BioSentinel: Validating Sensitivity of Yeast Biosensors to Deep Space Relevant Radiation.

With the imminent human exploration of deep space, it is more important than ever to understand the biological risks of deep space radiation exposure. The BioSentinel mission will be the first biological payload to study the effects of radiation beyond low Earth orbit in 50 years. This study is the last in a collection of articles about the BioSentinel biological CubeSat mission, where budding yeast cells will be used to investigate the response of a biological organism to long-term, low-dose deep space radiation. In this study, we define the methodology for detecting the biological response to space-like radiation using simulated deep space radiation and a metabolic indicator dye reduction assay. We show that there is a dose-dependent decrease in yeast cell growth and metabolism in response to space-like radiation, and this effect is significantly more pronounced in a strain of yeast that is deficient in DNA damage repair (rad51Δ) compared with a wild-type strain. Furthermore, we demonstrate the use of flight-like instrumentation after exposure to space-like ionizing radiation. Our findings will inform the development of novel and improved biosensors and technologies for future missions to deep space.

BioSentinel: A Biofluidic Nanosatellite Monitoring Microbial Growth and Activity in Deep Space.

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.

EcAMSat spaceflight measurements of the role of σs in antibiotic resistance of stationary phase Escherichia coli in microgravity.

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.

Payload hardware and experimental protocol development to enable future testing of the effect of space microgravity on the resistance to gentamicin of uropathogenic Escherichia coli and its σs-deficient mutant.

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.

Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.

Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFß2hi and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER- breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

Development path and current status of the NANIVID: a new device for cancer cell studies.

Cancer cells create a unique microenvironment in vivo that enables migration to distant organs. To better understand the tumor micro-environment, special tools and devices are required to monitor the interactions between different cell types and the effects of particular chemical gradients. Our study presents the design and optimization of a versatile chemotaxis device, the nano-intravital device (NANIVID), which consists of etched and bonded glass substrates that create a soluble factor reservoir. The device contains a customized hydrogel blend that is loaded with epidermal growth factor (EGF), which diffuses from the outlet to create a chemotactic gradient that can be sustained for many hours in order to attract specific cells to the device. A microelectrode array is under development for quantification of cell collection and will be incorporated into future device generations. Additionally, the NANIVID can be modified to generate gradients of other soluble factors in order to initiate controlled changes to the microenvironment including the induction of hypoxia, manipulation of extracellular matrix stiffness, etc. The focus of the article is to present the design and optimization of the device towards wide ranging applications of cancer cell dynamics in vitro and, ultimately, implantation for in vivo investigations.

One-dimensional self-assembly of mouse embryonic stem cells using an array of hydrogel microstrands.

The ability of embryonic stem (ES) cells to self-renew indefinitely and to differentiate into multiple cell lineages holds promise for advances in modeling disease progression, screening drugs and treating diseases. To realize these potentials, it is imperative to study self-assembly in an embryonic microenvironment, as this may increase our understanding of ES cell maintenance and differentiation. In this study, we synthesized an array of one-dimensional alginate gel microstrands and aqueous microstrands through an SU-8 filter device by means of capillary action. Furthermore, we investigated self-assembly behaviors and differentiation potentials of mouse ES cells cultured in microstrands of varying diameters. We found that microstrands with an aqueous interior facilitated high density cell culture and formed compact microtissue structures, while microstrands with gelled interiors promote smaller cell aggregate structures. In particular, we noticed that ES cells collected from one-dimensional aqueous microstrands favored the differentiation towards cell lineages of endoderm and mesoderm, whereas those from gelled microstrands preferred to differentiate into ectoderm and mesoderm lineages. In addition to providing a "liquid-like" tubular microenvironment to understand one-dimensional self-assembly process of ES cells, this alginate hydrogel microstrand system also offers an alternative way to manipulate the stem cell fate-decision using bioengineered microenvironments.