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Small satellite technologies, particularly CubeSats, are enabling breakthrough research in space. Over the past 15 years, NASA Ames Research Center has developed and flown half a dozen biological CubeSats in low Earth orbit (LEO) to conduct space biology and astrobiology research investigating the effects of the space environment on microbiological organisms. These studies of the impacts of radiation and reduced gravity on cellular processes include dose-dependent interactions with antimicrobial drugs, measurements of gene expression and signaling, and assessment of radiation damage. BioSentinel, the newest addition to this series, will be the first deep space biological CubeSat, its heliocentric orbit extending far beyond the radiation-shielded environment of low Earth orbit. BioSentinel's 4U biosensing payload, the first living biology space experiment ever conducted beyond the Earth-Moon system, will use a microbial bioassay to assess repair of radiation-induced DNA damage in eukaryotic cells over a duration of 6-12 months. Part of a special collection of articles focused on BioSentinel and its science mission, this article describes the design, development, and testing of the biosensing payload's microfluidics and optical systems, highlighting improvements relative to previous CubeSat life-support and bioanalytical measurement technologies.

The E. coli AntiMicrobial Satellite (EcAMSat) was a successful spaceflight mission that performed an autonomous experiment to investigate the effects of microgravity on the antibiotic resistance of uropathogenic E. coli; the latest in a series of biology-focused CubeSats developed at NASA Ames Research Center. While maintaining the bacteria at 37 °C, the payload delivered growth medium, different doses of the antibiotic gentamicin, and finally the metabolic indicator alamarBlue to each of 48 microwells in the fluidic card. An LED and detector system was used to monitor the changes in absorbance in each experiment well at three wavelengths (470, 525, and 615 nm), from which the growth of cells and their metabolic activity could be measured. The success of the mission was in part due to leveraging lessons learned and proven technologies from previous bio-CubeSat missions, including modified spare hardware from the PharmaSat payload. This paper details the design and assembly of the biofluidic system payload; the modifications to the PharmaSat hardware required to run the EcAMSat experiment and the challenges those changes created; and finally, the testing approach employed to ensure the success of the payload during the flight campaign.

We report the results of the EcAMSat (Escherichia coli Antimicrobial Satellite) autonomous space flight experiment, investigating the role of σs in the development of antibiotic resistance in uropathogenic E. coli (UPEC) in microgravity (µ-g). The presence of σs, encoded by the rpoS gene, has been shown to increase antibiotic resistance in Earth gravity, but it was unknown if this effect occurs in µ-g. Two strains, wildtype (WT) UPEC and its isogenic ΔrpoS mutant, were grown to stationary phase aboard EcAMSat, an 11-kg small satellite, and in a parallel ground-based control experiment; cell growth rates for the two strains were found to be unaltered by µ-g. After starvation for over 24 h, stationary-phase cells were incubated with three doses of gentamicin (Gm), a common treatment for urinary tract infections (which have been reported in astronauts). Cellular metabolic activity was measured optically using the redox-based indicator alamarBlue (aB): both strains exhibited slower metabolism in µ-g, consistent with results from previous smallsat missions. The results also showed that µ-g did not enhance UPEC resistance to Gm; in fact, both strains were more susceptible to Gm in µ-g. It was also found, via a second ground-control experiment, that multi-week storage in the payload hardware stressed the cells, potentially obscuring small differential effects of the antibiotic between WT and mutant and/or between µ-g and ground. Overall, results showed that the ∆rpoS mutant was 34–37% less metabolically active than the WT for four different sets of conditions: ground without Gm, ground with Gm; µ-g without Gm, µ-g with Gm. We conclude therefore that the rpoS gene and its downstream products are important therapeutic targets for treating bacterial infections in space, much as they are on the ground.

Human immune response is compromised and bacteria can become more antibiotic resistant in space microgravity (MG). We report that under low-shear modeled microgravity (LSMMG), stationary-phase uropathogenic Escherichia coli (UPEC) become more resistant to gentamicin (Gm), and that this increase is dependent on the presence of σs (a transcription regulator encoded by the rpoS gene). UPEC causes urinary tract infections (UTIs), reported to afflict astronauts; Gm is a standard treatment, so these findings could impact astronaut health. Because LSMMG findings can differ from MG, we report preparations to examine UPEC's Gm sensitivity during spaceflight using the E. coli Anti-Microbial Satellite (EcAMSat) as a free-flying "nanosatellite" in low Earth orbit. Within EcAMSat's payload, a 48-microwell fluidic card contains and supports study of bacterial cultures at constant temperature; optical absorbance changes in cell suspensions are made at three wavelengths for each microwell and a fluid-delivery system provides growth medium and predefined Gm concentrations. Performance characterization is reported here for spaceflight prototypes of this payload system. Using conventional microtiter plates, we show that Alamar Blue (AB) absorbance changes can assess the Gm effect on E. coli viability, permitting telemetric transfer of the spaceflight data to Earth. Laboratory results using payload prototypes are consistent with wellplate and flask findings of differential sensitivity of UPEC and its ∆rpoS strain to Gm. if σs plays the same role in space MG as in LSMMG and Earth gravity, countermeasures discovered in recent Earth studies (aimed at weakening the UPEC antioxidant defense) to control UPEC infections would prove useful also in space flights. Further, EcAMSat results should clarify inconsistencies from previous space experiments on bacterial antibiotic sensitivity and other issues.

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Procedure used for RT-qPCR in the WetLab-2 study

Steps needed to prepare reagents to ship to vendor to manufacture lyophilized qPCR and RT-qPCR Reagents

The O/OREOS (Organism/Organic Exposure to Orbital Stresses) nanosatellite is the first science demonstration spacecraft and flight mission of the NASA Astrobiology Small- Payloads Program (ASP). O/OREOS was launched successfully on November 19, 2010, to a high-inclination (72 deg), 650-km Earth orbit aboard a US Air Force Minotaur IV rocket from Kodiak, Alaska. O/OREOS consists of 3 conjoined cubesat (each 1000 cu cm) modules: (i) a control bus; (ii) the Space Environment Survivability of Living Organisms (SESLO) experiment; and (iii) the Space Environment Viability of Organics (SEVO) experiment. Among the innovative aspects of the O/OREOS mission are a real-time analysis of the photostability of organics and biomarkers and the collection of data on the survival and metabolic activity for microorganisms at 3 times during the 6-month mission. We report on the spacecraft characteristics, payload capabilities, and present operational phase and flight data from the O/OREOS mission. The science and technology rationale of O/OREOS supports NASA0s scientific exploration program by investigating the local space environment as well as space biology relevant to Moon and Mars missions. It also serves as a precursor for experiments on small satellites, the International Space Station (ISS), future free-flyers and lunar surface exposure facilities.

The interactions of numerous abiotic and biotic factors experienced by sea turtle embryos during incubation affect their survival. In this study we determined the hatching and emergence success of green turtles Chelonia mydas from nests on 4 beaches on the Galapagos Islands, one of the most important rookeries for green turtles in the eastern Pacific Ocean. Mean (±SD) hatching and emergence success for the 1039 nests examined were 46.0 ± 33.4 and 45.6 ± 33.4%, respectively. These values are relatively low compared to other green turtle populations worldwide. We evaluated the effects of beach, year, day of oviposition, carapace length and width of female, nest position, nest habitat, and nest chamber depth on hatching and emergence success with binomial generalized additive models with fixed effects. We found variation in hatching and emergence success was significant among beaches, years, day of oviposition, and nest habitat. Predation by feral pigs and beetles and destruction of earlier nests by nesting females were the most important causes of embryo mortality. Efforts to keep threats at minimum levels, particularly controlling pigs near Isabela beaches, should be considered a major conservation objective. This study highlights important differences among beaches within a rookery and emphasizes the need to continue improving management strategies to protect green turtles and their critical habitats. Quantitative information provided here can be used as a basis for long-term studies in the Gala- pagos and for comparison to other sea turtles rookeries.

Impacts on sea turtle populations from fisheries bycatch, climate change, habitat destruction and poaching have been widely discussed, but little is known about boat strikes as a potential threat to sea turtles. Here we present data on Pacific green turtles (Chelonia mydas) that suffered boat strikes in the Galapagos Marine Reserve at nesting beaches at Isabela Island and from foraging sites at San Cristobal Island from 2008 to 2011. Tourism in Galapagos has increased to more than 180,000 visitors a year and the boat traffic within the Marine Reserve poses a significant risk to sea turtles. Boat strikes were most frequent at foraging sites close to Puerto Baquerizo Moreno, a commercial and tourism port, where incidence varied between 16 and 20%. Incidence at nesting beaches was much lower with 3.7% of 4198 turtles showing injuries resulting from boat strikes. However, at the nesting beaches most of the impacts (59%) had occurred during the same nesting season while only 5% of the sea turtles observed at the foraging sites suffered their injuries during the study period. No data on survival rates from boat strike exist, however it is clear that many turtles die from the trauma caused by the impact. This report focuses on only the survivors; therefore the results represent a minimum estimate. Our study clearly shows that the overall threat from boat strikes has an important effect on the Galapagos green turtle population. Conservation management including speed limits in key turtle habitats are needed to improve their survival in the Galapagos Marine Reserve.

We report the first telemetered spaceflight science results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment, executed by one of the two 10 cm cube-format payloads aboard the 5.5 kg Organism/Organic Exposure to Orbital Stresses (O/OREOS) free-flying nanosatellite. The O/OREOS spacecraft was launched successfully to a 72° inclination, 650 km Earth orbit on 19 November 2010. This satellite provides access to the radiation environment of space in relatively weak regions of Earth's protective magnetosphere as it passes close to the north and south magnetic poles; the total dose rate is about 15 times that in the orbit of the International Space Station. The SESLO experiment measures the long-term survival, germination, and growth responses, including metabolic activity, of Bacillus subtilis spores exposed to the microgravity, ionizing radiation, and heavy-ion bombardment of its high-inclination orbit. Six microwells containing wild-type (168) and six more containing radiation-sensitive mutant (WN1087) strains of dried B. subtilis spores were rehydrated with nutrient medium after 14 days in space to allow the spores to germinate and grow. Similarly, the same distribution of organisms in a different set of microwells was rehydrated with nutrient medium after 97 days in space. The nutrient medium included the redox dye Alamar blue, which changes color in response to cellular metabolic activity. Three-color transmitted intensity measurements of all microwells were telemetered to Earth within days of each of the 48 h growth experiments. We report here on the evaluation and interpretation of these spaceflight data in comparison to delayed-synchronous laboratory ground control experiments.

Calyptogena gallardoiis a vesicomyid bivalve inhabiting a methane seep area located at a depth of 740–870 m off the Bay of Concepción, Chile. Vesicomyids host chemoautotrophic sulphide-oxidizing endosymbiont bacteria and are always found associated to reducing environments. In this study, the gonadal structure and the gametes produced byC. gallardoiare described. Light microscopy is used to examine serial histological sections of the gonads, and scanning electron microscopy is used to visualize the external morphology of gametes. The gonads of both males and females are organized in ramified tubular acini. In males, mature sperm are stored near genital openings in acini lined with a secretor epithelium that resembles a seminal receptacle. Spermatozoids have bullet-like heads with an average length of 30.3±2.6 μm (mean±1 SD). In females, the mature oocytes are driven toward the genital opening through evacuator conduits lined by ‘paddle’ cilia. The average diameter of oogonias is 11.6±2.5 μm and that of mature oocytes is 273.8±23.1 μm, making the size of the mature oocyte among the largest reported for bivalves. In addition,C. gallardoiis shown to have external sexual dimorphism. Shells of males are significantly smaller and more elongated with sloping postero-dorsal margin compared with shells of females. The data are discussed in the context of available information on reproductive biology of vesicomyids.

Although ambient levels of estradiol and synthesis of the osteoblast growth factor IGF-I are inversely related in vivo, estradiol has little or no direct effect on igf1 gene expression in rat osteoblasts in vitro. Rather, estradiol suppresses the effect of hormones that enhance igf1 expression through protein kinase A dependent activation of CCAAT enhancer binding protein (C/EBP) transcription factors. We show here that inhibition of C/EBP activity by estradiol relates to the level of estrogen receptor alpha (ERalpha) expression, and that a complex between hormone-activated ERalpha and C/EBPdelta inhibits transcription by each factor. Protein fragmentation, co-immunoprecipitation, and gene expression studies identified domains for physical and functional interactions between ERalpha and C/EBPdelta. Whereas ERalpha and fragments comprising its various domains associated with C/EBPdelta, only ERalpha fragment A/B alone replicated the suppressive effect of intact ERalpha on endogenous C/EBPdelta activity. Complementary studies showed that several carboxyl regions of C/EBPdelta cooperatively inhibit ERalpha dependent transcription. Therefore, multiple domains of C/EBPdelta and ERalpha can physically interact to alter gene expression in osteoblasts in selective ways that depend on variations in the local hormone environment. Their combined effects on one important gene target, igf1, may help to determine the balance in the overall rates of bone formation.

Background Abundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses. Results Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested. Conclusions These results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.

The mechanisms that couple cell cycle progression with the organization of the peripheral cytoskeleton are poorly understood. In Saccharomyces cerevisiae, the Swe1 protein has been shown previously to phosphorylate and inactivate the cyclin-dependent kinase, Cdc28, thereby delaying the onset of mitosis. The nim1-related protein kinase, Hsl1, induces entry into mitosis by negatively regulating Swe1. We have found that Hsl1 physically associates with the septin cytoskeleton in vivo and that Hsl1 kinase activity depends on proper septin function. Genetic analysis indicates that two additional Hsl1-related kinases, Kcc4 and Gin4, act redundantly with Hsl1 to regulate Swe1. Kcc4, like Hsl1 and Gin4, was found to localize to the bud neck in a septin-dependent fashion. Interestingly, hsl1 kcc4 gin4 triple mutants develop a cellular morphology extremely similar to that of septin mutants. Consistent with the idea that Hsl1, Kcc4, and Gin4 link entry into mitosis to proper septin organization, we find that septin mutants incubated at the restrictive temperature trigger a Swe1-dependent mitotic delay that is necessary to maintain cell viability. These results reveal for the first time how cells monitor the organization of their cytoskeleton and demonstrate the existence of a cell cycle checkpoint that responds to defects in the peripheral cytoskeleton. Moreover, Hsl1, Kcc4, and Gin4 have homologs in higher eukaryotes, suggesting that the regulation of Swe1/Wee1 by this class of kinases is highly conserved.

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

We designed, built, tested, space-qualified, launched, and collected telemetered data from low Earth orbit from Pharma- Sat, a 5.1-kg free flying "nanosatellite" that supported microbial growth in 48 microfluidic wells, dosed microbes with multiple concentrations of a pharmaceutical agent, and monitored microbial growth and metabolic activity using a dedicated 3-color optical absorbance system at each microwell. The PharmaSat nanosatellite comprised a structure approximately 10 x 10 x 35 cm, including triple-junction solar cells, bidirectional communications, power-generation and energy- storage system, and a sealed payload 1.2-L containment vessel that housed the biological organisms along with the fluidic, optical, thermal, sensor, and electronic subsystems. Growth curves for S. cerevisiae (Brewer's yeast) were obtained for multiple concentrations of the antifungal drug voriconazole in the microgravity conditions of low Earth orbit. Corresponding terrestrial control experiments were conducted for comparison.

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

We designed, built, tested, space-qualified, launched, and downlinked bioanalytical data from a fully autonomous free-flying space satellite that supports microorganism growth in multiple fluidic wells and monitors gene expression via fluorescence. GeneSat-1 (total mass: 4.4 kg) includes solar cells, integrated spacecraft "bus" module (power/batteries/control/bi-directional communications), and an insulated pressure vessel housing the biofluidic, optical, thermal, and sensor subsystems. Data were obtained over ~ 100 hr following nutrient introduction that re-animated two strains of E. coli, with culture growth tracked via light scattering and green fluorescent protein expression monitored in each of nine bacteria-containing fluidic wells. Stability of temperature, pressure, and relative humidity were monitored by multiple sensors, as were radiation events and acceleration in three axes. Data were telemetered to Earth over the course of several weeks during and after the biological growth-and-analysis process.

Variations in transforming growth factor beta (TGF-beta) activity depend on the expression of specific receptors in normal as well as transformed cells. For example, in addition to mutations in TGF-beta type II receptor (TbetaRII) that abrogate normal TGF-beta function, its expression decreases during the transition from replication to extracellular matrix production, or in response to other growth regulators in bone. Therefore, to understand how TbetaRII expression is controlled, we cloned the rat TbetaRII gene promoter and defined basic aspects of its structure and activity. Among several cis-acting elements, mutations within an upstream E-box that specifically binds USF nuclear factors or a downstream Sp1 binding site significantly reduced TbetaRII promoter activity in primary cultures of fetal rat osteoblasts. Treatment with bone morphogenetic protein 2 (BMP-2), which induces further osteoblast differentiation, significantly reduced cell surface TbetaRII. However, BMP-2 did not alter TbetaRII promoter activity, steady state TbetaRII mRNA, or total TbetaRII protein, but caused an intracellular relocation of TbetaRII. Select transcriptional elements thus regulate TbetaRII gene expression, whereas post-translational events controlled by BMP-2 rapidly modify the amount of TbetaRII protein on the bone cell surface. Consequently, several processes can alter functional TbetaRII levels in order to regulate the biological effects of this important growth factor.