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NASA NTRS2010

Oscillating Cell Culture Bioreactor

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

Freed, Lisa E.↗
NASA NTRS2011

Mapping Oyster Reef Habitats in Mobile Bay

Oyster reefs around the world are declining rapidly, and although they haven t received as much attention as coral reefs, they are just as important to their local ecosystems and economies. Oyster reefs provide habitats for many species of fish, invertebrates, and crustaceans, as well as the next generations of oysters. Oysters are also harvested from many of these reefs and are an important segment of many local economies, including that of Mobile Bay, where oysters rank in the top five commercial marine species both by landed weight and by dollar value. Although the remaining Mobile Bay oyster reefs are some of the least degraded in the world, projected climate change could have dramatic effects on the health of these important ecosystems. The viability of oyster reefs depends on water depth and temperature, appropriate pH and salinity levels, and the amount of dissolved oxygen in the water. Projected increases in sea level, changes in precipitation and runoff patterns, and changes in pH resulting from increases in the amount of carbon dioxide dissolved in the oceans could all affect the viability of oyster reefs in the future. Human activities such as dredging and unsustainable harvesting practices are also adversely impacting the oyster reefs. Fortunately, several projects are already under way to help rebuild or support existing or previously existing oyster reefs. The success of these projects will depend on the local effects of climate change on the current and potential habitats and man s ability to recognize and halt unsustainable harvesting practices. As the extent and health of the reefs changes, it will have impacts on the Mobile Bay ecosystem and economy, changing the resources available to the people who live there and to the rest of the country, since Mobile Bay is an important national source of seafood. This project identified potential climate change impacts on the oyster reefs of Mobile Bay, including the possible addition of newly viable habitats in the southeastern regions of the Bay.

Bolte, Danielle↗
NASA NTRS2006

Automated Synthesis of Architecture of Avionic Systems

The Architecture Synthesis Tool (AST) is software that automatically synthesizes software and hardware architectures of avionic systems. The AST is expected to be most helpful during initial formulation of an avionic-system design, when system requirements change frequently and manual modification of architecture is time-consuming and susceptible to error. The AST comprises two parts: (1) an architecture generator, which utilizes a genetic algorithm to create a multitude of architectures; and (2) a functionality evaluator, which analyzes the architectures for viability, rejecting most of the non-viable ones. The functionality evaluator generates and uses a viability tree a hierarchy representing functions and components that perform the functions such that the system as a whole performs system-level functions representing the requirements for the system as specified by a user. Architectures that survive the functionality evaluator are further evaluated by the selection process of the genetic algorithm. Architectures found to be most promising to satisfy the user s requirements and to perform optimally are selected as parents to the next generation of architectures. The foregoing process is iterated as many times as the user desires. The final output is one or a few viable architectures that satisfy the user s requirements.

Chau, Savio↗
NASA NTRS2011

Report of the Horizontal Launch Study

A study of horizontal launch concepts has been conducted. This study, jointly sponsored by the Defense Advanced Research Projects Agency (DARPA) and the National Aeronautics and Space Administration (NASA) was tasked to estimate the economic and technical viability of horizontal launch approaches. The study team identified the key parameters and critical technologies which determine mission viability and reported on the state of the art of critical technologies, along with objectives for technology development.

Wilhite, Alan W.↗
NASA NTRS2011

Assessment of Resistance of Bacillus Horneckiae Endospores to UV Radiation and Function of Their Extraneous Layer in Resistance

Spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. The spore's resistance might be due to their metabolically dormant state, and/or by the presence of a series of protective structures that encase the interior-most compartment, the core, which houses the spore chromosome. These spores have multiple layers surrounding the cell that are not found in vegetative cells, and some species have an outer layer of proteins and glycoproteins termed the "exosporium" or a fibrous "extraneous layer" (EL). Bacillus horneckiae is an EL-producing novel sporeformer isolated from a Phoenix spacecraft assembly clean room, and it has previously demonstrated resistance to UV radiation up to 1000 J/m(sup 2). The EL appears to bind B. horneckiae spores into large aggregations, or biofilms, and may confer some UV resistance to the spores. Multiple culturing and purification schemes were tried to achieve high purity spores because vegetative cells would skew UV resistance results. An ethanol-based purification scheme produced high purity spores. Selective removal of the EL from spores was attempted with two schemes: a chemical extraction method and physical extraction (sonication). Results from survival rates in the presence and absence of the external layer will provide a new understanding of the role of biofilms and passive resistance that may favor survival of biological systems in aggressive extra-terrestrial environments. The chemical extraction method decreased viable counts of spores and lead to an inconclusive change UV resistance relative to non-extracted spores. The physical extraction method lead to non-aggregated spores and did not alter viability; however, it produced UV resistance profiles similar to non-extracted spores. In addition to the EL-removal study, samples of B. horneckiae spores dried on aluminum coupons and exposed to increasing UV (200-400 nm range) levels (0 to 8.0 x 105 kJ/m(sup 2)) were tested for viability, which indicated that the maximum UV exposure level that still resulted in viable spores was 5.0 x 10? kJ/m(sup 2).

resistance↗
NASA NTRS2014

The Nuclear Cryogenic Propulsion Stage

Nuclear Thermal Propulsion (NTP) development efforts in the United States have demonstrated the technical viability and performance potential of NTP systems. For example, Project Rover (1955 - 1973) completed 22 high power rocket reactor tests. Peak performances included operating at an average hydrogen exhaust temperature of 2550 K and a peak fuel power density of 5200 MW/m3 (Pewee test), operating at a thrust of 930 kN (Phoebus-2A test), and operating for 62.7 minutes in a single burn (NRX-A6 test). Results from Project Rover indicated that an NTP system with a high thrust-to-weight ratio and a specific impulse greater than 900 s would be feasible. Excellent results were also obtained by the former Soviet Union. Although historical programs had promising results, many factors would affect the development of a 21st century nuclear thermal rocket (NTR). Test facilities built in the US during Project Rover no longer exist. However, advances in analytical techniques, the ability to utilize or adapt existing facilities and infrastructure, and the ability to develop a limited number of new test facilities may enable affordable development, qualification, and utilization of a Nuclear Cryogenic Propulsion Stage (NCPS). Bead-loaded graphite fuel was utilized throughout the Rover/NERVA program, and coated graphite composite fuel (tested in the Nuclear Furnace) and cermet fuel both show potential for even higher performance than that demonstrated in the Rover/NERVA engine tests.. NASA's NCPS project was initiated in October, 2011, with the goal of assessing the affordability and viability of an NCPS. FY 2014 activities are focused on fabrication and test (non-nuclear) of both coated graphite composite fuel elements and cermet fuel elements. Additional activities include developing a pre-conceptual design of the NCPS stage and evaluating affordable strategies for NCPS development, qualification, and utilization. NCPS stage designs are focused on supporting human Mars missions. The NCPS is being designed to readily integrate with the Space Launch System (SLS). A wide range of strategies for enabling affordable NCPS development, qualification, and utilization should be considered. These include multiple test and demonstration strategies (both ground and in-space), multiple potential test sites, and multiple engine designs. Two potential NCPS fuels are currently under consideration - coated graphite composite fuel and tungsten cermet fuel. During 2014 a representative, partial length (approximately 16") coated graphite composite fuel element with prototypic depleted uranium loading is being fabricated at Oak Ridge National Laboratory (ORNL). In addition, a representative, partial length (approximately 16") cermet fuel element with prototypic depleted uranium loading is being fabricated at Marshall Space Flight Center (MSFC). During the development process small samples (approximately 3" length) will be tested in the Compact Fuel Element Environmental Tester (CFEET) at high temperature (approximately 2800 K) in a hydrogen environment to help ensure that basic fuel design and manufacturing process are adequate and have been performed correctly. Once designs and processes have been developed, longer fuel element segments will be fabricated and tested in the Nuclear Thermal Rocket Element Environmental Simulator (NTREE) at high temperature (approximately 2800 K) and in flowing hydrogen.

Houts, Michael G.↗
NASA NTRS2012

Survival of B. Horneckiae Spores Under Ground-simulated Space Conditions

To prevent forward contamination and maintain the scientific integrity of future life detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated habitats, spore-forming microbes are highly resistant to various physical and chemical conditions, which include ionizing and UV radiation, desiccation and oxidative stress, and the harsh environment of outer space or planetary surfaces. Recently a radiation resistant, spore forming bacterial isolate, Bacillus horneckiae, was isolated from a clean room of the Kennedy Space Center where the Phoenix spacecraft was assembled. The exceptionally high tolerance of extreme conditions demonstrated by sporeforming bacteria highlighted the need to assess the viability of these microbes in situ (in real) space. The proposed BOSS (Biofilm Organisms Surfing Space) project aims to understand the mechanisms by which biofilm forming organisms, such as B. horneckiae, will potentially be able to withstand harsh space conditions. As previously stated, the spore producing ability of these species gives them increased survivability to harsh conditions. Some of the spores will have the protective exosporium layer artificially removed before the test to determine if the existence of this layer significantly changes the survivability during the mission. In preparation for that experiment, we analyzed spores which were exposed during a ground simulation, the EXPOSE R2 Biofilm Organisms Surfing Space (BOSS). Previous to exposure, spores were deposited onto spacecraft grade aluminum coupons in a spore suspension calculated to contain between 10(exp 7) and 10(exp 8) spores. This precursor series will be used to establish a baseline survivability function for comparison with the future flight tests during EXPOSE-R. For each coupon, a 10% polyvinyl alcohol (PVA) film was applied and peeled from the coupon to recover the spores. One hundred μl of sterile 10% PVA was applied to the surface of the coupon and allowed to dry for 1 hour at 37 C. The films were then removed using sterile scalpel and forceps and placed into a glass test tube containing 2 milliliters of sterile deionized water. The PVA film process was then repeated on each coupon one additional time to ensure recovery of the majority of spores. The second PVA film was added in the same glass tube as in the previous round. If the spores remained 100% viable, the test tubes should now contain between 5 X 10(exp 6) and 5 X 10(exp 7) spores per millimeter; however, it is expected that some loss of viability has occurred. In order to assess this loss, the number of colony forming, viable spores was counted. To count the colony forming units (CFUs), the spore containing solution was diluted in a process of 10-fold serial dilution by mixing successive solutions in a 100 microliter spore suspension to 900 microliter deionized H2O ratio. A sample dilution series revealed that 10(exp -3) and 10(exp -4) concentrations would be necessary for an accurate CFU count to be taken. For those two concentrations, a spread on a TSA plate was prepared and incubated at 32 C. For the samples exposed to UV radiation, the cell survivability was too low to establish a count from 100 microliter spread plating. Instead, no dilutions were performed and the entire 2 milliliter spore suspension was plated and incubated at 32 C. The plate's CFU counts were taken at 24 hours and 48 hours from the time of plating. At the end of the CFU counting the total surviving spores in each sample were calculated based on the number of CFUs that were observed per 100 microliters, or per 2 milliliters for the UV irradiated samples. The results of these calculations are shown in Figures 1 and 2.

cleanroom↗
NASA NTRS2015

Space-to-Space Power Beaming Enabling High Performance Rapid Geocentric Orbit Transfer

The use of electric propulsion is more prevalent than ever, with industry pursuing all electric orbit transfers. Electric propulsion provides high mass utilization through efficient propellant transfer. However, the transfer times become detrimental as the delta V transitions from near-impulsive to low-thrust. Increasing power and therefore thrust has diminishing returns as the increasing mass of the power system limits the potential acceleration of the spacecraft. By using space-to-space power beaming, the power system can be decoupled from the spacecraft and allow significantly higher spacecraft alpha (W/kg) and therefore enable significantly higher accelerations while maintaining high performance. This project assesses the efficacy of space-to-space power beaming to enable rapid orbit transfer while maintaining high mass utilization. Concept assessment requires integrated techniques for low-thrust orbit transfer steering laws, efficient large-scale rectenna systems, and satellite constellation configuration optimization. This project includes the development of an integrated tool with implementation of IPOPT, Q-Law, and power-beaming models. The results highlight the viability of the concept, limits and paths to infusion, and comparison to state-of-the-art capabilities. The results indicate the viability of power beaming for what may be the only approach for achieving the desired transit times with high specific impulse.

Dankanich, John W.↗
NASA NTRS2015

EcAMSat: Effect of Space-Flight on Antibiotic Resistance of a Pathogenic Bacterium and its Genetic Basis

Human immune response is compromised in space and incidence of urinary tract infections (UTI) in astronauts has been reported. We have found that the causative agent of UTI, the uropathogenic Escherichia coli, becomes more resistant to gentamicin (Gm), which is commonly used to treat this disease, under modeled microgravity conditions (MMG), the increase being controlled by the stress response master regulator, σs. While the wild type bacterium becomes virtually invincible under MMG, the strain missing this sigma factor barely survives. We report here preparatory ground work for testing this finding in space flight on a nanosatellite. We have shown that the effect of Gm treatment on culture viability is directly correlated to increased Alamar Blue (AB) reduction; we have identified conditions to keep the experimental elements - the bacterial cultures, Gm, and AB - in a state of viability and potency to permit successful spaceflight experimentation given the necessary constraints. Spaceflight kinetics of AB reduction will be transmitted from the satellite via telemetry. The PharmaSat hardware previously used for space experimentation with yeast was modified to permit studies with bacteria by reducing the filter pore size and increasing fluidics volume to enable more fluid exchanges. Several verification tests have been run using the nanosatellite's flight software and prototype hardware. Cells were grown to stationary phase to induce the σs-controlled stress resistance and treated with Gm. Without Gm, the mutant took longer than the wild type to reduce the AB; this time difference increased almost 8 fold at 55 μg/mL Gm concentration. Thus, using flight hardware the mutant shows similarly increased sensitivity to Gm compared to the wild type to that found in our pilot microtiter plate experiments. Previous inflight experiments have given contradictory results concerning bacterial antibiotic resistance; none has yet explored the involvement of specific genes in this phenomenon. With our system ready to fly in late 2015/early 2016, these questions can be approached

uropathogenic E. coli↗
NASA NTRS2016

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

mechanical loading↗
NASA NTRS2017

A Review of the Proposed K (sub Isi) Offset-Secant Method for Size-Independent Linear-Elastic Toughness Evaluation

The proposed size-independent linear-elastic fracture toughness, K (sub Isi), for potential inclusion in ASTM E399 targets a consistent 0.5 millimeters crack extension for all specimen sizes through an offset secant that is a function of the specimen ligament length. The K (sub Isi) method also includes an increase in allowable deformation, and the removal of the P (sub max)/P (sub Q) criterion. A finite element study of the K (sub Isi) test method confirms the viability of the increased deformation limit, but has also revealed a few areas of concern. Findings: 1. The deformation limit, b (sub o) greater than or equal to 1.1 times (K (sub I) divided by delta (sub ys) squared) maintains a K-dominant crack tip field with limited plastic contribution to the fracture energy; 2. The three dimensional effects on compliance and the shape of the force versus CMOD (Crack-Mouth Opening Displacement) trace are significant compared to a plane strain assumption; 3. The non-linearity in the force versus CMOD trace at deformations higher than the current limit of 2.5 times (K (sub I) divided by delta (sub ys) squared) is sufficient to introduce error or even "false calls" regarding crack extension when using a constant offset secant line. This issue is more significant for specimens with W (width) greater than or equal to 2 inches; 4. A non-linear plasticity correction factor in the offset secant may improve the viability of the method at deformations between 2.5 times (K (sub I) divided by delta (sub ys) squared) and 1.1 times (K (sub I) divided by delta (sub ys) squared).

James, Mark↗
NASA NTRS2017

Unmanned Aircraft Systems (UAS) Integration in the National Airspace System (NAS) Project KDP-C Review

The topics discussed are the UAS-NAS project life-cycle and ARMD thrust flow down, as well as the UAS environments and how we operate in those environments. NASA's Armstrong Flight Research Center at Edwards, CA, is leading a project designed to help integrate unmanned air vehicles into the world around us. The Unmanned Aircraft Systems Integration in the National Airspace System project, or UAS in the NAS, will contribute capabilities designed to reduce technical barriers related to safety and operational challenges associated with enabling routine UAS access to the NAS. The project falls under the Integrated Systems Research Program office managed at NASA Headquarters by the agency's Aeronautics Research Mission Directorate. NASA's four aeronautics research centers - Armstrong, Ames Research Center, Langley Research Center, and Glenn Research Center - are part of the technology development project. With the use and diversity of unmanned aircraft growing rapidly, new uses for these vehicles are constantly being considered. Unmanned aircraft promise new ways of increasing efficiency, reducing costs, enhancing safety and saving lives 460265main_ED10-0132-16_full.jpg Unmanned aircraft systems such as NASA's Global Hawks (above) and Predator B named Ikhana (below), along with numerous other unmanned aircraft systems large and small, are the prime focus of the UAS in the NAS effort to integrate them into the national airspace. Credits: NASA Photos 710580main_ED07-0243-37_full.jpg The UAS in the NAS project envisions performance-based routine access to all segments of the national airspace for all unmanned aircraft system classes, once all safety-related and technical barriers are overcome. The project will provide critical data to such key stakeholders and customers as the Federal Aviation Administration and RTCA Special Committee 203 (formerly the Radio Technical Commission for Aeronautics) by conducting integrated, relevant system-level tests to adequately address safety and operational challenges of national airspace access by unmanned aircraft systems, or UAS. In the process, the project will work with other key stakeholders to define necessary deliverables and products to help enable such access. Within the project, NASA is focusing on five sub-projects. These five focus areas include assurance of safe separation of unmanned aircraft from manned aircraft when flying in the national airspace; safety-critical command and control systems and radio frequencies to enable safe operation of UAS; human factors issues for ground control stations; airworthiness certification standards for UAS avionics and integrated tests and evaluation designed to determine the viability of emerging UAS technology. Five Focus Areas of the UAS Integration in the NAS Project Separation Assurance Provide an assessment of how planned Next Generation Air Transportation System (NextGen) separation assurance systems, with different functional allocations, perform for UAS in mixed operations with manned aircraft Assess the applicability to UAS and the performance of NASA NextGen separation assurance systems in flight tests with realistic latencies and uncertain trajectories Assess functional allocations ranging from today's ground-based, controller-provided aircraft separation to fully autonomous airborne self-separation Communications Develop data and rationale to obtain appropriate frequency spectrum allocations to enable safe and efficient operation of UAS in the NAS Develop and validate candidate secure safety-critical command and control system/subsystem test equipment for UAS that complies with UAS international/national frequency regulations, standards and recommended practices and minimum operational and aviation system performance standards for UAS Perform analysis to support recommendations for integration of safety-critical command and control systems and air traffic control communications to ensure safe and efficient operation of UAS in the NAS Human Systems Integration Develop a research test bed and database to provide data and proof of concept for GCS - ground control station - operations in the NAS Coordinate with standards organizations to develop human-factors guidelines for GCS operation in the NAS Certification Define a UAS classification scheme and approach to determining Federal Aviation Regulation airworthiness requirements applicable to all UAS digital avionics Provide hazard and risk-related data to support development of type design criteria and best development practices Integrated Tests and Evaluation Integrate and test mature concepts from technical elements to demonstrate and test viability Evaluate the performance of technology development in a relevant environment (full-mission, human-in-the-loop simulations and flight tests)

outreach↗
NASA NTRS2017

Growth and Survival of Perchlorate-Reducing Bacteria in Media Containing Elevated Perchlorate Concentrations and UV-C Conditions

Introduction: The identification of perchlorate (ClO4(-)) on Mars has led to the possibility that complete redox couples are available for microbial metabolism in contemporary surface environments. Perchlorate-reducing bacteria (PRB) utilize ClO4(-) and chlorate (ClO3(-)) as terminal electron acceptors due to the high reduction potential. Additionally, ClO4(-) salts have been suggested as a possible source of brines on Mars and spectral evidence indicates that the hydration of ClO4(-) salts in the regolith of Martian is linked to the surface recurring slope lineae (RSL). For these reasons PRB may serve as analog organisms for possible life on Mars. However, there is very little information on the viability of PRB in aqueous environments that contain high levels of perchlorate Microorganisms on or near the surface of Mars, such as in the RSL, would potentially be exposed to high-salinity and high ultraviolet radiation environments. Under these extreme conditions, microorganisms must possess mechanisms for maintaining continued high genome fidelity. To assess possible microbial viability in contemporary Mars analog environments we are investigating the tolerance of two PRB strains in aqueous conditions under high UV-C conditions and high ClO4(-) concentrations.

Bywaters, K. F.↗
NASA NTRS2017

Building Better Biosensors for Exploration into Deep-Space, Using Humanized Yeast

1.BioSentinel is 1 of 13 secondary payloads hitching a ride beyond Low Earth Orbit on Exploration Mission 1 (EM-1), set to launch from NASAs Space Launch System in 2019. EM-1 is our first opportunity to investigate the effects of the deep space environment on a eukaryotic biological system, the budding yeast S. cerevisiae. Though separated by a billion years of evolution we share hundreds of genes important for basic cell function, including responses to DNA damage. Thus, yeast is an ideal biosensor for detecting typesextent of damage induced by deep-space radiation.We will fly desiccated cells, then rehydrate to wake them up when the automated payload is ready to initiate the experiment. Rehydration solution contains SC (Synthetic Complete) media and alamarBlue, an indicator for changes in growth and metabolism. Telemetry of LED readings will then allow us to detect how cells respond throughout the mission. The desiccation-rehydration process can be extremely damaging to cells, and can severely diminish our ability to accurately measure and model cellular responses to deep-space radiation. The aim of this study is to develop a better biosensor: yeast strains that are more resistant to desiccation stress. We will over-express known cellular protectants, including hydrophilin Sip18, the protein disaggregase Hsp104, and thioredoxin Trx2, a responder to oxidative stress, then measure cell viability after desiccation to determine which factors improve stress tolerance. Over-expression of SIP18 in wine yeast starter cultures was previously reported to increase viability following desiccation stress by up to 70. Thus, we expect similar improvements in our space-yeast strains. By designing better yeast biosensors we can better prepare for and mitigate the potential dangers of deep-space radiation for future missions.This work is funded by NASAs AES program.

biosensor↗
NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗
NASA NTRS2016

Pathfinder Technology Demonstrator: GlobalStar Testing and Results

The communications subsystem of a spacecraft is typically a SWaP (size, weight, and power) intensive subsystem in a SWaP constrained environment such as a CubeSat. Use of a satellite-based communication system, such as GlobalStars duplex GSP-1720 radio is a low SWaP potentially game-changing low-cost communication subsystem solution that was evaluated for feasibility for the NASA Pathfinder Technology Demonstrator (PTD) project. The PTD project is a series of 6U CubeSat missions to flight demonstrate and characterize novel small satellite payloads in low Earth orbit. GlobalStar is a low Earth orbit satellite constellation for satellite phone and low-speed data communications, and the GSP-1720 is their single board duplex radio most commonly used in satellite phones and shipment tracking devices. The PTD project tested the GSP-1720 to characterize its viability for flight using NASA GEVS (General Environmental Verification Standard) vibration and thermal vacuum levels, as well as testing the uplink-downlink connectivity, data throughput, and file transfer capabilities. This presentation will present the results of the environmental and capability testing of the GSP-1720 performed at NASA Ames Research Center, as well as the viability for CubeSat use in LEO.

satellite communication↗
NASA NTRS2017

Biosentinel: Improving Desiccation Tolerance of Yeast Biosensors for Deep-Space Missions

BioSentinel is one of 13 secondary payloads to be deployed on Exploration Mission 1 (EM-1) in 2019. We will use the budding yeast Saccharomyces cerevisiae as a biosensor to determine how deep-space radiation affects living organisms and to potentially quantify radiation levels through radiation damage analysis. Radiation can damage DNA through double strand breaks (DSBs), which can normally be repaired by homologous recombination. Two yeast strains will be air-dried and stored in microfluidic cards within the payload: a wild-type control strain and a radiation sensitive rad51 mutant that is deficient in DSB repairs. Throughout the mission, the microfluidic cards will be rehydrated with growth medium and an indicator dye. Growth rates of each strain will be measured through LED detection of the reduction of the indicator dye, which correlates with DNA repair and the amount of radiation damage accumulated. Results from BioSentinel will be compared to analog experiments on the ISS and on Earth. It is well known that desiccation can damage yeast cells and decrease viability over time. We performed a screen for desiccation-tolerant rad51 strains. We selected 20 re-isolates of rad51 and ran a weekly screen for desiccation-tolerant mutants for five weeks. Our data shows that viability decreases over time, confirming previous research findings. Isolates L2, L5 and L14 indicate desiccation tolerance and are candidates for whole-genome sequencing. More time is needed to determine whether a specific strain is truly desiccation tolerant. Furthermore, we conducted an intracellular trehalose assay to test how intracellular trehalose concentrations affect or protect the mutant strains against desiccation stress. S. cerevisiae cell and reagent concentrations from a previously established intracellular trehalose protocol did not yield significant absorbance measurements, so we tested varying cell and reagent concentrations and determined proper concentrations for successful protocol use.

BioSentinel↗
NASA NTRS2016

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesionattachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

Juran, C. M.↗