Clover development during spaceflight: a model system.

The development of legume root nodules was studied as a model system for the examination of gravitational effects on plant root development. In order to examine whether rhizobial association with clover roots can be achieved in microgravity, experiments were performed aboard the KC-135 parabolic aircraft and aboard the sounding rocket mission Consort 3. Binding of rhizobia to roots and the initial stages of root nodule development successfully occurred in microgravity. Seedling germination experiments were performed in the sliding block device, the Materials Dispersion Apparatus, aboard STS-37. When significant hydration of the seeds was achieved, normal rates of germination and seedling development were observed.     

Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.     

Comparison of the effects of spaceflight and hindlimb-suspension on rat pituitary vasopressin and brainstem norepinephrine content.

To compare actual spaceflight to ground-based simulation (hindlimb-suspension), we measured the norepinephrine (NE) content in A1, A2, A5 and A6 (locus coeruleus) and the vasopressin content in the neurohypophysial system. The experimental period was of 9 days' duration. The NE content in the locus coeruleus decreased significantly in rats flown for 9 days (67%, p < 0.001), but showed no significant changes after hindlimb-suspension. These results demonstrated that suspended rats adapted better to weightlessness-simulation than flown rats to actual microgravity. In rats flown aboard SLS-1, the vasopressin content was significantly increased in the posterior pituitary (71%, p < 0.01), and was decreased in the hypothalamus (49%, p < 0.05). In 9-day suspended rats pituitary vasopressin levels were unchanged, while in the hypothalamus a significant decrease was noted (21%, p < 0.05). It was concluded that spaceflight changes in pituitary vasopressin levels and in the locus coeruleus NE content were consistent with a stress reaction, occurring during and/or after landing. These results confirmed that hindlimb-suspension model constitutes a valid and less stressful [correction of lesstressful] ground-based simulation of microgravity in rats.     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

Quantitative interpretation of heavy ions effects: models for the biological effects of heavy ions.

Heavy ions are an important part of space radiation. Although they contribute only about 1 percent in number the fraction in terms of energy deposited is much higher. Also the quality of radiation is different from the other components since the LET is generally quite high. This poses the problem of Relative Biological Effectiveness (RBE). It is considerably more important in space than on earth because shielding measures are costly and sometimes not even feasible. Radiation hazards appear to be the limiting factor In long term space flights and their evaluation constitutes a major task. There is still no general agreement about RBE of earthbound radiation, and even less concerning the biological weighting of very heavy and very energetic ions in space. Because of the lack of experimental data--particularly for risk estimates in humans-- theoretical approaches may be very helpful in this respect and provide the only means to judge the radiation protection situation in outer space. In order to be useful careful checks of their consistency are necessary. This paper summarizes some of the more common approaches in a critical manner. The unhappy conclusion at the end will be that at present it is not possible to understand even heavy ion action on survival quantitatively with an acceptable precision.     

Overwintering in the Antarctica as an analog for long term manned spaceflight.

As a part of Polar Psychological Project planned by SCAR group on Antarctic-Space-Related-Human-Factors-Research, a series of psychological tests were conducted on the Japanese wintering-over personnel. The main purpose of the research was to study the behavioral characteristics, such as depression, insomnia, anxiety, and hostility, which might develop during the course of wintering-over period, and it involves a detailed study of the individuals and interpersonal relationship.     

Formation and vesiculation of biomembranes during spaceflight.

Shuttle flight, sounding rocket flight, and parabolic flight experiments demonstrate the formation of bilayer membrane vesicles (liposomes) in reduced gravity, following the dilution of detergent from detergent-phospholipid mixed micelles. The reduction in detergent concentration initiates assembly of bilayer membrane sheets, which are sensitive to solution disturbances. An increase in disturbances by forced dilution results in small diameter liposomes (< 150 nm), in both ground and flight samples. In the absence of forced dilution, liposomes remain small at 1-g, but exhibit much larger diameters at 0-g (1000-2000 nm). Our spaceflight data reveal that membrane assembly and vesiculation are strongly influenced by gravity-induced solution disturbances (e.g., convection currents), which limit vesicle diameter.     

The effects of HGMFs on the plant gravisensing system.

High Gradient Magnetic Fields (HGMFs) offer new opportunities for studying the gravitropic system of plants. However, it is necessary to analyze the influence that HGMF can have on cellular processes and structures that may not be related to amyloplasts displacement. This paper considers possible HGMF effects on plants, which may accompany HGMF stimulation of amyloplasts and contribute to the mechanisms of the HGMF-induced curvature.     

The C.E.B.A.S.-Minimodule: behaviour of an artificial aquatic ecological system during spaceflight.

The C.E.B.A.S.-Minimodule, a closed aquatic ecosystem integrated into a middeck locker and consisting of a Zoological (animal tanks), a Botanical (plant bioreactor), a Microbial (bacteria filter) and an Electronic Component (data acquisition/control system) was flown on the STS-89 spaceshuttle mission in January 1998 for 9 days. Preflight the plant bioreactor was loaded with 53 g of Ceratophyllum demersum (coontail) and the animal tanks with 4 adult pregnant females of the fish, Xiphophorus helleri (sword-tails), 200 juveniles of the same species less than 1 week of age, 38 large and 30 juvenile Biomphalaria glabrata water snails. The filter compartment was filled with 200 g of lava grain inoculated with laboratory strains of ammonia-oxidizing bacteria. A ground reference was undertaken with the same biological setup with a delay of 4 d. After an adaptation period of 5 d the system was closed and integrated into the spaceshuttle one day before launch. Video recordings of the animals were automatically taken for 10 minutes in 2-hour periods; the tapes were changed daily by the astronauts. The chemical and physical data for the aquatic system were within the expected range and were closely comparable in comparison to the ground reference. After 9 d under space conditions, the plant biomass increased to 117 g. The plants were all found in very good condition. All 4 adult female fish were retrieved in a good physiological condition. The juvenile fishes had a survival rate of about 33%. Almost 97% of the snails had survived and produced more than 250 neonates and 40 spawning packs. All samples were distributed according to a defined schedule and satisfied all scientific needs of the involved 12 principal investigators. This was the first successful spaceflight of an artificial aquatic ecosystem containing vertebrates, invertebrates, higher plants and microorganisms self-sustained by its inhabitants only. C.E.B.A.S. in a modified form and biological setup is a promising candidate for the early space station utilization as a first midterm experiment.     

Neuritogenesis: a model for space radiation effects on the central nervous system.

Pivotal to the astronauts' functional integrity and survival during long space flights are the strategies to deal with space radiations. The majority of the cellular studies in this area emphasize simple endpoints such as growth related events which, although useful to understand the nature of primary cell injury, have poor predictive value for extrapolation to more complex tissues such as the central nervous system (CNS). In order to assess the radiation damage on neural cell populations, we developed an in vitro model in which neuronal differentiation, neurite extension, and synaptogenesis occur under controlled conditions. The model exploits chick embryo neural explants to study the effects of radiations on neuritogenesis. In addition, neurobiological problems associated with long-term space flights are discussed.     

Improvements in the re-flight of spaceflight experiments on plant tropisms

In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.     

Comparison of the effects of two models for perpendicular diffusion on cosmic-ray latitudinal gradients

We compare the effects of two different models for perpendicular diffusion on the latitudinal gradients of galactic cosmic ray protons during solar minimum conditions. These two models correspond to the newly developed non-linear guiding center theory [Matthaeus, W.H., Qin, G., Bieber, J.W., Zank, G.P. Nonlinear collisionless perpendicular diffusion of charged particles. Astrophys. J. Lett., 590 (1), L53–L56, 2003] and the theory based on a velocity correlation function approach [Bieber, J.W., Matthaeus, W.H. Perpendicular diffusion and drift at intermediate cosmic-ray energies. Astrophys. J., 485 (2) 655–659, 1997]. In this ab initio study a steady-state two-dimensional numerical modulation model is used which incorporates a state-of-the-art turbulence model. We show that the non-linear guiding center theory predicts a mean free path that has a rigidity dependence that better accounts for the latitudinal gradients measured by Ulysses during its first fast latitude scan in 1994/1995.     

Comparative study of spermatogonial survival after x-ray exposure, high LET (HZE) irradiation or spaceflight.

Spermatogonial cell loss has been observed in rats flown on Space Lab 3, Cosmos 1887, Cosmos 2044 and in mice following irradiation with X-ray or with high energy (HZE) particle beams. Spermatogonial loss is determined by cell counting in maturation stage 6 seminiferous [correction of seminferous] tubules. With the exception of Iron, laboratory irradiation experiments (with mice) revealed a similar pattern of spermatogonial loss proportional to the radiation dose at levels less than 0.1 Gy. Helium and Argon irradiation resulted in a 5% loss of spermatogonia after only 0.01 Gy exposure. However, significant spermatogonial loss (45%) occured at this radiation level with Iron particle beams. The loss of spermatogonia during each space flight was less than 10% when compared to control (non-flight) animals. This loss, although small, was significant. Although radiation may be a contributing factor in the loss of spermatogonia during space flight, exposure levels, as determined by dosimetry, were not significant to account for the total cell loss observed.     

Growth protocols for etiolated soybeans germinated within BRIC-60 canisters under spaceflight conditions.

As part of the GENEX (Gene Expression) spaceflight experiment, protocols were developed to optimize the inflight germination and subsequent growth of 192 soybean (Glycine max cv McCall) seeds during STS-87. We describe a method which provided uniform growth and development of etiolated seedlings while eliminating root and shoot restrictions for short-term (4-7 day) experiments. Final seedling growth morphologies and the gaseous CO2 and ethylene levels present both on the last day in space and at the time of recovery within the spaceflight and ground control BRIC-60 canisters are presented.     

Embryogenesis, hatching and larval development of Artemia during orbital spaceflight.

Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degrees of development can take place in this microgravity environment.     

Influence of different atmospheric models and seeing effects on the nonlinear calibration

This paper describes a technique intended to improve calibration of vector magnetograms achieved by the spectral line Fei 5324.19 Å which was first described in detail by Hagyard and Kineke [Hagyard, M.J., Kineke, J.I., Improved method for calibrating filter vector magnetographs, Solar Phys., 158, 11–28, 1995.]. The technique attempts to compensate for nonlinear behavior of the measured linear and circular polarizations of the line as functions of inclination angle and field strength. This method depends on different atmospheric models. To the three different atmospheric models, the VAL-C quiet photosphere model, the Ding and Fang penumbra model and Allen umbral model, the circular polarization signal is directly linearly proportional to the longitudinal field strength in the range of 800 G, 1000 G and 1300 G, respectively. For the transverse field, however, this linear relationship holds over much smaller ranges which are 200 G, 300 G and 200 G. The improved method improves the accuracy in calibrating the polarization signals over a wide range of field strengths and inclination angles. With the seeing effects decreasing in the magnetic observation, the longitudinal fields and the transverse ones are underestimated. The differences in the zero-iterative longitudinal and transverse field are relatively minor, but differences in the multi-iterative longitudinal and transverse field can be as high as 260 G and 500 G. The improved calibration method is more sensitive to the seeing effects variation than the former calibration method.     

A ground-based study for a shuttle BRIC experiment on gravity effects on gene expression.

A BRIC (Biological Research In a Canister) experiment to investigate the effects of reduced gravity at the molecular level using Arabidopsis has been initiated. In preparation for a space flight experiment, a series of ground-based studies were conducted. Results from these studies indicate that: 1) up to 20,000 seeds can be germinated on a 100 mm diameter Petri plate, 2) nylon membrane is the best surface for recovery of plant material after freezing, 3) depending on the age of the seedlings at the time of freezing, 20 to 40 g of tissue can be obtained from Petri plates that fit in a single canister; 4) tissue from one canister yields adequate amounts of RNA to perform differential display to isolate gravity-regulated genes. Our results indicate that the proposed BRIC experiment is feasible and can provide valuable information on the possible effects of microgravity on gene regulation.     

Animal research facility for Space Station Freedom.

An integrated animal research facility is planned by NASA for Space Station Freedom which will permit long-term, man-tended experiments on the effects of space conditions on vertebrates. The key element in this facility is a standard type animal habitat which supports and maintains the animals under full bioisolation during transport and during the experiment. A holding unit accommodates the habitats with animals to be maintained at zero gravity; and a centrifuge, those to be maintained at artificial gravity for control purposes or for gravity threshold studies. A glovebox permits handling of the animals for experimental purposes and for transfer to a clean habitat. These facilities are described, and the aspects of environmental control, monitoring, and bioisolation are discussed.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.