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1.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
2.  The Effects of Simulated Microgravity on Intervertebral Disc Degeneration 
Astronauts experience back pain, particularly low back pain, during and after spaceflight. Recent studies have described histological and biochemical changes in rat intervertebral discs after space travel, but there is still no in vitro model to investigate the effects of microgravity on disc metabolism.
To study the effects of microgravity on disc degeneration and to establish an in vitro simulated microgravity study model
Discs were cultured in static and rotating conditions in bioreactor, and the characteristics of disc degeneration were evaluated
The mice discs were cultured in a rotating wall vessel bioreactor where the microgravity condition was simulated. Intervertebral discs were cultured in static and microgravity condition. Histology, biochemistry, and immunohistochemical assays were performed to evaluate the characteristics of the discs in microgravity condition.
Intervertebral discs cultured in rotating bioreactors were found to develop changes of disc degeneration manifested by reduced red Safranin-o staining within the annulus fibrosus, downregulated GAG content and GAG/Hypro ratio, increased MMP-3 expression, and upregulated apoptosis.
We conclude that simulated microgravity induces the molecular changes of disc degeneration. The rotating bioreactor model will provide a foundation to investigate the effects of microgravity on disc metabolism.
PMCID: PMC3612270  PMID: 23537452
intervertebral disc; microgravity; disc degeneration; apoptosis; extracellular matrix
3.  Characterizing the Effects of VPA, VC and RCCS on Rabbit Keratocytes onto Decellularized Bovine Cornea 
PLoS ONE  2012;7(11):e50114.
To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH4OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
PMCID: PMC3510233  PMID: 23209652
4.  Ground-Based Facilities for Simulation of Microgravity: Organism-Specific Recommendations for Their Use, and Recommended Terminology 
Astrobiology  2013;13(1):1-17.
Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature. Key Words: 2-D clinostat—3-D clinostat—Gravity—Magnetic levitation—Random positioning machine—Simulated microgravity—Space biology. Astrobiology 13, 1–17.
PMCID: PMC3549630  PMID: 23252378
5.  Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station 
The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.
PMCID: PMC3650649  PMID: 19609626
Immune response; Space flight; Microgravity
6.  Role and Regulation of σs in General Resistance Conferred by Low-Shear Simulated Microgravity in Escherichia coli 
Journal of Bacteriology  2004;186(24):8207-8212.
Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes. Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant. Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts. As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet. The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is σs dependent, irrespective of the growth phase. However, SMG-induced increased resistance was dependent on σs only in stationary phase, being independent of this sigma factor in exponential phase. σs concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells. While SMG affected σs synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on σs levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency. Since σs regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.
PMCID: PMC532419  PMID: 15576768
7.  Enhancement of Osteogenic Differentiation and Proliferation in Human Mesenchymal Stem Cells by a Modified Low Intensity Ultrasound Stimulation under Simulated Microgravity 
PLoS ONE  2013;8(9):e73914.
Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs). Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC) in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG) and treated with LIPUS at 30mW/cm2 for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01) and ALP activity by 22% (p<0.01), while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001) and mineralization by 45% (p<0.001) in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.
PMCID: PMC3772078  PMID: 24069248
8.  Impact of simulated microgravity on the normal developmental time line of an animal-bacteria symbiosis 
Scientific Reports  2013;3:1340.
The microgravity environment during space flight imposes numerous adverse effects on animal and microbial physiology. It is unclear, however, how microgravity impacts those cellular interactions between mutualistic microbes and their hosts. Here, we used the symbiosis between the host squid Euprymna scolopes and its luminescent bacterium Vibrio fischeri as a model system. We examined the impact of simulated microgravity on the timeline of bacteria-induced development in the host light organ, the site of the symbiosis. To simulate the microgravity environment, host squid and symbiosis-competent bacteria were incubated together in high-aspect ratio rotating wall vessel bioreactors and examined throughout the early stages of the bacteria-induced morphogenesis. The host innate immune response was suppressed under simulated microgravity; however, there was an acceleration of bacteria-induced apoptosis and regression in the host tissues. These results suggest that the space flight environment may alter the cellular interactions between animal hosts and their natural healthy microbiome.
PMCID: PMC3581829  PMID: 23439280
9.  Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings 
BMC Plant Biology  2013;13:124.
Cell growth and cell proliferation are intimately linked in the presence of Earth’s gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter.
We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells.
In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.
PMCID: PMC3847623  PMID: 24006876
10.  Nitrogen metabolism in haloarchaea 
Saline Systems  2008;4:9.
The nitrogen cycle (N-cycle), principally supported by prokaryotes, involves different redox reactions mainly focused on assimilatory purposes or respiratory processes for energy conservation. As the N-cycle has important environmental implications, this biogeochemical cycle has become a major research topic during the last few years. However, although N-cycle metabolic pathways have been studied extensively in Bacteria or Eukarya, relatively little is known in the Archaea. Halophilic Archaea are the predominant microorganisms in hot and hypersaline environments such as salted lakes, hot springs or salted ponds. Consequently, the denitrifying haloarchaea that sustain the nitrogen cycle under these conditions have emerged as an important target for research aimed at understanding microbial life in these extreme environments.
The haloarchaeon Haloferax mediterranei was isolated 20 years ago from Santa Pola salted ponds (Alicante, Spain). It was described as a denitrifier and it is also able to grow using NO3-, NO2- or NH4+ as inorganic nitrogen sources. This review summarizes the advances that have been made in understanding the N-cycle in halophilic archaea using Hfx mediterranei as a haloarchaeal model. The results obtained show that this microorganism could be very attractive for bioremediation applications in those areas where high salt, nitrate and nitrite concentrations are found in ground waters and soils.
PMCID: PMC2483277  PMID: 18593475
11.  Morphological and Physiological Changes in Mature In Vitro Neuronal Networks towards Exposure to Short-, Middle- or Long-Term Simulated Microgravity 
PLoS ONE  2013;8(9):e73857.
One of the objectives of the current international space programmes is to investigate the possible effects of the space environment on the crew health. The aim of this work was to assess the particular effects of simulated microgravity on mature primary neuronal networks and specially their plasticity and connectivity. For this purpose, primary mouse neurons were first grown for 10 days as a dense network before being placed in the Random Positioning Machine (RPM), simulating microgravity. These cultures were then used to investigate the impact of short- (1 h), middle- (24 h) and long-term (10 days) exposure to microgravity at the level of neurite network density, cell morphology and motility as well as cytoskeleton properties in established two-dimensional mature neuronal networks. Image processing analysis of dense neuronal networks exposed to simulated microgravity and their subsequent recovery under ground conditions revealed different neuronal responses depending on the duration period of exposure. After short- and middle-term exposures to simulated microgravity, changes in neurite network, neuron morphology and viability were observed with significant alterations followed by fast recovery processes. Long exposure to simulated microgravity revealed a high adaptation of single neurons to the new gravity conditions as well as a partial adaptation of neuronal networks. This latter was concomitant to an increase of apoptosis. However, neurons and neuronal networks exposed for long-term to simulated microgravity required longer recovery time to re-adapt to the ground gravity. In conclusion, a clear modulation in neuronal plasticity was evidenced through morphological and physiological changes in primary neuronal cultures during and after simulated microgravity exposure. These changes were dependent on the duration of exposure to microgravity.
PMCID: PMC3774774  PMID: 24066080
12.  Simulated Microgravity Compromises Mouse Oocyte Maturation by Disrupting Meiotic Spindle Organization and Inducing Cytoplasmic Blebbing 
PLoS ONE  2011;6(7):e22214.
In the present study, we discovered that mouse oocyte maturation was inhibited by simulated microgravity via disturbing spindle organization. We cultured mouse oocytes under microgravity condition simulated by NASA's rotary cell culture system, examined the maturation rate and observed the spindle morphology (organization of cytoskeleton) during the mouse oocytes meiotic maturation. While the rate of germinal vesicle breakdown did not differ between 1 g gravity and simulated microgravity, rate of oocyte maturation decreased significantly in simulated microgravity. The rate of maturation was 8.94% in simulated microgravity and was 73.0% in 1 g gravity. The results show that the maturation of mouse oocytes in vitro was inhibited by the simulated microgravity. The spindle morphology observation shows that the microtubules and chromosomes can not form a complete spindle during oocyte meiotic maturation under simulated microgravity. And the disorder of γ-tubulin may partially result in disorganization of microtubules under simulated microgravity. These observations suggest that the meiotic spindle organization is gravity dependent. Although the spindle organization was disrupted by simulated microgravity, the function and organization of microfilaments were not pronouncedly affected by simulated microgravity. And we found that simulated microgravity induced oocytes cytoplasmic blebbing via an unknown mechanism. Transmission electron microscope detection showed that the components of the blebs were identified with the cytoplasm. Collectively, these results indicated that the simulated microgravity inhibits mouse oocyte maturation via disturbing spindle organization and inducing cytoplasmic blebbing.
PMCID: PMC3135614  PMID: 21765954
13.  The Brazilian Research and Teaching Center in Biomedicine and Aerospace Biomedical Engineering 
Hippokratia  2008;12(Suppl 1):32-36.
The recent engagement of Brazil in the construction and utilization of the International Space Station has motivated several Brazilian research institutions and universities to establish study centers related to Space Sciences. The Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS) is no exception.
Method: The University initiated in 1993 the first degree course training students to operate commercial aircraft in South America (the School of Aeronautical Sciences. A further step was the decision to build the first Brazilian laboratory dedicated to the conduct of experiments in ground-based microgravity simulation. Established in 1998, the Microgravity Laboratory, which was located in the Instituto de Pesquisas Cientificas e Tecnologicas (IPCT), was supported by the Schools of Medicine, Aeronautical Sciences and Electrical Engineering/Biomedical Engineering. At the end of 2006, the Microgravity Laboratory became a Center and was transferred to the School of Engineering.
Results: The principal activities of the Microgravity Centre are the development of research projects related to human physiology before, during and after ground-based microgravity simulation and parabolic flights, to aviation medicine in the 21st century and to aerospace biomedical engineering.
Conclusion: The history of Brazilian, and why not say worldwide, space science should unquestionably go through PUCRS. As time passes, the pioneering spirit of our University in the aerospace area has become undeniable. This is due to the group of professionals, students, technicians and staff in general that have once worked or are still working in the Center of Microgravity, a group of faculty and students that excel in their undeniable technical-scientific qualifications.
PMCID: PMC2577397  PMID: 19048090
Microgravity; space life sciences; research center; space biomedicine
14.  NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure 
PLoS ONE  2011;6(11):e26603.
The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.
PMCID: PMC3212516  PMID: 22096490
15.  Space Microbiology 
Summary: The responses of microorganisms (viruses, bacterial cells, bacterial and fungal spores, and lichens) to selected factors of space (microgravity, galactic cosmic radiation, solar UV radiation, and space vacuum) were determined in space and laboratory simulation experiments. In general, microorganisms tend to thrive in the space flight environment in terms of enhanced growth parameters and a demonstrated ability to proliferate in the presence of normally inhibitory levels of antibiotics. The mechanisms responsible for the observed biological responses, however, are not yet fully understood. A hypothesized interaction of microgravity with radiation-induced DNA repair processes was experimentally refuted. The survival of microorganisms in outer space was investigated to tackle questions on the upper boundary of the biosphere and on the likelihood of interplanetary transport of microorganisms. It was found that extraterrestrial solar UV radiation was the most deleterious factor of space. Among all organisms tested, only lichens (Rhizocarpon geographicum and Xanthoria elegans) maintained full viability after 2 weeks in outer space, whereas all other test systems were inactivated by orders of magnitude. Using optical filters and spores of Bacillus subtilis as a biological UV dosimeter, it was found that the current ozone layer reduces the biological effectiveness of solar UV by 3 orders of magnitude. If shielded against solar UV, spores of B. subtilis were capable of surviving in space for up to 6 years, especially if embedded in clay or meteorite powder (artificial meteorites). The data support the likelihood of interplanetary transfer of microorganisms within meteorites, the so-called lithopanspermia hypothesis.
PMCID: PMC2832349  PMID: 20197502
16.  Proteomic Analysis of Mouse Hypothalamus under Simulated Microgravity 
Neurochemical research  2008;33(11):2335-2341.
Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.
PMCID: PMC2740374  PMID: 18473167
Brain; Hypothalamus; Microgravity
17.  The effect of low shear force on the virulence potential of Yersinia pestis: new aspects that space-like growth conditions and the final frontier can teach us about a formidable pathogen 
Manned space exploration has created a need to evaluate the effects of space-like stress (SLS) on pathogenic and opportunistic microbes. Interestingly, several Gram-negative enteric pathogens, e.g., Salmonella enterica serovar Typhimurium, have revealed a transient hyper-virulent phenotype following simulated microgravity (SMG) or actual space flight exposures. We have explored the virulence potential of Yersinia pestis KIM/D27 (YP) following exposure to mechanical low shear forces associated with SMG. Our experimental results demonstrated that SMG-grown YP was decreased in its induced HeLa cell cytotoxicity, suggesting that SMG somehow compromises T3SS functions. This was confirmed by an actual reduced amount of effector protein production and secretion through the T3SS injectisome. Also, SMG-grown YP proliferated less than their NG-grown counterparts did during an 8-h macrophage infection. Presently, we are evaluating the influence of SMG on various KIM/D27 mutant strains to further understanding of our initial phenomenology described above. Taken together, characterizing YP grown under the low shear forces of SMG can provide new insights into its pathogenesis and potentially uncover new targets that could be exploited for the development of novel antimicrobials as well as potential live-attenuated vaccines.
PMCID: PMC3417468  PMID: 22919696
simulated microgravity; Yersinia pestis; type three secretion system; high aspect ratio vessel; low shear forces
18.  Suboptimal evolutionary novel environments promote singular altered gravity responses of transcriptome during Drosophila metamorphosis 
Previous experiments have shown that the reduced gravity aboard the International Space Station (ISS) causes important alterations in Drosophila gene expression. These changes were shown to be intimately linked to environmental space-flight related constraints.
Here, we use an array of different techniques for ground-based simulation of microgravity effects to assess the effect of suboptimal environmental conditions on the gene expression of Drosophila in reduced gravity. A global and integrative analysis, using “gene expression dynamics inspector” (GEDI) self-organizing maps, reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. Although the genes that are affected are different in each simulation technique, we find that the same gene ontology groups, including at least one large multigene family related with behavior, stress response or organogenesis, are over represented in each case.
These results suggest that the transcriptome as a whole can be finely tuned to gravity force. In optimum environmental conditions, the alteration of gravity has only mild effects on gene expression but when environmental conditions are far from optimal, the gene expression must be tuned greatly and effects become more robust, probably linked to the lack of experience of organisms exposed to evolutionary novel environments such as a gravitational free one.
PMCID: PMC3716659  PMID: 23806134
Evolutionary genomics; Gene family evolution; Microgravity-hypergravity; Magnetic levitation; Gene expression; Microarray
19.  Haloarchaeal-Type β-Ketothiolases Involved in Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthesis in Haloferax mediterranei 
Applied and Environmental Microbiology  2013;79(17):5104-5111.
The key enzymes for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis in haloarchaea have been identified except the β-ketothiolase(s), which condense two acetyl coenzyme A (acetyl-CoA) molecules to acetoacetyl-CoA, or one acetyl-CoA and one propionyl-CoA to 3-ketovaleryl-CoA. Whole-genome analysis has revealed eight potential β-ketothiolase genes in the haloarchaeon Haloferax mediterranei, among which the PHBV-specific BktB and PhaA were identified by gene knockout and complementation analysis. Unlike all known bacterial counterparts encoded by a single gene, the haloarchaeal PhaA that was involved in acetoacetyl-CoA generation, was composed of two different types of subunits (PhaAα and PhaAβ) and encoded by the cotranscribed HFX_1023 (phaAα) and HFX_1022 (phaAβ) genes. Similarly, the BktB that was involved in generation of acetoacetyl-CoA and 3-ketovaleryl-CoA, was also composed of two different types of subunits (BktBα and BktBβ) and encoded by cotranscribed HFX_6004 (bktBα) and HFX_6003 (bktBβ). BktBα and PhaAα were the catalytic subunits and determined substrate specificities of BktB and PhaA, respectively. Their catalytic triad “Ser-His-His” was distinct from the bacterial “Cys-His-Cys.” BktBβ and PhaAβ both contained an oligosaccharide-binding fold domain, which was essential for the β-ketothiolase activity. Interestingly, BktBβ and PhaAβ were functionally interchangeable, although PhaAβ preferred functioning with PhaAα. In addition, BktB showed biotechnological potential for the production of PHBV with the desired 3-hydroxyvalerate fraction in haloarchaea. This is the first report of the haloarchaeal type of PHBV-specific β-ketothiolases, which are distinct from their bacterial counterparts in both subunit composition and catalytic residues.
PMCID: PMC3753943  PMID: 23793631
20.  Characterization of CRISPR RNA Biogenesis and Cas6 Cleavage-Mediated Inhibition of a Provirus in the Haloarchaeon Haloferax mediterranei 
Journal of Bacteriology  2013;195(4):867-875.
The adaptive immune system comprising CRISPR (clustered regularly interspaced short palindromic repeats) arrays and cas (CRISPR-associated) genes has been discovered in a wide range of bacteria and archaea and has recently attracted comprehensive investigations. However, the subtype I-B CRISPR-Cas system in haloarchaea has been less characterized. Here, we investigated Cas6-mediated RNA processing in Haloferax mediterranei. The Cas6 cleavage site, as well as the CRISPR transcription start site, was experimentally determined, and processing of CRISPR transcripts was detected with a progressively increasing pattern from early log to stationary phase. With genetic approaches, we discovered that the lack of Cas1, Cas3, or Cas4 unexpectedly resulted in a decrease of CRISPR transcripts, while Cas5, Cas6, and Cas7 were found to be essential in stabilizing mature CRISPR RNA (crRNA). Intriguingly, we observed a CRISPR- and Cas3-independent inhibition of a defective provirus, in which the putative Cascade (CRISPR-associated complex for antiviral defense) proteins (Cas5, Cas6, Cas7, and Cas8b) were indispensably required. A sequence carried by a proviral transcript was found to be homologous to the CRISPR repeat RNA and vulnerable to Cas6-mediated cleavage, implying a distinct interference mechanism that may account for this unusual inhibition. These results provide fundamental information for the subtype I-B CRISPR-Cas system in halophilic archaea and suggest diversified mechanisms and multiple physiological functions for the CRISPR-Cas system.
PMCID: PMC3562093  PMID: 23243301
21.  Radiation-associated cardiovascular risks for future deep-space missions 
Journal of Radiation Research  2014;55(Suppl 1):i37-i39.
Background: During the future Moon and Mars missions, astronauts will be exposed to space radiation (IR) for extended time. The majority of space flight-associated risks identified for the cardiovascular (CV) system to date were determined shortly after low Earth orbit (LEO) short- and long-duration space flights that include: serious cardiac dysrhythmias, compromised orthostatic CV response and manifestation of previously asymptomatic CV disease. Further ground-based experiments using a surrogate model of microgravity supported the space flight data for significant cardiac remodeling due to prolonged exposure to microgravity. These symptoms were determined to be a consequence of adaptation to microgravity that could be ameliorated by a post-mission exercise program, and were not identified as risk factors that were causatively related to space IR. Long-term degenerative effects of cosmic IR during and after space flights on CV system are unknown.
It was suggested that due to GCR, each cell in an astronaut's body will be traversed by 1H every 3 days, helium (2He) nuclei every few weeks and high charge and energy (HZE) nuclei (e.g. 28Si, 56Fe) every few months. Despite the fact that only 1% of GCR is composed of ions heavier than helium, ∼41% of the IR dose-equivalent is predicted to be HZE particles with 13% being from 56Fe particles, only. During an exploration-class space mission to Mars, astronauts will not have access to comprehensive healthcare services for a period of at least 2–3 years. Since the majority of experienced astronauts are middle-aged (average age is 46, and the range is 33–58 years), they are at risk for developing serious CV events which could be life-threatening for the astronaut and mission-threatening for NASA. Therefore, it is important to evaluate the effects and potential CV risks caused by space IR. We hypothesized that: (i) low-dose space IR-induced biological responses may be long-lasting and are IR type-dependent; (ii) IR may increase CV risks in the aging heart (IR + AGING model) and affect the heart recovery after an adverse CV event, such as acute myocardial infarct (IR + AGING + AMI model).
Methods: Eight- to 9-month-old C57BL/6N male mice were IR once with proton (1H) 50 cGy, 1 GeV/n or iron (56Fe) 15 cGy, 1 GeV/n. We evaluated IR-induced biological tissue responses—underlying molecular mechanisms, calcium handling, signal transduction, gene expression and cardiac fibrosis. Cardiac function was assessed by echocardiography (ECHO) and hemodynamic measurements (HEMO) as detailed in Fig. 1. AMI was induced by ligation of left anterior descending coronary artery 1 and 3 months post-IR as detailed in Fig. 2.Fig. 1.Radiation + aging model. Fig. 2.Radiation + aging model + adverse CV event model.
Results: In the IR + AGING model, cardiac function was not different among the control and 1H-IR group, whereas left ventricular end-diastolic pressure (LVEDP) was significantly increased in 56Fe mice 1 and 3 months post-IR. There was a small but statistically significant (P < 0.04) improvement of ejection fraction % (EF%) in 1H-IR vs control mice. One month post-IR, compared with control, 1H- and 56Fe-IR hearts had a significant up-regulation of sarcolemmal Na+–Ca2+ exchanger (NCX) (∼200% P<0.007), sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a, >200% increases, P < 0.02) and 400% decreases in p-p38 MAPK (P < 0.05), suggesting activation of compensatory mechanisms in [Ca2+]i handling in these hearts. By 3 months, compared with control, 1H- and 56Fe-IR hearts had 200–500% (P < 0.02) decreases in SERCA2a and more than 200% decreases in p-Creb-1 (P < 0.02), suggesting reduced capacity in intracellular [Ca2+]i handling. These data suggest that dysfunction in [Ca2+]i handling combined with LVEDP increase after 56Fe-IR may arise from the excessive demand on the heart due to prolonged activation of compensatory mechanisms that lead to changes in SERCA2a and p-Creb1 levels. This may represent a possible intracellular mechanism of heart failure in development in 56Fe-IR hearts.
In the IR + AGING + AMI model, no mortality was observed among three different groups 1 or 3 months post-IR and up to 28 days post-AMI. However, 1 month post-IR and 28 days post-AMI, the infarct size was significantly smaller in 56Fe-IR (p < 0.003) and 1H-IR (p = n.s.) vs control-IR mice, suggesting that at 1 month, 56Fe-IR primes the heart to recover better after AMI. In contrast, 3 months post AMI, 1H-AMI mice had a better cardiac functional recovery compared with control-AMI and 56Fe-AMI mice. The ejection fraction (EF%) was most decreased in 56Fe-AMI mice (56Fe-AMI vs 1H-AMI: 18 vs 48%, P < 0.007, ∼65–70% pre-AMI EF% for all groups). There was a 2- to 4-fold increase in LVEDP in 56Fe-AMI vs 1H-AMI (P < 0.04), suggesting that 56Fe-AMI hearts developed cardiac de-compensation. Western blots showed that 3 days post-AMI, compared with control- and 1H-IR-AMI mice, 56Fe-IR-AMI hearts had a 4- to 7-fold (P < 0.04) decreases in p-Akt (Thr308), p-Erk1/2 (P < 0.007) and ∼2-fold (P < 0.01) increase in phosphorylated ribosomal protein S6 kinase (p-S6k, a readout for mTORC1 pathway activation), suggesting decreased survival and angiogenesis signaling and decreased autophagy in these hearts. Seven days post-AMI, the levels of p-pErk1/2 were comparable between all three treatment conditions. However, in 56Fe-IR-AMI hearts, the p-Akt (Thr308) levels remained 4-fold decreased. Additionally, here was a 3-fold (P<0.05) decrease in p-S6k levels and >10-fold increase in p-p38 MAPK level in 56Fe vs control and 1H-IR-AMI hearts, suggesting continuous decreases in the survival, proliferation and angiogenesis signaling (p-Akt and p-S6k) and increase in the apoptotic signaling (p-p38 MAPK) up to Day 7 post-AMI in 56Fe-IR-AMI mice.
In summary, our results revealed that by 1 and 3 months post-IR in IR + AGING, 56Fe-IR but not 1H-IR mice had worse cardiac function. Further, a single 1H-IR 3 months prior to AMI improved, whereas 56Fe-IR worsened, recovery from AMI recovery. Our data in the IR + AGING and IR + AGING + AMI groups strongly suggest that low-dose HZE particle IR (56Fe) have long-lasting negative effect on heart homeostasis during normal aging, and present a significant CV risk for recovery after adverse CV event, such as AMI.
PMCID: PMC3941505  PMID: 24585984
HZE; iron; proton; low-dose; cardiovascular risks; Ca2+
22.  Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station 
PLoS ONE  2014;9(3):e91814.
The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.
PMCID: PMC3950288  PMID: 24618597
23.  Spherical particles of halophilic archaea correlate with exposure to low water activity – implications for microbial survival in fluid inclusions of ancient halite 
Geobiology  2012;10(5):424-433.
Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (aw). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of aw ≤ 0.75, but exposure to buffered 4 m LiCl (aw ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering aw should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.
PMCID: PMC3495301  PMID: 22804926
24.  Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica 
Saline Systems  2010;6:9.
The halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.
With the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements.
We discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.
PMCID: PMC2939530  PMID: 20727166
25.  Novel Insights into the Diversity of Catabolic Metabolism from Ten Haloarchaeal Genomes 
PLoS ONE  2011;6(5):e20237.
The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis.
Methodology/Principal Findings
We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses.
These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.
PMCID: PMC3102087  PMID: 21633497

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