Radiation-induced apoptosis in scid mice spleen after low dose irradiation.

To assess the radioadaptive response of the whole body system in mice, we examined the temporal effect of low dose priming as an indicator of challenging irradiation-induced apoptosis through a p53 tumor suppressor protein- mediated signal transduction pathway. The p53 protein also plays an important role both in cell cycle control and DNA repair through cellular signal transduction. Using severe combined immunodeficiency mice defective in DNA-dependent protein kinase catalytic subunit, we examined the role of DNA-dependent protein kinase activity in radioadaptation induced by low dose irradiation. Specific pathogen free 5-week-old female severe combined immunodeficiency mice and the parental mice (CB- 17 Icr +/+) were irradiated with X-ray at 3.0 Gy at 1, 2, 3 or 4 weeks after the conditioning irradiation at 0.15, 0.30, 0.45 or 0.60 Gy. The mice spleens were fixed for immunohistochemistry 12 h after the challenging irradiation. The p53-dependent apoptosis related Bax proteins on formalin-fixed paraffin-embedded sections were stained by the avidin-biotin peroxidase complex method. The apoptosis incidence in the sections was measured by hematoxylin-eosin staining. The frequency of Bax- and apoptosis-positive cells increased up to 12 h after the challenging irradiation in the spleen of both mice. However, these cells were not observed after a low dose irradiation at 0.15-0.60 Gy. When pre-irradiation at 0.45 Gy 2 weeks before the challenging irradiation at 3.0 Gy was performed, Bax accumulation and apoptosis induced by challenging irradiation were depressed in the spleens of CB-17 Icr +/+ mice, but not in severe combined immunodeficiency mice. These data suggest that DNA-dependent protein kinase might play a major role in radioadaptation induced by pre-irradiation with a low dose in mice spleen. We expect that the present findings will provide useful information in the health care of space crews.     

Cultures of human liver cells in simulated microgravity environment.

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.     

Radiation effects in nematodes: results from IML-1 experiments.

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA Biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Right sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.     

Fertilization and development of eggs of the South African clawed toad, Xenopus laevis, on sounding rockets in space.

Egg rotation and centrifugation experiments strongly suggest a role for gravity in the determination of the spatial structure of amphibian embryos. Decisive experiments can only be made in Space. Eggs of Xenopus laevis, the South African clawed toad, were the first vertebrate eggs which were successfully fertilized on Sounding Rockets in Space. Unfixed, newly fertilized eggs survived reentry, and a reasonable number showed a seemingly normal gastrulation but died between gastrulation and neurulation. Only a few reached the larval stage, but these developed abnormally. In the future, we intend to test whether this abnormal morphogenesis is due to reentry perturbations, or due to a real microgravity effect, through perturbation of the reinitiation of meiosis and other processes, or started by later sperm penetration.     

GNSS single frequency ionospheric range delay corrections: NeQuick data ingestion technique

A study of the performance of the NeQuick model and the Klobuchar model for GNSS single frequency range delay correction on a global scale was done using data for moderate solar activity. In this study NeQuick was used in the way intended for Galileo. This study is to assess the performance of the two models at each ionospheric geographic region during moderate solar activity as previously published studies were concentrated only on high solar activity. The results obtained showed that NeQuick outperformed Klobuchar for the whole year at the three geographical regions of the ionosphere. In terms of monthly root mean square of mismodeling, NeQuick outperformed Klobuchar by 15 TECU or more at low-latitudes, 5 TEC or more at mid-latitudes, and 1 TECU or more at high-latitudes.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.     

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.     

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.     

Early development of fern gametophytes in microgravity.

Dormant spores of the fern Ceratopteris richardii were flown on Shuttle mission STS-93 to evaluate the effects of micro-g on their development and on their pattern of gene expression. Prior to flight the spores were sterilized and sown into one of two environments: (1) Microscope slides in a video-microscopy module; and (2) Petri dishes. All spores were then stored in darkness until use. Spore germination was initiated on orbit after exposure to light. For the spores on microscope slides, cell level changes were recorded through the clear spore coat of the spores by video microscopy. After their exposure to light, spores in petri dishes were frozen in orbit at four different time points during which on earth gravity fixes the polarity of their development. Spores were then stored frozen in Biological Research in Canister units until recovery on earth. The RNAs from these cells and from 1-g control cells were extracted and analyzed on earth after flight to assay changes in gene expression. Video microscopy results revealed that the germinated spores developed normally in microgravity, although the polarity of their development, which is guided by gravity on earth, was random in space. Differential Display-PCR analyses of RNA extracted from space-flown cells showed that there was about a 5% change in the pattern of gene expression between cells developing in micro-g compared to those developing on earth.     

Parathyroid hormone-related protein is a gravisensor in lung and bone cell biology.

Parathyroid Hormone-related Protein (PTHrP) has been shown to be essential for the development and homeostatic regulation of lung and bone. Since both lung and bone structure and function are affected by microgravity, we hypothesized that 0 x g down-regulates PTHrP signaling. To test this hypothesis, we suspended lung and bone cells in the simulated microgravity environment of a Rotating Wall Vessel Bioreactor, which simulates microgravity, for up to 72 hours. During the first 8 hours of exposure to simulated 0 x g, PTHrP expression fell precipitously, decreasing by 80-90%; during the subsequent 64 hours, PTHrP expression remained at this newly established level of expression. PTHrP production decreased from 12 pg/ml/hour to 1 pg/ml/hour in culture medium from microgravity-exposed cells. The cells were then recultured at unit gravity for 24 hours, and PTHrP expression and production returned to normal levels. Based on these findings, we have obtained bones from rats flown in space for 2 weeks (Mission STS-58, SL-2). Analysis of PTHrP expression by femurs and tibias from these animals (n=5) revealed that PTHrP expression was 60% lower than in bones from control ground-based rats. Interestingly, there were no differences in PTHrP expression by parietal bone from space-exposed versus ground-based animals, indicating that the effect of weightlessness on PTHrP expression is due to the unweighting of weight-bearing bones. This finding is consistent with other studies of microgravity-induced osteoporosis. The loss of the PTHrP signaling mechanism may be corrected using chemical agents that up-regulate this pathway. In conclusion, PTHrP represents a stretch-sensitive paracrine signaling mechanism that may sense gravity.     

Mass exchange in an experimental new-generation life support system model based on biological regeneration of environment.

An experimental model of a biological life support system was used to evaluate qualitative and quantitative parameters of the internal mass exchange. The photosynthesizing unit included the higher plant component (wheat and radish), and the heterotrophic unit consisted of a soil-like substrate, California worms, mushrooms and microbial microflora. The gas mass exchange involved evolution of oxygen by the photosynthesizing component and its uptake by the heterotroph component along with the formation and maintaining of the SLS structure, growth of mushrooms and California worms, human respiration, and some other processes. Human presence in the system in the form of "virtual human" that at regular intervals took part in the respirative gas exchange during the experiment. Experimental data demonstrated good oxygen/carbon dioxide balance, and the closure of the cycles of these gases was almost complete. The water cycle was nearly 100% closed. The main components in the water mass exchange were transpiration water and the watering solution with mineral elements. Human consumption of the edible plant biomass (grains and roots) was simulated by processing these products by a unique physicochemical method of oxidizing them to inorganic mineral compounds, which were then returned into the system and fully assimilated by the plants. The oxidation was achieved by "wet combustion" of organic biomass, using hydrogen peroxide following a special procedure, which does not require high temperature and pressure. Hydrogen peroxide is produced from the water inside the system. The closure of the cycle was estimated for individual elements and compounds. Stoichiometric proportions are given for the main components included in the experimental model of the system. Approaches to the mathematical modeling of the cycling processes are discussed, using the data of the experimental model. Nitrogen, as a representative of biogenic elements, shows an almost 100% closure of the cycle inside the system. The proposed experimental model of a biological system is discussed as a candidate for potential application in the investigations aimed at creating ecosystems with largely closed cycles of the internal mass exchange. The formation and maintenance of sustainable cycling of vitally important chemical elements and compounds in biological life support systems (BLSS) is an extremely pressing problem. To attain the stable functioning of biological life support systems (BLSS) and to maintain a high degree of closure of material cycles in than, it is essential to understand the character of mass exchange processes and stoichiometnc proportions of the initial and synthesized components of the system.     

Red light-induced suppression of gravitropism in moss protonemata.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.     

Genetic changes in mammalian cells transformed by helium ions.

Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9-10 keV/micrometer). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells.     

Comparison of three soil-like substrate production techniques for a bioregenerative life support system

It is very important to recycle the inedible biomass of higher plants to improve the closure of bioregenerative life support system (BLSS). Processing candidate higher plant residues into the soil-like substrate (SLS) as the plant growth medium is a promising way to achieve. In this study, three different processing techniques of SLSs, using residues of wheat and rice as feedstock, were compared. As for the first traditional technique, SLS1 was obtained by successive conversion of wheat straw by oyster mushrooms and worms. In the other two methods, SLSs were produced with aerobic fermentation (SLS2) or anaerobic fermentation (SLS3) followed by worm conversion. The changes in SLS cellulose, lignin, available elements and pH were measured during the production processes. The maturity was evaluated by the value of C/N. The fertilities were compared in terms of available elements contents and lettuce productivities. The results indicated that the second technique was optimal, whose process cycle was 30 days less than that of SLS1. The total cellulose and lignin degradation of SLS2, achieved 98.6% and 93.1% during the 93-days-processing, and the lettuce productivity reached 12.0 g m⁻² day⁻¹.     

Effects of 40Ar and 56Fe ions on retinal photoreceptor cells of the rabbit: implications for manned missions to Mars.

Losses of photoreceptor cells (rods) from the retinas of New Zealand white (NZW) rabbits were detectable within 2 years after localized acute irradiation of optic and proximal tissues with > or = 7 Gy of 530 MeV u-1 40Ar ions or > or = 2 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition. Those limits were determined only from an analysis of variance of dose groups because the shapes of the dose response curves at early post-irradiation times are not known, a concern being addressed by experiments in progress. Losses of photoreceptor cells for the period 0.5-2.5 years post-irradiation, determined by provisional linear regression analysis, were approximately 1.7% Gy-1 and 2.5% Gy-1 for 40Ar and 56Fe ions, respectively.     

Simultaneous exposure of mammalian cells to heavy ions and X-rays.

Crews of space missions are exposed to a mixed radiation field, including sparsely and densely ionizing radiation. To determine the biological effectiveness of mixed high-/low-LET radiation fields, mammalian cells were exposed in vitro simultaneously to X-rays and heavy ions, accelerated at the HIMAC accelerator. X-ray doses ranged from 1 to 11 Gy. At the same time, cells were exposed to either 40Ar (550 MeV/n, 86 keV/micrometers), 28Si (100 MeV/n, 150 keV/micrometers), or 56Fe (115 MeV/n, 442 keV/micrometers) ions. Survival was measured in hamster V79 fibroblasts. Structural aberrations in chromosome 2 were measured by chemical-induced premature chromosome condensation combined with fluorescence in situ hybridization in isolated human lymphocytes. For argon and silicon experiments, measured damage in the mixed radiation field was consistent with the value expected using an additive function for low- and high-LET separated data. A small deviation from a simple additive function is observed with very high-LET iron ions combined to X-rays.     

Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells.

The effects of microgravity on Jurkat cells--a T-lymphoid cell line--was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a or 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.     

Integration of In-Situ Resource Utilization into lunar/Mars exploration through field analogs

The ability to extract and process resources at the site of exploration into useful products such as propellants, life support and power system consumables, and radiation and rocket exhaust plume debris shielding, known as In-Situ Resource Utilization or ISRU, has the potential to significantly reduce the launch mass, risk, and cost of robotic and human exploration of space. The incorporation of ISRU into missions can also significantly influence technology selection and system development in other areas such as power, life support, and propulsion. For example, the ability to extract or produce large amounts of oxygen and/or water in-situ could minimize the need to completely close life support air and water processing system cycles, change thermal and radiation protection of habitats, and influence propellant selection for ascent vehicles and surface propulsive hoppers. While concepts and even laboratory work on evaluating and developing ISRU techniques such as oxygen extraction from lunar regolith have been going on since before the Apollo 11 Moon landing, no ISRU system has ever flown in space, and only recently have ISRU technologies been developed at a scale and at a system level that is relevant to actual robotic and human mission applications. Because ISRU hardware and systems have never been demonstrated or utilized before on robotic or human missions, architecture and mission planners and surface system hardware developers are hesitant to rely on ISRU products and services that are critical to mission and system implementation success. To build confidence in ISRU systems for future missions and assess how ISRU systems can best influence and integrate with other surface system elements, NASA, with international partners, are performing analog field tests to understand how to take advantage of ISRU capabilities and benefits with the minimum of risk associated with introducing this game-changing approach to exploration. This paper will describe and review the results of four analog field tests (Moses Lake in 6/08, Mauna Kea in 11/08, Flagstaff in 9/09, and Mauna Kea in 1/10) that have begun the process of integrating ISRU into robotic and human exploration systems and missions, and propose future ISRU-related analog field test activities that can be performed in collaboration with non-US space agencies.     

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.