UV photobiochemistry under space conditions

The response of spores of Bacillus subtilis, cells of Deinococcus radiodurans and conidia of Aspergillus ochraceus to actual and simulated space conditions (UV in combination with long-term exposure to extremely dry conditions, including vacuum) has been studied: The following effects have been analyzed: decrease of viability, occurrence of DNA double strand breaks, formation of DNA-protein cross-links and DNA-DNA cross-links. All organisms show an increased sensitivity to UV light in extreme dryness (dry argon or vacuum) compared to an irradiation in aqueous suspension. The UV irradiation leads in all cases to a variety of DNA lesions. Very conspicuous is the occurrence of double strand breaks. Most of these double strand breaks are produced by incomplete repair of other lesions, especially base damages. The increase in DNA lesions can be correlated to the loss in viability. The specific response of the chromosomal DNA to UV irradiation in extreme dryness, however, varies from species to species and depends on the state of dehydration. The formation of DNA double strand breaks and DNA-protein cross-links prevails in the case of B. subtilis spores. In cells of Deinococcus radiodurans DNA-DNA cross-links often predominate, in conidia of Aspergillus ochraceus double strand breaks. The results obtained by direct exposure to space conditions (EURECA mission and D2 mission) largely agree with the laboratory data.     

Survival under space vacuum — Biochemical aspects

Exposure to vacuum predominantly causes the removal of water. As a consequence hydrophobic bonds (e.g. of membranes and proteins) are disrupted and metabolism practically comes to a complete halt. Removal of hydrate water also causes substantial changes regarding the structure of DNA (A-structure likely prevails). Some organisms, however, especially bacterial spores and fungal conidia are so well adapted to extreme dryness that substantial fractions of these organisms survive several months of vacuum even at room temperature. In these organisms some vacuum-induced alterations occur that are not readily reversed by readdition of water; mutations become evident and the amount of DNA covalently bound to protein is drastically increased. The mechanisms of these processes and their possible repair are not yet clear. There is evidence that chemical reactions (e.g. dehydration reactions) are involved although they likely proceed at an extremely low rate. Using the dehydration of serin by vacuum as a model system (the resulting amino acrylic acid is converted into pyruvic acid and ammonia after reexposure to water) we could establish that about 3 out of 100 000 serins are finally converted into pyruvic acid after exposure to 10⁻⁶ Torr for 1 week at 55°C. In dry Ar the corresponding rate is only about 1.5.     

ERA-experiment "Space Biochemistry".

The general goal of the experiment was to study the response of anhydrobiotic (metabolically dormant) microorganisms (spores of Bacillus subtilis, cells of Deinococcus radiodurans, conidia of Aspergillus species) and cellular constituents (plasmid DNA, proteins, purple membranes, amino acids, urea) to the extremely dehydrating conditions of open space, in some cases in combination with irradiation by solar UV-light. Methods of investigation included viability tests, analysis of DNA damages (strand breaks, DNA-protein cross-links) and analysis of chemical effects by spectroscopic, electrophoretic and chromatographic methods. The decrease in viability of the microorganisms was as expected from simulation experiments in the laboratory. Accordingly, it could be correlated with the increase in DNA damages. The purple membranes, amino acids and urea were not measurably effected by the dehydrating condition of open space (in the dark). Plasmid DNA, however, suffered a significant amount of strand breaks under these conditions. The response of these biomolecules to high fluences of short wavelength solar UV-light is very complex. Only a brief survey can be given in this paper. The data on the relatively good survival of some of the microorganisms call for strict observance of COSPAR Planetary Protection Regulations during interplanetary space missions.     

UV photobiochemistry of anhydrobiotic organisms at extremely low temperatures

Spores of Bacillus subtilis (TKJ 3412), cells of Deinococcus radiodurans R₁ (wild type) and conidia of Aspergillus ochraceus (strain 3174) have been UV irradiated (254 nm) in the dry state (3% relative humidity, argon) or in aqueous suspension at room temperature, at −55°C to −70°C and at −165°C to −170°C. The following effects have been analyzed: decrease in viability, occurrence of DNA strand breaks (pulsed-field gel electrophoresis) and production of DNA-protein cross-links (membrane filter method). The loss in viability is usually more pronounced at around −70°C than at room temperature, but it is lowest around −170°C. The kind of prevailing DNA damage varies from organism to organism. The amount of UV induced DNA-protein cross-link products steadily decreases with the temperature and is lowest at −170°C. The decrease in highly polymeric DNA by double strand breaks follows no universal pattern. The observed hypersensitivity of the three very different species at −70°C can therefore not be simply explained on the basis of the number of DNA lesions analyzed in the course of this work. We suggest that also the changing state of cellular water below and above about −130°C significantly contributes to the change in photosensitivity.     

Biochemical constraints for survival under Martian conditions.

A wide variety of terrestrial organisms, the so-called "anhydrobiotes," has learned to survive in a state of extreme dehydration in dry environments. Strategies for survival include the accumulation of certain polyols and nonreducing saccharides, which help to prevent damage to membranes and proteins, but at low water partial pressure DNA is also progressively damaged by various lesions, including strand breaks and cross-linking to proteins. These lesions, if they are not too numerous, can be repaired before the first replication step after rehydration, but long-term exposure to dry conditions finally diminishes the chances of survival as these lesions accumulate. If an organism has no chance to repair the accumulated DNA damage during intermittent periods of active life, survival will not exceed a few decades. The restriction of survival by dryness-induced DNA lesions is corroborated by new data on conidia of Aspergillus and the free plasmid pBR 322. Our results will be discussed with respect to the chance of finding dormant life or biochemical fossils on the surface of Mars.     

Survival in extreme dryness and DNA-single-strand breaks.

A wide variety of organisms (the so-called "anhydrobiotes') is able to survive long periods of time in a state of utmost dehydration and can thus survive in extremely dry environments including artificially imposed or space vacuum. Known strategies of survival include the accumulation of certain polyols, especially disaccharides, which help prevent damage to membranes and proteins. Here we report that DNA in vacuum-dried spores is damaged to a very substantial degree by processes leading to DNA strand breaks. Most of these lesions are obviously repaired during germination, but extensive damage to DNA and enzymes after long exposure times (months to years) finally diminish the chances of survival.     

DNA fragmentation by charged particle tracks.

High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons (60Co) or 125 keV/micrometers nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.     

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.     

Radiosensitivity to high energy iron ions is influenced by heterozygosity for Atm, Rad9 and Brca1

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Atm, Rad9 and Brca1 on cell oncogenic transformation and cell survival induced by 1 GeV/n⁵⁶Fe ions. Our results show that cells heterozygous for both Atm and Rad9 or Atm and Brca1 have high survival rates and are more sensitive to transformation by high energy iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose–response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation.     

基于优先级赤字轮询调度的WAIC网络延迟分析

航空电子机内无线通信（WAIC）在降低飞机重量和节省成本等方面的优势让其在航空电子系统的应用上具有可观的前景。为了研究基于802.11的WAIC网络的传输延迟并保证其可靠性,提出了一种优先级赤字轮询调度（PDRR）的介质访问控制（MAC）协议。首先,通过确定性网络演算方法为MAC层协议的活动建立了到达曲线和服务曲线模型。其次,充分考虑无线通信物理层的特点和所结合信道反转方法,给出了WAIC网络流量调度最坏情况下的端到端延迟的评价方法,可以发现信道反转后稳定的信道容量提供了较为保守的延迟界限。最后,通过案例分析对比了高优先级的WAIC节点与普通优先级节点的延迟界限以及信道反转的影响。结果表明:高优先级节点比普通优先级节点具有更好的实时性,并且可以通过增加平均信噪比来改善传输的延迟界限。      

Testing anti-fungal activity of a soil-like substrate for growing plants in bioregenerative life support systems

The object of this research is to study a soil-like substrate (SLS) to grow plants in a Bioregenerative Life Support System (BLSS). Wheat and rice straw were used as raw materials to prepare SLS. Anti-fungal activity of SLS using test cultures of Bipolaris sorokiniana, a plant-pathogenic fungus which causes wheat root rot was studied. Experiments were conducted with SLS samples, using natural soil and sand as controls. Infecting the substrates, was performed at two levels: the first level was done with wheat seeds carrying B. sorokiniana and the second level with seeds and additional conidia of B. sorokiniana from an outside source. We measured wheat disease incidence and severity in two crop plantings. Lowest disease incidence values were obtained from the second planting, SLS: 26% and 41% at the first and the second infection levels, respectively. For soil the values were 60% and 82%, respectively, and for sand they were 67% and 74%, respectively. Wheat root rot in the second crop planting on SLS, at both infection levels was considerably less severe (9% and 13%, respectively) than on natural soil (20% and 33%) and sand (22% and 32%). SLS significantly suppressed the germination of B. sorokiniana conidia. Conidia germination was 5% in aqueous SLS suspension, and 18% in clean water. No significant differences were found regarding the impact on conidia germination between the SLS samples obtained from wheat and rice straw. The anti-fungal activity in SLS increased because of the presence of worms. SLS also contained bacteria stimulating and inhibiting B. sorokiniana growth.     

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.     

Survival of microorganisms in space: A review

Spores of $\text{Bacillus subtilis}$ were exposed to selected factors of space (vacuum, solar UV radiation, heavy ions of cosmic radiation), and their response was studied after recovery. These investigations were supplemented by ground-based studies under simulated space conditions. The vacuum of space did not inactivate the spores. However, vacuum-induced structural changes in the DNA, and probably in the proteins, caused a supersensitivity to solar UV radiation. This phenomenon is caused by the production of specific photoproducts in DNA and protein, which cannot be removed by normal cellular repair processes. In vegetative bacterial cells, exposed to vacuum, cell dehydration led to damage of the cell membrane, which could be partly repaired during subsequent incubation. The high local effectiveness of the cosmic heavy ions further decreases the chance that spores can survive for any length of time in space. Nonetheless, a spore travelling through space and protected from ultraviolet radiation could possibly survive an interplanetary journey. Such a situation favors panspermia as a possible explanation for the origin of life.     

Alkylating agent (MNU)-induced mutation in space environment.

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.     

Low-dose carbon ion irradiation effects on DNA damage and oxidative stress in the mouse testis

To investigate the effects of low-dose carbon ion irradiation on reproductive system of mice, the testes of outbred Kunming strain mice were whole-body irradiated with 0, 0.05, 0.1, 0.5 and 1 Gy, respectively. We measured DNA double-strand breaks (DNA DSBs) and oxidative stress parameters including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and testis weight and sperm count at 12 h, 21 d and 35 d after irradiation in mouse testis. At 12 h postirradiation, a significant increase in DNA DSB level but no pronounced alterations in MDA content or SOD activity were observed in 0.5 and 1 Gy groups compared with the control group. At 21 d postirradiation, there was a significant reduction in sperm count and distinct enhancements of DSB level and MDA content in 0.5 and 1 Gy groups in comparison with control. At 35 d postirradiation, the levels of DNA DSBs and MDA, and SOD activity returned to the baseline except for the MDA content in 1 Gy (P < 0.05), while extreme falls of sperm count were still observed in 0.5 (P < 0.01) and 1 Gy (P < 0.01) groups. For the 0.05 or 0.1 Gy group, no differences were found in DNA DSB level and MDA content between control and at 12 h, 21 d and 35 d after irradiation, indicating that lower doses of carbon ion irradiation have no significant influence on spermatogenesis processes. In this study, male germ cells irradiated with over 0.5 Gy of carbon ions are difficult to repair completely marked by the sperm count. Furthermore, these data suggest that the deleterious effects may be chronic or delayed in reproductive system after whole-body exposure to acute high-dose carbon ions.     

Induction and rejoining of DNA double-strand breaks in CHO cells after heavy ion irradiation.

DNA double-strand breaks (DSBs) are the crucial events ultimately leading to cell inactivation. Aimed at understanding the biological action of the charged particle component of cosmic radiation, the induction of DSBs and their repairability was evaluated in Chinese hamster ovary (CHO-K1) cells after exposure to accelerated particles. Irradiations were performed with various ion species including O, Ni and Ca, covering a LET range from 20 to 2000 keV/micrometer. DSBs were determined for plateau-phase cells using the electrophoretic elution of radiation-induced DNA fragments in a static electric field combined with fluorescence scanning of ethidium bromide stained gels. Assuming a DSB yield of 22 DSB per Gy per cell, as derived from X-irradiation, cross-sections for DSB production were calculated from the corresponding fluence-effect curves at a fraction of 0.7 of DNA retained. The same ordinate was used as a reference for the calculation of relative biological efficiency (RBE) for DSB induction. At low LETs (< or = 20 keV/micrometer) RBE values slightly above unity were obtained, but a decrease of RBE was observed with increasing LET. In the region of 100-200 keV/micrometer the RBE for initial DSB induction was clearly below unity. Rejoining of DSBs was assessed by measuring the fraction of DNA retained following post-irradiation incubation of cells under culture conditions. After exposure to Ca ions, DSB rejoining was considerably impaired compared to X-rays.     

Microsatellite instability in human mammary epithelial cells transformed by heavy ions

We analyzed DNA and proteins obtained from normal and transformed human mammary epithelial cells for studying the neoplastic transformation by high-LET irradiation in vitro. We also examined microsatellite instability in human mammary cells transformed to various stages of carcinogenesis, such as normal, growth variant and tumorigenic, using microsatellite marker D5S177 on the chromosome 5 and CY17 on the Chromosome 10. Microsatellite instabilities were detected in the tumorigenic stage. These results suggest that microsatellite instability may play a role in the progression of tumorigenecity. The cause of the genomic instability has been suggested as abnormalities of DNA-repair systems which may be due to one of the three reasons: 1) alterations of cell cycle regulating genes. 2) mutations in any of the DNA mismatch repair genes. 3) mutation in any of the DNA strand breaks repair genes. No abnormality of these genes and encoded proteins, however was found in the present studies. These studies thus suggest that the microsatellite instability is induced by an alternative mechanism.     

Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO₂ incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK⁻) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.     

航天器AIT中心微生物多样性分析

航天器AIT（总装、集成和测试）中心的洁净度控制是防止造成航天器正向污染的重要保障.利用高通量测序技术与传统培养法比较分析了中国某航天器AIT中心的空气微生物组成与多样性.基于高通量的测序分析结果显示:该AIT中心空气中优势细菌以芽孢杆菌属为主,相对丰度78.47%±1.59%;优势真菌为银耳目,相对丰度8.97%±0.93%;基于培养法获得的优势细菌为葡萄球菌属,且未获得真菌培养物.空气微生物的chao1指数、Simpson多样性指数以及Shannon多样性指数分析显示,该AIT中心空气中细菌的多样性水平均高于真菌.      

Induction and repair of DNA double-strand breaks in human fibroblasts after particle irradiation.

Two assay were employed to study the induction and repair of DNA double-strand breaks (dsbs) in normal human fibroblasts after exposure to particle radiation covering an LET range from 1 to 350 keV/micrometer. The hybridization assay allows measurement of absolute induction frequencies in defined regions of the genome and quantitates rejoining of correct DNA ends while the FAR assay determines all rejoining events, correct and incorrect. Assuming Poisson statistics for the number of breaks per DNA fragment investigated, and thus neglecting any clustering of breaks, we found the induction rate to decrease with increasing LET of the particles. RBE values compared to 225 kVp X-rays dropped to 0.48 for the highest LETs. Repair studies of X-ray-induced dsbs showed that almost all breaks (>95%) are rejoined after incubation times of 24 h while the frequency for correct rejoining is only 70%. Thus about 25% of the initially induced breaks are rejoined by the connection of incorrect DNA ends. Postirradiation incubation after particle irradiation showed less efficient total rejoining with increasing LET and an impaired ability for correct rejoining. The frequency for rejoining of incorrect DNA ends was found to be independent of LET. The possible biological significance of the different rejoining events is discussed.