Changes in Gene Expression of E. coli under Conditions of Modeled Reduced Gravity

Relatively few studies have examined bacterial responses to the reduced gravity conditions that are experienced by bacteria grown in space. In this study, whole genome expression of Escherichia coli K12 under clinorotation (which models some of the conditions found under reduced gravity) was analyzed. We hypothesized that phenotypic differences at cellular and population levels under clinorotation (hereafter referred to as modeled reduced gravity) are directly coupled to changes in gene expression. Further, we hypothesized that these responses may be due to indirect effects of these environmental conditions on nutrient accessibility for bacteria. Overall, 430 genes were identified as significantly different between modeled reduced gravity conditions and controls. Up-regulated genes included those involved in the starvation response (csiD, cspD, ygaF, gabDTP, ygiG, fliY, cysK) and redirecting metabolism under starvation (ddpX, acs, actP, gdhA); responses to multiple stresses, such as acid stress (asr, yhiW), osmotic stress (yehZYW), oxidative stress (katE, btuDE); biofilm formation (lldR, lamB, yneA, fadB, ydeY); curli biosynthesis (csgDEF), and lipid biosynthesis (yfbEFG). Our results support the previously proposed hypothesis that under conditions of modeled reduced gravity, zones of nutrient depletion develop around bacteria eliciting responses similar to entrance into stationary phase which is generally characterized by expression of starvation inducible genes and genes associated with multiple stress responses.     

The effect of simulated microgravity on bacteria from the mir space station

The effects of simulated microgravity on two bacterial isolates, Sphingobacterium thalpophilium and Ralstonia pickettii (formerly Burkholderia pickettii), originally recovered from water systems aboard the Mir space station were examined. These bacteria were inoculated into water, high and low concentrations of nutrient broth and subjected to simulated microgravity conditions. S. thalpophilium (which was motile and had flagella) showed no significant differences between simulated microgravity and the normal gravity control regardless of the method of enumeration and medium. In contrast, for R. pickettii (that was non-motile and lacked flagella), there were significantly higher numbers in high nutrient broth under simulated microgravity compared to normal gravity. Conversely, when R. pikkettii was inoculated into water (i.e., starvation conditions) significantly lower numbers were found under simulated microgravity compared to normal gravity. Responses to microgravity depended on the strain used (e.g., the motile strain exhibited no response to microgravity, while the non-motile strain did), the method of enumeration, and the nutrient concentration of the medium. Under oligotrophic conditions, non-motile cells may remain in geostationary orbit and deplete nutrients in their vicinity, while in high nutrient medium, resources surrounding the cell may be sufficient so that high growth is observed until nutrients becoming limiting.     

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.     

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.     

Numerical Simulation of Condensation of Bubbles under Microgravity Conditions by Moving Mesh Method in the Double-staggered Grid

A numerical 2D method for simulation of two-phase flows including phase change under microgravity conditions is presented in this paper, with a level set method being coupled with the moving mesh method in the double-staggered grid systems. When the grid lines bend very much in a curvilinear grid, great errors may be generated by using the collocated grid or the staggered grid. So the double-staggered grid was adopted in this paper. The level set method is used to track the liquid–vapor interface. The numerical analysis is fulfilled by solving the Navier–Stokes equations using the SIMPLER method, and the surface tension force is modeled by a continuum surface force approximation. A comparison of the numerical results obtained with different numerical strategies shows that the double-staggered grid moving-mesh method presented in this paper is more accurate than that used previously in the collocated grid system. Based on the method presented in this paper, the condensation of a single bubble in the cold water under different level of gravity is simulated. The results show that the condensation process under the normal gravity condition is different from the condensation process under microgravity conditions. The whole condensation time is much longer under the normal gravity than under the microgravity conditions.     

An experimental investigation into liquid jetting modes and break-up mechanisms conducted in a new reduced gravity facility

Liquid jets, important to many industrial applications including various drop-on-demand processes, are experimentally produced in reduced gravity conditions and analysed to determine the mode the liquid jet is operating in. Three physically different modes of liquid jetting are observed along with their associated break up mechanisms and discussed. Additionally, a) the theoretical transition between jetting and the onset of chaotic dripping is shown to be valid over a range of Re and We numbers in a reduced gravity environment; b) the theoretical transition between a convective and an absolute instability acting on a liquid jet does not appear valid for high Reynolds number jets operating in reduced gravity; c) for the first time, chaotic dripping has been observed and documented in reduced gravity; and, d) a transition is shown to exist in reduced gravity between chaotic dripping and quasi-steady growth. The work has applications to systems operating in both reduced gravity and in select systems operating in normal gravity. The work has been highly successful and has demonstrated the utility of the new Australian test facility in providing adequate test time and reduced gravity conditions to study sensitive phenomena under reduced gravity conditions.     

Numerical Simulation of Bubble Dynamics in Microgravity

A numerical method for the simulation of two-phase flows under microgravity conditions is presented in this paper. The level set method is combined with the moving mesh method in a collocated grid to capture the moving interfaces of the two-phase flow, and a SIMPLER-based method is employed to numerically solve the complete incompressible Navier-Stokes equations, and the surface tension force is modeled by a continuum surface force approximation. Based on the numerical results, the coalescence process of two bubbles under microgravity conditions (10^???2×g) is compared to that under normal gravity, and the effect of gravities on the bubbles coalescence dynamics is analyzed. It is showed that the velocity fields inside and around the bubbles under different gravity conditions are quite similar, but the strength of vortices behind the bubbles in the normal gravity is much stronger than that under microgravity conditions. It is also found that under microgravity conditions, the time for two bubbles coalescence is much longer, and the deformation of bubbles is much less, than that under the normal gravity.     

Influence of buoyancy convection on solute distribution in Pd40Ni40P20 alloy

The differences in the solute distribution in microstructure of Pd40Ni40P20 alloy solidified on board a Chinese retrievable satellite and on the ground were studied. In comparison with those crystallized under normal gravity conditions (1 g), it was found that the P content was lower, but the Pd content was higher in the primary phase in microgravity conditions ( g). In the eutectic region the P content, however, was increased but the Pd content was decreased. The differences in solute distribution crystallized under 1 g and g conditions show the influence of buoyancy convection on the mass transport coefficient in liquids under normal gravity conditions. © 1998 Chapman & Hall     

Short-term microgravity to isolate graviperception in cells

In the fall of 1991 a series of drop-tower experiments in ZARM (Bremen) was devoted to behavioural responses of unicellular organisms to step-type transition from normal gravity to microgravity. Modules for simultaneous 4-fold video-recording were incorporated into the flight capsule. In the course of 25 flights, 100 sets of experiments, each holding 100 to 200 cells, were flown under various conditions with a technical success rate of 94% and about 80% of the cells accessible to evaluation in the laboratory. A major goal of the experiments was the assessment of parameters of locomotion (velocity, orientation) in the absence of the gravity vector. The data show that in two species, Paramecium and Loxodes, the properties of steady-state microgravity-swimming correspond to horizontal swimming under 1g-conditions. In a third species, Didinium, microgravity-swimming velocity exceeds 1 g-horizontal rates. The data are in agreement with an electrophysiological hypothesis of graviperception in cells.     

Ions and water transmembrane transport in nervous and testicular cultured cells in low gravity conditions

Aim of the present study was to investigate on the possible alterations induced by on ground modeled microgravity on ion-water transport proteins at cellular level. For the purpose we used astrocytes, C₆ line, neurons (NT₂ line from human teratocarcinoma) and testicular cells (germ cells, Sertoli cells, and Leydig cells; primary cultures from trypsinised prepuberal pig testes). Modeled microgravity was achieved by a desktop 3D Random Positioning Machine, cultures were kept rotating for 30′, 1h and 24h. After 30′, immunopositivity for the antibodies to Na⁺/K⁺ATPase and Na⁺/K⁺/Cl^? co-transporters was greatly diminished, the plasma membrane appeared to be altered, and the mitochondria inner cristae were disrupted. Immunostaining to the antibody to the water channel aquaporin 4 was very bright. After 1h at random rotation immunostaining for the heat shock protein Hsp27 was visible, After 24h, immunostaining for the ion transport proteins was again like that of the controls, plasma membrane and the mitochondria were again normal. Immunostaining for aquaporin 4 become again similar to that of the controls. We conclude that low gravity induces only transient alterations in the cell’s transmembrane ion-water transport: the cells are able to adapt to the gravity vector changes in few hours.     

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.     

Heat transfer enhancement of nanofluids in rotary blade coupling of four-wheel-drive vehicles

Summary This study adds CuO and Al₂O₃ nano particles and antifoam respectively into cooling engine oil. A comparison is made between their heat transfer performance and that of oil without adding such substances. The experimental platform is a real-time four-wheel-drive (4WD) transmisson system. It adopts advanced rotary blade coupling (RBC), where a high local temperature occurs easily at high rotating speed. Therefore, it is imperative to improve the heat transfer efficiency. Any resolution to such problems requires a thorough understanding of the thermal behavior of the rotating flow field within the power transmission system. The experiment measures the temperature distribution of RBC exterior at four different rotating speeds (400rpm, 800rpm, 1200rpm and 1600rpm), simulating the conditions of a real car at different rotating speeds and investigating the optimum possible compositions of a nanofluid for higher heat transfer performance.     

Bubble rupture in a vibrated liquid under microgravity

The response of an air bubble surrounded by a liquid in a sealed cell submitted to vibrations was investigated experimentally under microgravity conditions and compared to experiments under normal gravity conditions. As in normal gravity [1], it was observed that the bubble split into smaller parts when the acceleration of the vibrations reached a threshold. This threshold in microgravity is substantially smaller than that in normal gravity. Experimental results are presented in terms of an acceleration based Bond number which has been found to characterize the bubble behaviour in the laboratory experiments [1].     

A Hypergravity Environment Induced by Centrifugation Alters Plant Cell Proliferation and Growth in an Opposite Way to Microgravity

Seeds of Arabidopsis thaliana were exposed to hypergravity environments (2g and 6g) and germinated during centrifugation. Seedlings grew for 2 and 4 days before fixation. In all cases, comparisons were performed against an internal (subjected to rotational vibrations and other factors of the machine) and an external control at 1g. On seedlings grown in hypergravity the total length and the root length were measured. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation, which were quantified by counting the number of cells per millimeter in the specific cell files, and cell growth, which were appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus. The expression of cyclin B1, a key regulator of entry in mitosis, was assessed by the use of a CYCB1:GUS genetic construction. The results showed significant differences in some of these parameters when comparing the 1g internal rotational control with the 1g external control, indicating that the machine by itself was a source of alterations. When the effect of hypergravity was isolated from other environmental factors, by comparing the experimental conditions with the rotational control, cell proliferation appeared depleted, cell growth was increased and there was an enhanced expression of cyclin B1. The functional meaning of these effects is that cell proliferation and cell growth, which are strictly associated functions under normal 1g ground conditions, are uncoupled under hypergravity. This uncoupling was also described by us in previous experiments as an effect of microgravity, but in an opposite way. Furthermore, root meristems appear thicker in hypergravity-treated than in control samples, which can be related to changes in the cell wall induced by altered gravity.     

A bulk electrolysis Raman spectroelectrochemical cell using a rotating electrode

A fast bulk electrolysis Raman spectroelectrochemical cell is described. The cell employs a large-area platinum gauze and disk assembly which can be rotated at speeds up to 5,000 rpm. The complete electrolysis of a 5-mL solution can be achieved in less than 6 min using a 2,000 rpm rotation rate. The resonance Raman spectrum of (TPP*+)Cu(II) was collected in situ in this cell.     

Investigation of the effect of impeller speed on granules formed using a PMA-1 high shear granulator

Impeller speed was varied from 300 to 1500?rpm during the wet high shear granulation of a placebo formulation using a new vertical shaft PharmaMATRIX-1 granulator. The resulting granules were extensively analysed for differences caused by the varying impeller speed with emphasis on flowability. Microscopy showed that initial granules were formed primarily from microcrystalline cellulose at all tested impeller speeds. At low impeller speed of 300?rpm in the "bumpy" flow regime, forces from the impeller were insufficient to incorporate all the components of the formulation into the granules and to promote granule growth to a size that significantly improved flowability. The "roping" flow regime at higher impeller speeds promoted granule growth to a median particle size of at least 100 μm that improved the flowability of the mixture. Particle size distribution measurements and advanced indicators based on avalanching behavior, however, showed that an impeller speed of 700?rpm produced the largest fraction of optimal granules with the best flowability potential. This impeller speed allowed good development of "roping" flow for sufficient mixing, collision rates and kinetic energy for collisions while minimizing excessive centrifugal forces that promote buildup around the bowl perimeter.     

Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1?g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11?days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1?g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71?kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.     

Thyroid Cells Exposed to Simulated Microgravity Conditions – Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.