three-dimensional aggregates under microgravity. The approximately 40-fold up-regulation of ISG15 cannot yet be explained in detail, but strongly suggests that ISGylation, an alternative form of posttranslational modification, plays a role in longterm cultures.     

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.     

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.     

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.     

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.     

On VLSI interconnect optimization and linear ordering problem

Some well-known VLSI interconnect optimizations problems for timing, power and cross-coupling noise immunity share a property that enables mapping them into a specialized Linear Ordering Problem (LOP). Unlike the general LOP problem which is NP-complete, this paper proves that the specialized one has a closed-form solution. Let f(x,y):?²→? be symmetric, non-negative, defined for x≥0 and y≥0, and let f(x,y) be twice differentiable, satisfying ? ² f(x,y)/?x?y<0. Let π be a permutation of {1,…,n}. The specialized LOP comprises n objects, each associated with a real value parameter r _i , 1≤i≤n, and a cost f(r _i ,r _j ) associated to any two objects if |π(i)?π(j)|=1,1≤i,j≤n, and f(r _i ,r _j )=0 otherwise. We show that the permutation π which minimizes \(\sum_{i= 1}^{n - 1} f( r_{\pi^{ - 1}( i )},r_{\pi^{ - 1}( i + 1 )} )\), called “symmetric hill”, is determined upfront by the relations between the parameter values r _i .     

Electrically Charged Droplets in Microgravity

In this work, the interaction between electrically charged droplets in microgravity is considered. During the 22 s of microgravity brought by a parabolic flight, water droplets with a radius r ∈ [0.41 ? 0.97] mm were released one in front of the other. A high-speed camera allowed studying their interaction in the focal plane. The trajectories of the droplets are well adjusted by a punctual charge model. In some experiments, a physical contact between the charged droplets was observed. These collisions are studied via a phase diagram comparing the droplet Weber number, We, and the collision parameter, χ. By comparing these collisions to experiments involving neutral droplets, we deduce how the collision diagram is affected by electric charges. In particular, we show that the criterion for an impact between two droplets is no more χ < 1.     

Effect of a protruding rod-supported disk on the drag of a nose-controlled cylinder

The drag C _x of a cylinder of diameter D with a front protruding disk supported on a rod of length l has been studied as a function of the relative distance l/D under the conditions of high (supersonic) flight velocities. It is established that the optimum (minimum) drug C _x exists, the value of which agrees with the results of numerical simulations.     

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.     

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.     

Hexagonal-Type Ising Nanowire with Core/Shell Structure Designed with Half-Integer Spins: Compensation Behaviors and Phase Diagrams in the Temperature and Interaction Planes

The effective field theory with correlation is used to investigate the magnetic behaviors of the mixed spin-1/2 and spin-3/2 hexagonal Ising nanowire (HIN) with core/shell in the crystal field. The total magnetization as a function of the temperature is used to describe the compensation behaviors of the system, and the N-, Q-, P-, R-, and S-type compensation types are given. The dependence of the phase diagrams on interaction parameters is studied in detail and presented the phase diagrams in the six different planes, namely (J ₁, Δ, T), (J _C, Δ, T), (J _S, Δ, T), (J ₁, J _C, T), (J ₁, J _S, T) and (J _C, J _S, T).Besides, the system exhibit second-order phase transition and first-order phase transitions, which can be found via the variations of the total magnetization with the crystal field in the mixed spin-1/2 and spin-3/2 HIN.     

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.