ALBERT

Description: Responses of animals exposed to microgravity during in-space experiments were observed via available video recording stored in the NASA Ames Life Sciences Data Archive. These documented observations of animal behavior, as well as the range and level of activities during spaceflight, demonstrate that weightlessness conditions and the extreme novelty of the surroundings may exert damaging psychological stresses on the inhabitants. In response to a recognized need for in-flight animals to improve their wellbeing we propose to reduce such stresses by shaping and interrelating structures and surroundings to satisfying vital physiological needs of inhabitants. A Rodent Habitat Hardware System (RHHS) based housing facility incorporating a tubing network system, to maintain and monitor rodent health environment with advanced accessories has been proposed. Placing mice in a tubing-configured environment creates more natural space-restricted nesting environment for rodents, thereby facilitating a more comfortable transition to living in microgravity. A sectional tubing structure of the RHHS environment will be more beneficial under microgravity conditions than the provision of a larger space area that is currently utilized. The new tubing configuration was found suitable for further incorporation of innovative monitoring technology and accessories in the animal holding habitat unit which allow to monitor in real-time monitoring of valuable health related biological parameters under weightlessness environment of spaceflight.

Description: A coupling between geomagnetic activity and the human nervous system's function was identified by virtue of continuous monitoring of heart rate variability (HRV) and the time-varying geomagnetic field over a 31-day period in a group of 10 individuals who went about their normal day-to-day lives. A time series correlation analysis identified a response of the group's autonomic nervous systems to various dynamic changes in the solar, cosmic ray, and ambient magnetic field. Correlation coefficients and p values were calculated between the HRV variables and environmental measures during three distinct time periods of environmental activity. There were significant correlations between the group's HRV and solar wind speed, Kp, Ap, solar radio flux, cosmic ray counts, Schumann resonance power, and the total variations in the magnetic field. In addition, the time series data were time synchronized and normalized, after which all circadian rhythms were removed. It was found that the participants' HRV rhythms synchronized across the 31-day period at a period of approximately 2.5 days, even though all participants were in separate locations. Overall, this suggests that daily autonomic nervous system activity not only responds to changes in solar and geomagnetic activity, but is synchronized with the time-varying magnetic fields associated with geomagnetic field-line resonances and Schumann resonances.

Description: The detrimental effects of mechanical unloading in microgravity, including the musculo-skeletal system, are well documented. However, the effects of mechanical unloading on joint health and the interaction between bone and cartilage specifically, are less well known. Our ongoing studies with the mouse bone model have identified the failure of normal stem cell-based tissue regeneration, in addition to tissue degeneration, as a significant concern for long-duration spaceflight, especially in the mesenchymal and hematopoietic tissue lineages. Furthermore, we have identified the cell cycle arrest molecule, CDKN1ap21, as specifically up-regulated during spaceflight exposure and localized to osteoprecursors on the bone surface and chondroprogenitors in articular cartilage that are both required for normal tissue regeneration. The 30-day BionM1 and 37-day Rodent Research 1 (RR1) missions enabled the possibility of studying these effects in long-duration microgravity experiments. We hypothesized that the inhibition of stem cell-based tissue regeneration in short-duration spaceflight would continue during long-duration spaceflight resulting in significant tissue alterations and we specifically studied the hip joint (pelvis and proximal femur) to elucidate these effects. To test this hypothesis we analyzed bone and bone marrow stem cells using techniques including high-resolution Microcomputed Tomography (MicroCT), in-vivo differentiation and migration assays, and whole transcriptome expression profiling. We found that exposure to spaceflight for 30 days results in a significant decrease in bone volume fraction (-31), trabecular thickness (-14) and trabecular number (-20). Similar decrements in bone volume fraction (-27), trabecular number (-13) and trabecular thickness (-17) were found in female mice exposed to 37 days spaceflight. Furthermore, high-resolution MicroCT and immunohistochemical analysis of spaceflight tissues revealed a severe disruption of the epiphyseal boundary, resulting in endochondral ossification of the femoral head and perforation of articular cartilage by bone. This suggests that spaceflight in microgravity may cause rapid induction of an aging-like phenotype with signs of osteoarthritic disease in the hip joint. Microarray analysis also revealed that the top pathways altered during spaceflight include activation of matrix metalloproteinases, oxidative stress signaling and inflammation in both whole bone tissue and isolated bone marrow stem cells. In conclusion, the observed inhibition of stem cell-based tissue regeneration persists during long-duration spaceflight. Furthermore, spaceflight mice exhibit disruption of the epiphyseal boundary and endochondral ossification of the femoral head, and an inhibition of stem cell based tissue regeneration, which, taken together, may indicate onset of an accelerated aging phenotype with signs of osteoarthritic disease.

Description: Broad tissue degeneration and the failure of normal tissue regenerative processes in microgravity because of mechanical unloading are increasing concerns for sustaining life in space as the duration of future flight missions increases. Work in our laboratory has identified normal adult stem cell-based tissue regenerative processes, such as the formation of new bone, cartilage, and immune cells, as being particularly sensitive to the stresses of mechanical unloading in microgravity. Our studies have also identified the inhibition of differentiation of marrow mesenchymal stem cells and activation of CDKN1ap21-mediated cell cycle arrest in proliferative osteoprecursor cells on the bone surface as potential mechanisms for spaceflight-induced skeletal changes. This finding, in combination with the role of CDKN1ap21 as a suppressor of mammalian tissue regeneration, suggests that this gene could be responsible for suppressing stem cell-based tissue regeneration in response to disuse. In this work, we hypothesized that CDKN1ap21 regulates regenerative bone formation in response to alterations in mechanical load and tested this hypothesis by studying the skeletal phenotype and stem cell regenerative ability of juvenile (4-11 weeks old) and adult (18 weeks-12 months old) p21 (--) knockout (KO) mice. Additionally, we analyzed bone micro-architectural properties, bone formation rates and differentiation capacity of bone marrow stem cells (BMSCs) from male and female KO mice exposed to hindlimb unloading (HU) for 15-30 days. We found that juvenile KO mice exhibited increased femoral trabecular and cortical bone formation, whilst three-point bending of the tibias from KO mice showed decreased bone stiffness. Conversely, adult KO mice exhibited no significant differences in micro-architectural properties compared to WT (wild-type) but woven bone structure was indicative of rapid bone remodeling. Furthermore, cortical bone properties showed similar characteristics to aged bone, including increased cross-sectional area and perimeter, whilst three-point bending showed increased stiffness and toughness. Interestingly, in-vitro, KO mice exhibited increased differentiation and mineralized nodule formation in osteoblastogenesis assays compared to WT. Preliminary results from CDKN1ap21 KO mice subjected to HU suggest altered sensitivity to mechanical unloading resulting in decreased cortical thickness compared to WT mice. However, KO mice subjected to short and long-duration HU show increased in-vitro differentiation potential of BMSCs to from form mature, mineral-forming osteoblasts, indicating maintenance of regenerative potential. Analysis of bone formation rates, cell proliferation rates and key genes of interest are currently underway. These results indicate a novel role for CDKN1ap21 in load-dependent osteoprogenitor proliferation and differentiation and that deletion of CDKN1ap21 results in an age-dependent release of osteoblast proliferation inhibition and increased bone formation and turnover.

Description: In support of air revitalization system sorbent selection for future space missions, Ames Research Center (ARC) has performed CO2 capacity tests on various sorbents to complement structural strength tests from Marshall Space Flight Center (MSFC). The materials of interest are: Grace Davison Grade 544 13x, Honeywell UOP APG III, VSA-10, BASF 13x, and Grace Davison Grade 522 5A. Each sorbents CO2 capacity was measured using a Micromeritics ASAP 2020 Physisorption Volumetric Analysis machine to produce 0C, 10C, 25C, 50C, and 75C isotherms. These datasets were then extrapolated using Langmuir 3-Site and Toth isotherm models to compare with previously measured capacity data from MSFC using a thermogravimetric analysis approach. The modeling and extrapolation from ARC data correlated well with data measured at MSFC.

Description: Limits and guidelines are set on microbial counts in produce to protect the consumer. Different agencies make specifications, which constitute when a product becomes unsafe for human consumption. Producers design their procedures to comply with the limits, but they are responsible creating their own internal standards. The limits and guidelines are summarized here to be applied to assess the microbial safety of the NASA Veggie Program.

Description: As the world's space agencies and commercial entities continue to expand beyond Low Earth Orbit (LEO), novel approaches to carry out biomedical experiments with animals are required to address the challenge of adaptation to space flight and new planetary environments. The extended time and distance of space travel along with reduced involvement of Earth-based mission support increases the cumulative impact of the risks encountered in space. To respond to these challenges, it becomes increasingly important to develop the capability to manage an organism's self-regulatory control system, which would enable survival in extraterrestrial environments. To significantly reduce the risk to animals on future long duration space missions, we propose the use of metabolically flexible animal models as "pathfinders," which are capable of tolerating the environmental extremes exhibited in spaceflight, including altered gravity, exposure to space radiation, chemically reactive planetary environments and temperature extremes. In this report we survey several of the pivotal metabolic flexibility studies and discuss the importance of utilizing animal models with metabolic flexibility with particular attention given to the ability to suppress the organism's metabolism in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of these factors to minimize damage caused by exposure to spaceflight and extreme planetary environments. Examples of microorganisms and animal models with dormancy capabilities suitable for space research are considered in the context of their survivability under hostile or deadly environments outside of Earth. Potential steps toward implementation of metabolic control technology in spaceflight architecture and its benefits for animal experiments and manned space exploration missions are discussed.

Description: Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Description: So you want to conduct human spaceflight research aboard the International Space Station (ISS)? Once your spaceflight research aboard the ISS is proposal is funded.... the real work begins. Because resources are so limited for ISS research, it is necessary to maximize the work being done, while at the same time, minimizing the resources spent. Astronauts may be presented with over 30 human research experiments and select, on average approximately 15 in which to participate. In order to conduct this many studies, ISSMP uses the study requirements provided by the principle investigator to integrate all of this work into the astronauts' complement. The most important thing for investigators to convey to the ISSMP team is their RESEARCH REQUIREMENTS. Requirements are captured in the Experiment document. This document is the official record of how, what, where and when data will be collected. One common mistake that investigators make is not taking this document seriously, but when push comes to shove, if a research requirement is not in this document....it will not get done. The research requirements are then integrated to form a complement of research for each astronaut. What do we mean by integration? Many experiments have overlapping requirements; blood draws, behavioral surveys, heart rate measurement. Where possible, these measures are combined to reduce redundancy and save crew time. Investigators can access these data via data sharing agreements. More examples of how ISS research is integrated will be presented. There are additional limitations commonly associated with human spaceflight research that will also be discussed. Large/heavy hardware, invasive procedures, and toxic reagents are extremely difficult to implement on the ISS. There are strict limits placed on the amount of blood that can be drawn from crew members during (and immediately after) spaceflight. These limits are based on 30-day rolling accumulations. We have recently had to start restricting studies due to this limit. The NASA Human Research Program (HRP) provides extensive support, via ISSMP, to help investigators cope with all of the intricacies of conducting human spaceflight research. This presentation will help you take the best advantage of that support.

Description: Spectrum is a multispectral fluorescence imager designed for capturing in vivo genetic expression in a variety of biological organisms, providing a capability that does not currently exist on the International Space Station (ISS). Researching organisms that have been transformed with in vivo reporter genes ligated with fluorescent proteins allows the scientific community to further understand the fundamental biological responses of these organisms when subjected to space environments. Model organisms that may utilize multispectral imaging on the ISS include unicellular organisms (e.g. Saccharomyces cerevisiae), plants (e.g. Arabidopsis thaliana), and invertebrates (e.g. Caenorhabditis elegans).

Description: Continued space bioscience research onboard the International Space Station (ISS) and future long-duration flight missions to the Moon or Mars will require the ability to conduct on-orbit molecular analysis of biological samples independently from Earth. In the last year two new molecular analytic technologies have been installed and the technologies demonstrated onboard the ISS: The Sample Prep Module (SPM) WetLab-2 (WL2) qRT-PCR toolbox and the Oxford Nanopore MinIon Biomolecule Sequencer. Here we describe protocol development and integration into existing ISS technology for end-to-end on-orbit biological sample processing and molecular analysis with real time results generated utilizing only field offline analytic software. For this experiment we isolated primary cells from bone marrow flushes of wild type B6129SF2 mice (Jackson Labs) long bones. The cell isolate was then processed using the SPM to produce total 147nanograms of RNA. The total RNA was purified to only messenger RNA (mRNA) and transferred to Smartcycler Thermocycle ISS kit consumable tube using Eppendorf gel loading pipette tips for further processing. Complementary first strand cDNA was synthesized using OLIGO dT priming followed by addition of SuperScript II Reverse Transcriptase and thermal cycling as per manufacturers instruction. All thermal cycling was conducted using the ISS WetLab-2 Cephid Smarcycler real time thermal cycler. Our protocol takes advantage of mRNAs native poly(A) tail, synthesized in vivo to protect the mRNA from degradation by endonucleases, to eliminate end-prep for adapter ligation. The adapted library is purified using MyOne C1 Streptavidin beads before elution in buffer. The pre-sequencing library is diluted in the loading buffer and injected into the MinIon sample port, drawn into the nanopore window by capillary action, and sequenced using the MinKnown software with local basecalling. The sequencing read produced 34.5 million events and local basecalling produced 117,301 successful reads. NCBI Blast of the data for the mouse genome resulted in 2,462 successful nucleotide collection matches (gene sequences) exceeding 70 homology. These results demonstrate the viability of this novel flight ready end-to-end sample analytic methodology and provide a real time homolog for flight experimentation utilizing supply kits and technologies that have already been demonstrated on ISS.

Description: System testing of the Carbon Dioxide Removal and Compression System (CRCS) has revealed that sufficient CO2 removal capability was not achieved with the designed system. Subsystem component analysis of the zeolite bed revealed that the sorbent material suffered significant degradation and CO2 loading capacity loss. In an effort to find the root cause of this degradation, various factors were investigated to try to reproduce the observed performance loss. These factors included contamination by vacuum pump oil, o-ring vacuum grease, loadingunloading procedures, and operations. This paper details the experiments that were performed and their results.

Description: Spaceflight environments and their associated conditions, such as microgravity and space radiation, cause many biological functions formerly considered to be standard to behave in nonstandard ways. Exposure to microgravity has shown to induce deleterious effects in stem cell-based tissue regeneration, leading to immune system and healing response impairments as well as muscle and bone density loss. Such risks must be mitigated in order for long-term human space exploration to proceed. Thus, our work seeks to explore mechanisms of stem cell-based tissue regeneration that experience changes in spaceflight environments. Cellular senescence is a process of inducing cell cycle arrest that can be initiated by various stimuli. This function is influenced by two major pathways, controlled by p53 and pRB tumor suppressor proteins. p53 activity targets the cyclin-dependent kinase inhibitor gene p21Cdkn1a in osteogenic cell cycle arrest. Under conditions of mechanical unloading, stem cell-based tissue regeneration has shown to be decreased in both proliferation and differentiation, as many cells are arrested in progenitor states. p21 has shown upregulation in expression under conditions of microgravity, suggesting its role in regenerative bone formation arrest in space. p21 levels are found to be elevated independent of p53, suggesting a decrease in proliferation and regeneration without apoptosis, but rather through cell cycle arrest alone. Thus, we hypothesize that p21 is a mediator of cellular senescence in bone marrow stem cells. Culturing of bone marrow stem cells from wild type and p21 knockout mice under osteoblastogenic conditions will be completed to explore the role of p21Cdkn1a in stem cell proliferation and maturation. We believe that decreases in somatic stem cell differentiation may occur after spaceflight due to signal pathway alterations that result in downstream inhibition of genes involved in differentiation, preventing tissue from repairing and regenerating normally.

Description: The ends of human chromosomes contain telomeres, or tandem arrays of repeating DNA sequences capped by multiple associated proteins that protect chromosomal ends from degradation. Telomeres function to preserve genomic stability by preventing natural chromosomal ends from being recognized as broken DNA double-strand breaks and triggering inappropriate DNA damage responses. Mounting evidence shows telomere length is an inherited trait that decreases with cellular division and normal aging. In addition, telomere length also appears to be influenced by other factors such as cellular oxidative stress, radiation and mechanical unloading of tissues as in microgravity. To measure these potential effects of the space environment on telomere lengths and cellular aging and regenerative potential we developed a novel telomere measurement approach based on nanopore sequencing of PCR amplified bar-coded chromosome termini. Specifically, telomeres can be directly enriched using barcode sequences ligated to the end of a free end- repaired telomere using the WetLab-2 facility SmartCycler on ISS. Prior to the ligation and amplification protocol a proteinase K digestion of capping proteins followed by a single 95-degree C heat denaturation of the protease is included. After digestion and bar-code ligation, PCR amplification will initiate with the ligated barcoded sequence, suppressing amplification of intra-genomic fragments and resulting in long read barcoded telomere amplicons including the nanopore motor protein sequences. Purified PCR amplicons are then used for nanopore sequencing library generation by simple addition of motor proteins and sequencing library is loaded into the MinION nanopore DNA-sequencer. Amplicon sequence reads from the nanopore device can be base-called quickly on ISS due to barcoding ligation and subsequent PCR amplification enhancing the telomere sequence resolution. If successfully implemented on ISS this technique will provide a novel means of measuring regenerative ability of somatic stem cells in astronauts, and of determining whether spaceflight in microgravity alters their telomere lengths and causes premature cellular aging.

Description: As human habitation and eventual colonization of space becomes an inevitable reality, there is a necessity to understand how organisms develop over the life span in the space environment. Microgravity, altered CO2, radiation and psychological stress are some of the key factors that could affect mammalian reproduction and development in space, however there is a paucity of information on this topic. Here we combine early (neonatal) in vivo spectroscopic imaging with an adult emotionality assay following a common obstetric complication (prenatal asphyxia) likely to occur during gestation in space. The neural metabolome is sensitive to alteration by degenerative changes and developmental disorders, thus we hypothesized that that early neonatal neurometabolite profiles can predict adult response to novelty. Late gestation fetal rats were exposed to moderate asphyxia by occluding the blood supply feeding one of the rats pair uterine horns for 15min. Blood supply to the opposite horn was not occluded (within-litter cesarean control). Further comparisons were made with vaginal (natural) birth controls. In one-week old neonates, we measured neurometabolites in three brain areas (i.e., striatum, prefrontal cortex, and hippocampus). Adult perinatally-asphyxiated offspring exhibited greater anxiety-like behavioral phenotypes (as measured the composite neurobehavioral assay involving open field activity, responses to novel object, quantification of fecal droppings, and resident-intruder tests of social behavior). Further, early neurometabolite profiles predicted adult responses. Non-invasive MRS screening of mammalian offspring is likely to advance ground-based space analogue studies informing mammalian reproduction in space, and achieving high-priority.

Description: Spaceflight imposes multiple stresses on biological systems resulting in genome-scale adaptations. Understanding these adaptations and their underlying molecular mechanisms is important to clarifying and reducing the risks associated with spaceflight. One such risk is infection by microbes present in spacecraft and their associated systems and inhabitants. This risk is compounded by results suggesting that some microbes may exhibit increased virulence after exposure to spaceflight conditions. The yeast, S. cerevisiae, is a powerful microbial model system, and it's response to spaceflight has been studied for decades. However, to date, these studies have utilized common lab strains. Yet studies on trait variation in S. cerevisiae demonstrate that these lab strains are not representative of wild yeast and instead respond to environmental stimuli in an atypical manner. Thus, it is not clear how transferable these results are to the wild S. cerevisiae strains likely to be encountered during spaceflight. To determine if diverse S. cerevisiae strains exhibit a conserved response to simulated microgravity, we will utilize a collection of 100 S. cerevisiae strains isolated from clinical, environmental and industrial settings. We will place selected S. cerevisiae strains in simulated microgravity using a high-aspect rotating vessel (HARV) and document their transcriptional response by RNA-sequencing and quantify similarities and differences between strains. Our research will have a strong impact on the understanding of how genetic diversity of microorganisms effects their response to spaceflight, and will serve as a platform for further studies.

Description: Electrochemical detection of biological molecules is a pertinent topic and application in many fields such as medicine, environmental spills, and life detection in space. Proteases, a class of molecules of interest in the search for life, catalyze the hydrolysis of peptides. Trypsin, a specific protease, was chosen to investigate an optimized enzyme detection system using electrochemistry. This study aims at providing the ideal functionalization of an electrode that can reliably detect a signal indicative of an enzymatic reaction from an Enceladus sample.

Description: Future space exploration and long duration space flight will pose an array of challenges to the health and wellbeing of astronauts. Since 2015, Fairchild Tropical Botanic Garden (FTBG), in partnership with NASA's Veggie team, has been testing edible crops for space flight potential through a series of citizen science experiments. FTBG's interest in classroom-based science projects, along with NASA's successful operation of the Veggie system aboard the International Space Station (ISS), led to a NASA-FTBG partnership that gave rise to the Growing Beyond Earth STEM Initiative (GBE). Established in 2015, GBE now involves 131 middle and high school classrooms in South Florida, all conducting simultaneous plant science experiments. The results of those experiments (both numeric and visual) are directly shared with the space food production researchers at KSC. Through this session, we will explore the successful classroom implementation and integration into the curriculum, how the data is being used and the impact of the project on participating researchers, teachers, and students. Participating schools were supplied with specialized LED-lit growth chambers, mimicking the Veggie system on ISS, for growing edible plants under similar physical and environmental constraints. Research protocols were provided by KSC scientists, while edible plant varieties were selected mainly by the botanists at FTBG. In a jointly-led professional development workshop, participating teachers were trained to conduct GBE experiments in their classrooms. Teachers were instructed to not only teach basic botany concepts, but to also demonstrate practical applications of math, physics and chemistry. As experiments were underway, students shared data on plant germination, growth, and health in an online spreadsheet. Results from the students research show a promising selection of new plant candidates for possible further testing. Over a two year period, more than 5000 South Florida students, ages 11 to 18, participated in GBE. Evaluation of the program shows an increased knowledge of and interest in science and science careers among students. The program has also boosted the demand for summer high school internships at FTBG, further developing expertise in plant research and science related to space exploration. Supported by a grant from NASA (NNX16AM32G) to Fairchild Tropical Botanic Garden.

Description: No abstract available

Description: NASA invests in professional coaching as a way to accelerate the development of its staff. The speaker shares one foundational human development model in coaching - the Six Streams - and applies it to the challenges that new scientists face. The speaker also describes how a new scientist can develop greater capabilities in the Six Streams so that they can become a more effective scientist and feel more satisfaction with their work.

Description: We hypothesize that DNA damage induced by high local energy deposition, occurring when cells are traversed by high-LET (Linear Energy Transfer) particles, can be experimentally modeled by exposing cells to high doses of low-LET. In this work, we validate such hypothesis by characterizing and correlating the time dependence of 53BP1 radiation-induced foci (RIF) for various doses and LET across 72 primary skin fibroblast from mice. This genetically diverse population allows us to understand how genetic may modulate the dose and LET relationship. The cohort was made on average from 3 males and 3 females belonging to 15 different strains of mice with various genetic backgrounds, including the collaborative cross (CC) genetic model (10 strains) and 5 reference mice strains. Cells were exposed to two fluences of three HZE (High Atomic Energy) particles (Si 350 megaelectronvolts per nucleon, Ar 350 megaelectronvolts per nucleon and Fe 600 megaelectronvolts per nucleon) and to 0.1, 1 and 4 grays from a 160 kilovolt X-ray. Individual radiation sensitivity was investigated by high throughput measurements of DNA repair kinetics for different doses of each radiation type. The 53BP1 RIF dose response to high-LET particles showed a linear dependency that matched the expected number of tracks per cell, clearly illustrating the fact that close-by DNA double strand breaks along tracks cluster within one single RIF. By comparing the slope of the high-LET dose curve to the expected number of tracks per cell we computed the number of remaining unrepaired tracks as a function of time post-irradiation. Results show that the percentage of unrepaired track over a 48 hours follow-up is higher as the LET increases across all strains. We also observe a strong correlation between the high dose repair kinetics following exposure to 160 kilovolts X-ray and the repair kinetics of high-LET tracks, with higher correlation with higher LET. At the in-vivo level for the 10-CC strains, we observe that drops in the number of T-cells and B-cells found in the blood of mice 24 hours after exposure to 0.1 gray of 320 kilovolts X-ray correlate well with slower DNA repair kinetics in skin cells exposed to X-ray. Overall, our results suggest that repair kinetics found in skin is a surrogate marker for in-vivo radiation sensitivity in other tissue, such as blood cells, and that such response is modulated by genetic variability.

Description: Exploration of the solar system is constrained by the cost of moving mass off Earth. Producing materials in situ will reduce the mass that must be delivered from earth. CO2 is abundant on Mars and manned spacecraft. On the ISS, NASA reacts excess CO2 with H2 to generate CH4 and H2O using the Sabatier System. The resulting water is recovered into the ISS, but the methane is vented to space. Thus, there is a capability need for systems that convert methane into valuable materials. Methanotrophic bacteria consume methane but these are poor synthetic biology platforms. Thus, there is a knowledge gap in utilizing methane in a robust and flexible synthetic biology platform. The yeast Pichia pastoris is a refined microbial factory that is used widely by industry because it efficiently secretes products. Pichia could produce a variety of useful products in space. Pichia does not consume methane but robustly consumes methanol, which is one enzymatic step removed from methane. Our goal is to engineer Pichia to consume methane thereby creating a powerful methane-consuming microbial factory.

Description: 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.

Description: Pre-flight groundbased testing done to prepare for the first Rodent Research mission validation flight, RR1 (Choi et al, 2016 PlosOne). We purified RNA and measured RIN values to assess quality of the samples. For protein, we measured liver enzyme activities. We tested protocol and methods of preservation to date. Here we present an overview of results related to tissue preservation from the RR1 validation mission and a summary of findings to date from investigators who received RR1 teissues various Biospecimen Sharing Program.

Description: Spaceflight imposes multiple stresses on biological systems resulting in genome-scale adaptations. Understanding these adaptations and their underlying molecular mechanisms is important to clarifying and reducing the risks associated with spaceflight. One such risk is infection by microbes present in spacecraft and their associated systems and inhabitants. This risk is compounded by results suggesting that some microbes may exhibit increased virulence after exposure to spaceflight conditions. The yeast, S. cerevisiae, is a powerful microbial model system, and its response to spaceflight has been studied for decades. However, to date, these studies have utilized common lab strains. Yet studies on trait variation in S. cerevisiae demonstrate that these lab strains are not representative of wild yeast and instead respond to environmental stimuli in an atypical manner. Thus, it is not clear how transferable these results are to the wild S. cerevisiae strains likely to be encountered during spaceflight. To determine if diverse S. cerevisiae strains exhibit a conserved response to simulated microgravity, we will utilize a collection of 100 S. cerevisiae strains isolated from clinical, environmental and industrial settings. We will place selected S. cerevisiae strains in simulated microgravity using a high-aspect rotating vessel (HARV) and document their transcriptional response by RNA-sequencing and quantify similarities and differences between strains. Our research will have a strong impact on the understanding of how genetic diversity of microorganisms effects their response to spaceflight, and will serve as a platform for further studies.

Description: This payload overview presentation will be presented at the POIWG on October 17th, 2017. It provides a high-level overview of Cell Science-02 operations.

Description: System testing of the Carbon Dioxide Removal and Compression System (CRCS) has revealed that sufficient CO2 removal capability was not achieved with the designed system. Subsystem component analysis of the zeolite bed revealed that the sorbent material suffered significant degradation and CO2 loading capacity loss. In an effort to find the root cause of this degradation, various factors were investigated to try to reproduce the observed performance loss. These factors included contamination by vacuum pump oil, o-ring vacuum grease, loading/unloading procedures, and operations. This paper details the experiments that were performed and their results.

Description: Despite centuries of scientific balloon flights, only a handful of experiments have produced biologically-relevant results. Yet unlike orbital spaceflight, it is much faster and cheaper to conduct biology research with balloons, sending specimens to the near space environment of Earths stratosphere. Samples can be loaded the morning of a launch and sometimes returned to the laboratory within one day after flying. The National Aeronautics and Space Administration (NASA) flies large, unmanned scientific balloons from all over the globe, with missions ranging from hours to weeks in duration. A payload in the middle portion of the stratosphere (approx. 35 km above sea level) will be exposed to an environment similar to the surface of Mars: temperatures generally around -36 C, atmospheric pressure at a thin 1 kPa, relative humidity levels 〈1%, and a harsh illumination of ultraviolet (UV) and cosmic radiation levels (about 100 W/sq m and 0.1 mGy/d, respectively) that can be obtained nowhere else on the surface of the Earth, including environmental chambers and particle accelerator facilities attempting to simulate space radiation effects. Considering the operational advantages of ballooning and the fidelity of space-like stressors in the stratosphere, researchers in aerobiology, astrobiology, and space biology can benefit from balloon flight experiments as an intermediary step on the extraterrestrial continuum (ground, low Earth orbit, and deep space studies). Our presentation targets biologists with no background or experience in scientific ballooning. We will provide an overview of large balloon operations, biology topics that can be uniquely addressed in the stratosphere, and a roadmap for developing payloads to fly with NASA.

Description: Social interactions are adaptive responses to environmental pressures that have evolved to facilitate the success of individual animals and their progeny. Quantifying social behavior in social animals is therefore one method of evaluating an animal's health, wellbeing and their adjustment to changes in their environment. The interaction between environment and animal can influence numerous other physiological and psychological responses that may enhance, deter or shift an animals social paradigm. For this study, we utilized flight video from the Rodent Research Hardware and Operations Validation mission (Rodent Research-1; RR1) on the International Space Station (ISS). Female mice spent 37 days in microgravity on the ISS and video was captured during the final 33 days. In a previous analysis of individual behavior, we also reported an observed spontaneous ambulatory behavior which we termed circling or 'race tracking,' and we anecdotally observed an increase in group organization around this behavior. In this analysis we further examined this behavior, and other social interactions, to determine if (1) animals joining in on this behavior were induced by other cohort members already participating in this circling behavior, (2) rates of joining varied by number already participating.

Description: An experiment investigated the impact of normobaric hypoxia induction on aircraft pilot performance to specifically evaluate the use of hypoxia as a method to induce mild cognitive impairment to explore human-autonomous systems integration opportunities. Results of this exploratory study show that the effect of 15,000 feet simulated altitude did not induce cognitive deficits as indicated by performance on written, computer-based, or simulated flight tasks. However, the subjective data demonstrated increased effort by the human test subject pilots to maintain equivalent performance in a flight simulation task. This study represents current research intended to add to the current knowledge of performance decrement and pilot workload assessment to improve automation support and increase aviation safety.

Description: DNA methylation (addition of methyl groups to cytosines which normally represses gene transcription) and changes in telomere length (TTAGGG repeats on the ends of chromosomes) are two molecular modifications that result from stress and could contribute to the long-term effects of intrauterine exposure to maternal stress on offspring behavioral outcomes. Here, we measured methylation of Brain-derived neurotrophic factor (Bdnf), a gene important in development and plasticity, and telomere length in the brains of adult rat male and female offspring whose mothers were exposed to unpredictable and variable stressors throughout gestation. Males exposed to prenatal stress had greater methylation (Bdnf IV) in the medial prefrontal cortex (mPFC) compared to non-stressed controls. Further, prenatally-stressed males had shorter telomeres than controls in the mPFC. This study provides the first evidence in a rodent model of an association between prenatal stress exposure and subsequent shorter brain telomere length. Together findings indicate a long-term impact of prenatal stress on DNA methylation and telomere biology with relevance for behavioral and health outcomes, and contribute to a growing literature linking stress to intergenerational epigenetic alterations and changes in telomere length.

Description: Future long-duration space exploration beyond low earth orbit will increase human exposure to space radiation and microgravity conditions as well as associated risks to skeletal health. In animal studies, radiation exposure (greater than 1 Gy) is associated with pathological changes in bone structure, enhanced bone resorption, reduced bone formation and decreased bone mineral density, which can lead to skeletal fragility. Definitive measurements and detection of bone loss typically require large and specialized equipment which can make their application to long duration space missions logistically challenging. Towards the goal of developing non-invasive and less complicated monitoring methods to predict astronauts' health during spaceflight, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (IR). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and IR (0.5 Gy 56Fe 600 MeV/n and 0.5 Gy 1H 150 MeV/n), euthanized at one and 11 days post-irradiation (IR). To determine the extent of bone loss, tibiae were harvested and cancellous microarchitecture in the proximal tibia quantified ex vivo using microcomputed tomography (microCT). Statistical analysis was performed using Student's t-test. At one day post-IR, expression of FGF18 in skin was significantly greater (3.8X) than sham-irradiated controls, but did not differ at 11 days post IR. Expression levels of other genes associated with antioxidant response (Nfe2l2, FoxO3 and Sod1) and the cell cycle (Trp53, Cdkn1a, Gadd45g) did not significantly differ between the control and IR groups at either time point. Radiation exposure resulted in a 27.0% increase in FGF18-positive hair follicles at one day post-IR and returned to basal levels at 11 days post-IR. A similar trend was observed from FGF18 gene expression analysis of skin. In bone (femora), there was an increase in the expression of the pro-osteoclastogenic cytokine, MCP-1, one day after IR compared to non-irradiated controls. FGF18 expression in skin and MCP- 1 expression in bone were found to be positively correlated (P less than 0.002, r=0.8779). Further, microcomputed tomography analysis of tibia from these animals showed reduced cancellous bone volume (-9.9%) at 11 days post- IR. These results suggest that measurements of early radiation induced changes in FGF18 gene expression in skin may have value for predicting subsequent loss of cancellous bone mass. Further research may lead to the development of a relatively simple diagnostic tool for bone loss, with the advantage that hair follicles and skin are relatively easy to acquire from human subjects.

Description: Upon atmospheric exitre-entry and during training, astronauts are subjected to temporary periods of hypergravity, which has been implicated in the activation of oxidative stress pathways contributing to mitochondrial dysfunction and neuronal degeneration. The pathogenesis of Parkinsons disease and other neurodegenerative disorders is associated with oxidative damage to neurons involved in dopamine systems of the brain. Our study aims to examine the effects of a hypergravitational developmental environment on the degeneration of dopaminergic systems in Drosophila melanogaster. Male and female flies (Gal4-UAS transgenic line) were hatched and raised to adulthood in centrifugal hypergravity (97rpm, 3g). The nuclear expression of the reporter, Green Fluorescent Protein (GFP) is driven by the dopaminergic enzyme tyrosine hydroxylase (TH) promoter, allowing for the targeted visualization of dopamine producing neurons. After being raised to adulthood and kept in hypergravity until 18 days of age, flies were dissected and the expression of TH was measured by fluorescence confocal microscopy. TH expression in the fly brains was used to obtain counts of healthy dopaminergic neurons for flies raised in chronic hypergravity and control groups. Dopaminergic neuron expression data were compared with those of previous studies that limited hypergravity exposure to late life in order to determine the flies adaptability to the gravitational environment when raised from hatching through adulthood. Overall, we observed a significant effect of chronic hypergravity exposure contributing to deficits in dopaminergic neuron expression (p 0.003). Flies raised in 3g had on average lower dopaminergic neuron counts (mean 97.7) when compared with flies raised in 1g (mean 122.8). We suspect these lower levels of TH expression are a result of oxidative dopaminergic cell loss in flies raised in hypergravity. In future studies, we hope to further elucidate the mechanism by which hypergravity-induced oxidative stress damages the dopaminergic neuronal system, as well as examining possible chemical countermeasures to the hypergravity-induced oxidative stress response in dopaminergic neurons in order to combat cell death and consequent mental and behavioral deficits.

Description: A kit for the characterization of chromosomal inversions using single-stranded probes that are either all identical or all complementary to a single-stranded chromatid is described. Reporter species are attached to oligonucleotide strands designed such that they may hybridize to portions of only one of a pair of single-stranded sister chromatids which may be prepared by the CO-FISH procedure. If an inversion has occurred, these marker probes will be detected on the second sister chromatid at the same location as the inversion on the first chromatid. The kit includes non-repetitive probes that are either all identical or all complementary to at least a portion of a target DNA sequence of only one DNA strand of only one chromatid and may in some embodiments include reagents suitable for performing CO-FISH and/or reagents for hybridizing the probes to the target DNA sequence.

Description: Problem statement: During spaceflight, astronauts are subjected to microgravity as well as radiation, both of which have adverse effects on bones, soft tissues and organs, possibly by shared mechanisms. For this reason there is a need to identify broad-spectrum countermeasures to protect multiple tissues from both insults.6.The spaceflight environment poses multiple challenges to homeostasis, including microgravity and ionizing radiation. Together, these factors contribute to cellular stress, and effects include increased generation of reactive oxygen species (ROS), oxidative and DNA damage, cell cycle arrest and cell senescence. We have shown that a purified diet supplemented with dried plum (DP, 25) conferred full protection of cancellous structure from the rapid bone loss caused by exposure to ionizing radiation (Schreurs et al. 2016). Based on these promising results for a new countermeasure to prevent space radiation induced-tissue damage, we will conduct additional studies to advance the potential countermeasure to a higher CRL level. We will test the DP diet for its ability to prevent bone loss caused by simulated microgravity as well as exposure to radiation. This will be achieved by exposing mice to each factor (simulated microgravity and radiation) alone and in combination. We hypothesize that spaceflight conditions lead to oxidative damage and bone loss, and that DP, a dietary additive rich in antioxidant and polyphenolic compounds, is an effective countermeasure for multiple tissues, including bone. To test this hypothesis we will accomplish the following aims: Aim 1 Determine if the antioxidant rich diet DP prevents simulated microgravity-induced bone loss. Aim 2 Determine if DP prevents simulated spaceflight-induced bone loss (microgravity and radiation combined). Aim 3 Determine if DP is effective as a countermeasure for adverse effects of simulated microgravity and radiation on non-skeletal tissues (brain, eye).

Description: The Microbial Ecology and Biogeochemistry Research Laboratory at NASA Ames Research Center focuses primarily on the nutrient cycling and diversity of complex microbial communities. NASA is interested in the composition and functioning of microbial mat communities as these processes fundamentally shape the form and function of these analogs for the earliest forms of life on Earth (3.6 billion years ago), and likely will on other planets as well. Aquaponics systems are supported by microbial communities who perform many complex ecosystem services, including cycling nitrogen. Microbes are integral to the stability and productivity of aquaponics systems, which are analogous to microbial communities in food production systems that are essential for building efficient life support systems for long-distance space travel. Students at Meadow Park Middle School created 10 parallel aquaponics systems and took temporal microbial samples to characterize whether any macro-ecology variables impacted or changed the microbial diversity of these systems. Students additionally created a website so that other classrooms can pursue similar projects in their own schools (https://go.nasa.gov/2uJhxmF). Our lab at NASA Ames has sequenced water samples from each of the 10 tanks at 3 timepoints using a MinION sequencer. MPMS students will be involved in the analysis of the bioinformatics data generated through this collaboration. Our ongoing collaboration aims to collect and analyze data in the classroom setting that has utility for research scientists, while involving students as collaborators in the research process.

Description: The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

Description: No abstract available

Description: We examined experimentally the effects of radiation andor simulated weightlessness by hindlimb unloading on bone and blood vessel function either after a short period or at a later time after transient exposures in adult male, C57Bl6J mice. In sum, recent findings from our studies show that in the short term, ionizing radiation and simulate weightlessness cause greater deficits in blood vessels when combined compared to either challenge alone. In the long term, heavy ion radiation, but not unloading, can lead to persistent, adverse consequences for bone and vessel function, possibly due to oxidative stress-related pathways.

Description: An eye movement-based methodology and assessment tool may be used to quantify many aspects of human dynamic visual processing using a relatively simple and short oculomotor task, noninvasive video-based eye tracking, and validated oculometric analysis techniques. By examining the eye movement responses to a task including a radially-organized appropriately randomized sequence of Rashbass-like step-ramp pursuit-tracking trials, distinct performance measurements may be generated that may be associated with, for example, pursuit initiation (e.g., latency and open-loop pursuit acceleration), steady-state tracking (e.g., gain, catch-up saccade amplitude, and the proportion of the steady-state response consisting of smooth movement), direction tuning (e.g., oblique effect amplitude, horizontal-vertical asymmetry, and direction noise), and speed tuning (e.g., speed responsiveness and noise). This quantitative approach may provide fast and results (e.g., a multi-dimensional set of oculometrics and a single scalar impairment index) that can be interpreted by one without a high degree of scientific sophistication or extensive training.

Description: No abstract available

Description: Growing plants in space will be an essential part of sustaining astronauts during long-range missions. During the summer of 2017, three female NASA interns, have been engaged in research relevant to food production in space, and will present their projects to an all female program known as Girls in STEM camp. Ayla Grandpre, a senior from Rocky Mountain College, has performed data mining and analysis of crop growth results gathered through Fairchild Botanical Gardens program, Growing Beyond Earth. Ninety plants were downselected to three for testing in controlled environment chambers at KSC. Ayla has also managed an experiment testing a modified hydroponics known as PONDS, to grow mizuna mustard greens and red robin cherry tomatoes. Emma Boehm, a senior from the University of Minnesota, has investigated methods to sterilize seeds and analyzed the most common microbial communities on seed surfaces. She has tested a bleach fuming method and an ethanol treatment. Emma has also tested Tokyo bekana Chinese cabbage seeds from four commercial seed vendors to identity differences in germination and growth variability. Lastly, Payton Barnwell, a junior from Florida Polytechnic University has shown that light recipes provided by LEDs can alter the growth and nutrition of 'Outredgeous' lettuce, Chinese cabbage, and Mizuna. The results of her light quality experiments will provide light recipe recommendations for space crops that grown in the Advanced Plant Habitat currently aboard the International Space Station.

Description: Commercial activated carbons from Calgon (207C and OVC) and Cabot Norit (RB2 and GCA 48) were evaluated for use in spacecraft trace contaminant control filters. The Polanyi potential plots of the activated carbons were compared using to those of Barnebey-Cheney Type BD, an untreated activated carbon with similar properties as the acid-treated Barnebey-Sutcliffe Type 3032 utilized in the TCCS. Their adsorptive capacities under dry conditions were measured in a closed loop system and the sorbents were ranked for their ability to remove common VOCs found in spacecraft cabin air. This comparison suggests that these sorbents can be ranked as GCA 48 207C, OVC RB2 for the compounds evaluated.

Description: Thigmomorphogenesis can be utilized to improve volume utilization efficiency in peppers (Capsicum annum cv. California Wonder), a candidate crop for fresh food production in space. The effect occurred primarily through a reduction in average plant height. Reductions in vegetative growth metrics during the juvenile growth phase (growth leading up to and including early anthesis) were not observed during the mature or fruiting phase, with the notable exception of reduced plant height. Early flower production and fruit set was reduced under MS; however, the total edible biomass was not reduced, with MS plants producing fewer but larger fruits. The overall reduction in plant height due to MS (Mechanical Stimulation) was sufficient to realize theoretical improvements in VUE (Volume Use Efficiency) for large vertical farming systems. The reduced heights observed could improve VUE in single tier spaceflight hardware (e.g., Veggie; Massa 2016 (this issue)) in that crops that would not normally fit in these spaceflight systems may be accommodated if MS can be applied. Although the potential for using MS to induce thigmomorphogenic phenotypes has long been appreciated, it is only recently that the growth systems themselves could take advantage of the modified crop architecture associated with MS. It is with this in mind that renewed attention should be given to developing procedures for environmentally modifying crops for spaceflight applications.

Description: An overview of NASA's plant research for bioregenerative life support is given, reviewing much of the work conducted at NASA's Kennedy Space Center over the past 25 years.

Description: Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

Description: Our current study aims to determine the molecular mechanisms that underlie these cardiac changes in response to spaceflight. The central hypothesis of our study is that long duration simulated weightlessness and subsequent recovery causes select and persistent changes in gene expression and oxidative defense-related pathways. In this study, we will first conduct general analyses of three-month old male and female animals, focusing on two key long-duration time points, (i.e. after 90 days of simulated weightlessness (HU) and after 90 days recovery from 90 days of HU. Both rat-specific gene arrays and qPCR will be performed focusing on genes already implicated in oxidative stress responses and cardiac disease. Gene expression analyses will be complemented by biochemical tests of frozen tissue lysates for select markers of oxidative damage.

Description: Considering the range of functions proteins perform, it is surprising they fold into a relatively small set of structures or "folds" that facilitate such function. One explanation is that only a minority were fit enough to emerge from Darwinian selection during the early evolution of life. Alternatively, perhaps only a fraction of all possible folds were trialed. Understanding proto-catalyst selection will aid understanding of the origins and early evolution of life. To investigate which explanation is correct, we study a protein evolved in vitro to bind ATP by Jack Szostak (Fig. 1). This protein adopts a fold which is absent from nature. We are testing whether this fold would have possessed the capability to evolve that would have been essential to survive natural selection on early Earth. Folds that couldn't improve their fitness and evolve to perform new functions would have been replaced by rivals that could. To determine whether the fold is evolvable, we are attempting to change the function of the protein by rationally redesigning to bind GTP. Two design strategies in the region of the nucleobase have been implemented to provide hydrogen bonding partners for the ligand i) an insertion ii) a MET to ASN mutation. Redesigns are being studied computationally at Ames Research Center including free energy of binding calculations. Binding affinities of promising redesigns are to be validated by experimental collaborators at ForteBio using Super Streptavidin Biosensors. If the fold is found to be non-evolvable, this may suggest that many structures were trialed, but the majority were pruned on the basis of their evolvability. Alternatively, if the fold is demonstrated to be evolvable, it would be difficult to explain its absence from nature without considering the possibility that the fold simply wasn't sampled on early Earth. This would not only further our understanding of the origins of life on Earth but also suggest a common phe-nomenon of proto-catalyst evolution.

Description: Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n=22), normally loaded HU Cage Pair-Fed controls (CPF; n=22), and Vivarium controls fed ad libitum (VIV; n=10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8+/- 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, and/or due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5% vs 3.0%, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system. Research supported by NNX15AB48G to JST.

Description: Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided data illuminating the deleterious biological response of bone to mechanical unloading. Down regulation of proliferative mechanisms within stem cell populations of the osteogenic niche is a suggested mechanism for loss of bone mass. However the intercellular communicative cues from osteoblasts and osteocytes in managing stem cell proliferation and osteogenic differentiation are largely unknown. In this investigation, MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells, are co-culture under dynamic tensile conditions and evaluated for phenotypic expression of biochemical signaling proteins influential in driving stem cell differentiation. MLO-Y4 and MC3T3-E1 were co-cultured on polyethersulfone membrane with a 0.45m porosity to permit soluble factor transfer and direct cell-cell gap junction signaling. Cyclic tensile stimulation was applied for 48 h at a frequency of 0.1Hz and strain of 0.1. Total Live cell counts indicate mechanical activation of MC3T3-E1s inhibits proliferation while MLO-Y4s increase in number. However, the percent of live MLO-Y4s within the population is low (46.3 total count, *p0.05, n4) suggesting a potential apoptotic signaling cascade. Immunofluorescence demonstrated that stimulation of co-cultures elicits increased gap junction communication. 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 suggest that an osteocyte-osteoblast signaling feedback mechanism may regulate mechanotransduction of an apoptotic cascade within osteocytes and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

Description: Microgravity is one of the most import factors in space flight where its impact on living biological organisms is concerned. Many different ailments have been reported in astronauts such as spaceflight related osteopenia, cardiovascular concerns, and loss of eye sight. In order to understand why g causes these issues we must understand what is happening at the most basic of biological structures, the cell. The work done in this report is a culmination of contributions made to a much larger project. The project seeks to understand how cellular physiology is changing in SMG conditions and use this knowledge to feed into a follow-up study on the genetic changes that are seen in SMG environments. Cells were imaged using confocal microscopy after 20hrs and 48hrs in a 3D clinostat called the Gravite. Lengths, widths, heights, and total cell areas were measured using an image analysis software package ImageJ. There were significant differences in lengths and widths of cell nuclei, and total area of cell coverage. The report then discusses some of the problems with the testing apparatus and how 3D printing technology may be used to create better sample holders for the 3D clinostat.

Description: Embodiments of the invention include capsules for containing medical implants and delivery systems for release of active biological substances into a host body. Delivery systems comprise a capsule comprising an interior enclosed by walls, and a source of active biological substances enclosed within the capsule interior, wherein the active biological substances are free to diffuse across the capsule walls. The capsule walls comprise a continuous mesh of biocompatible fibers and a seal region where two capsule walls overlap. The interior of the capsule is substantially isolated from the medium surrounding the capsule, except for diffusion of at least one species of molecule between the capsule interior and the ambient medium, and prevents cell migration into or out of the capsule. Methods for preparing and using the capsules and delivery systems are provided.

Description: The development and function of living tissues depends largely on interactions between cells that can vary in both time and space; however, temporal control of cell-cell interaction is experimentally challenging. By employing a micromachined silicon substrate with moving parts, herein is disclosed the dynamic regulation of cell-cell interactions via direct manipulation of adherent cells with micron-scale precision. The inventive devices and methods allow mechanical control of both tissue composition and spatial organization. The inventive device and methods enable the investigation of dynamic cell-cell interaction in a multitude of applications, such as intercellular communication, spanning embryogenesis, homeostasis, and pathogenic processes.

Description: GeneLab Strategic Plan goals for the GeneLab project for the International Space Life Science Working Group.

Description: The present invention is directed to methods of manufacturing bioactive gels from ECM material, i.e., gels which retain bioactivity, and can serve as scaffolds for preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. The manufacturing methods take advantage of a new recognition that bioactive gels from ECM material can be created by digesting particularized ECM material in an alkaline environment and neutralizing to provide bioactive gels.

Description: As the worlds space agencies and commercial entities continue to expand beyond Low Earth Orbit (LEO), novel approaches to carry out biomedical experiments with animals are required to address the challenge of adaptation to space flight and new planetary environments. The extended time and distance of space travel along with reduced involvement of Earth-based mission support increases the cumulative impact of the risks encountered in space. To respond to these challenges, it becomes increasingly important to develop the capability to manage an organisms self-regulatory control system, which would enable survival in extraterrestrial environments. To significantly reduce the risk to animals on future long duration space missions, we propose the use of metabolically flexible animal models as pathfinders, which are capable of tolerating the environmental extremes exhibited in spaceflight, including altered gravity, exposure to space radiation, chemically reactive planetary environments and temperature extremes.In this report we survey several of the pivotal metabolic flexibility studies and discuss the importance of utilizing animal models with metabolic flexibility with particular attention given to the ability to suppress the organism's metabolism in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of these factors to minimize damage caused by exposure to spaceflight and extreme planetary environments. Examples of microorganisms and animal models with dormancy capabilities suitable for space research are considered in the context of their survivability under hostile or deadly environments outside of Earth. Potential steps toward implementation of metabolic control technology in spaceflight architecture and its benefits for animal experiments and manned space exploration missions are discussed.

Description: Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

Description: A closed-loop food production system will be important to gain autonomy on long duration space missions. Crop growth experiments in the Veggie plant chamber aboard the International Space Station (ISS) are helping to identify methods and limitations of food production in space. Prior to flight, seeds are surface sterilized to reduce environmental and crew contamination risks.

Description: Transforming the Martian atmosphere into something suitable for plant life would require a scientific feat that we currently do not possess the means to achieve, but indoor agriculture with LEDs could be just the alternative. Previous research has shown that light recipes provided by LEDs can alter the growth and nutrition of a plant based on wavelengths emitted, and crops grown in space aboard the International Space Station would respond similarly. By testing various LED light recipes such as ratios of red, green, blue (RGB) wavelengths, along with white (W) and far red (FR) on flight approved crops, harvest data were analyzed for trends to determine best light conditions for plant growth. Crops of Outredgeous Lettuce, Tokyo Bekana Chinese cabbage, and Mizuna were grown for 28 days with harvests on 14, 21, and 28 days after planting. By collecting fresh mass, shoot dimensions, chlorophyll estimates, leaf areanumber, and dry mass, the overall differences per light treatment were compared. For Outredgeous lettuce, a recipe of W+FR LEDs yielded increases in biomass and size compared to RB LEDs alone. However, the RB recipe resulted in smaller plants with higher concentrations in phytonutrients. Overall, the RGB + FR treatment with ratios similar to sunlight provided a promising balance of optimized biomass, size, and nutrient content. The Chinese cabbage, which was grown under various ratios of W and B light, showed no differences between recipes, and exhibited similar physiological responses regardless of the light recipes that were tested. The Mizuna studies are still ongoing. Crops for ISS chambers and astronaut consumption are targeted based on biomass, physiology, and for human psychological benefit. The goal of this research is to provide light recipe recommendations for space crops that have been thoroughly tested on the ground. These results will serve a major benefit for astronauts growing crops in the Advanced Plant Habitat currently in orbit aboard the ISS. Future research studies include using LEDs to mimic and provide the solar spectrum in a controlled environment to take the next step in further optimizing crops for space.

Description: This is a set of guides for taking care of plants in Veggie. These were developed for crew autonomous gardening.

Description: No abstract available

Description: Why do we need a DNA sequencer to support the human exploration of space? (A) Operational environmental monitoring; (1) Identification of contaminating microbes, (2) Infectious disease diagnosis, (3) Reduce down mass (sample return for environmental monitoring, crew health, etc.). (B) Research; (1) Human, (2) Animal, (3) Microbes/Cell lines, (4) Plant. (C) Med Ops; (1) Response to countermeasures, (2) Radiation, (3) Real-time analysis can influence medical intervention. (C) Support astrobiology science investigations; (1) Technology superiorly suited to in situ nucleic acid-based life detection, (2) Functional testing for integration into robotics for extraplanetary exploration mission.

Description: At its core, the field of microbial experimental evolution seeks to elucidate the natural laws governing the history of microbial life by understanding its underlying driving mechanisms. However, observing evolution in nature is complex, as environmental conditions are difficult to control. Laboratory-based experiments for observing population evolution provide more control, but manually culturing and studying multiple generations of microorganisms can be time consuming, labor intensive, and prone to inconsistency. We have constructed a prototype, closed system device that automates the process of directed evolution experiments in microorganisms. It is compatible with any liquid microbial culture, including polycultures and field samples, provides flow control and adjustable agitation, continuously monitors optical density (OD), and can dynamically control environmental pressures such as ultraviolet-C (UV-C) radiation and temperature. Here, the results of the prototype are compared to iterative exposure and survival assays conducted using a traditional hood, UV-C lamp, and shutter system.

Description: Emergence of replicable genetic molecules was one of the marking points in the origin of life, evolution of which can be conceptualized as a walk through the space of all possible sequences. A theoretical concept of fitness landscape helps to understand evolutionary processes through assigning a value of fitness to each genotype. Then, evolution of a phenotype is viewed as a series of consecutive, single-point mutations. Natural selection biases evolution toward peaks of high fitness and away from valleys of low fitness. whereas neutral drift occurs in the sequence space without direction as mutations are introduced at random. Large networks of neutral or near-neutral mutations on a fitness landscape, especially for sufficiently long genomes, are possible or even inevitable. Their detection in experiments, however, has been elusive. Although a few near-neutral evolutionary pathways have been found, recent experimental evidence indicates landscapes consist of largely isolated islands. The generality of these results, however, is not clear, as the genome length or the fraction of functional molecules in the genotypic space might have been insufficient for the emergence of large, neutral networks. Thorough investigation on the structure of the fitness landscape is essential to understand the mechanisms of evolution of early genomes. RNA molecules are commonly assumed to play the pivotal role in the origin of genetic systems. They are widely believed to be early, if not the earliest, genetic and catalytic molecules, with abundant biochemical activities as aptamers and ribozymes, i.e. RNA molecules capable, respectively, to bind small molecules or catalyze chemical reactions. Here, we present results of our recent studies on the structure of the sequence space of RNA ligase ribozymes selected through in vitro evolution. Several hundred thousands of sequences active to a different degree were obtained by way of deep sequencing. Analysis of these sequences revealed several large clusters defined such that every sequence in a cluster can be reached from any other sequence in the same cluster through a series of single point mutations. Sequences in a single cluster appear to adopt more than one secondary structure. The mechanism of refolding within a single cluster was examined. To shed light on possible evolutionary paths in the space of ribozymes, the connectivity between clusters was investigated. The effect of length of RNA molecules on the structure of the fitness landscape and possible evolutionary paths was examined by way of comparing functional sequences of 20 and 80 nucleobases in length. It was found that sequences of different lengths shared secondary structure motifs that were presumed responsible for catalytic activity, with increasing complexity and global structural rearrangements emerging in longer molecules.

Description: Ion channels are pore-forming protein assemblies that mediate the transport of small ions across cell membranes. Otherwise, membrane bilayers would be almost impermeable to ions incapable to traverse the low dielectric constant, hydrophobic membrane core. Ion channels are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses, cardiac functions, muscle contraction and apoptosis. On the other extreme of biological complexity, viral ion channels (viroporins) influence many stages of the virus infection cycle either through regulating virus replication, such as entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. Ion channels were crucial components of protocells. Their emergence facilitated adaptation of nascent life to different environmental conditions. The earliest ion channels must have been much simpler than most of their modern ancestors. Viral channels are among only a few naturally occurring models to study the structure, function and evolution of primordial channels. Experimental studies of these properties are difficult and often unreliable. In principle, computational methods, and molecular dynamics (MD) simulations in particular, can aid in providing information about both the structure and the function of ion channels. However, MD suffers from its own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. It is, therefore, essential to determine the reliability of MD simulations. We propose to do so on the basis of two criteria. One is channel stability on time scales that extend for several microseconds or longer. The other is the ability to reproduce the measured ionic conductance as a function of applied voltage. If both the stability and the calculated ionic conductance are satisfactory it will greatly increase our confidence that the structure and the function of a channel are described sufficiently accurately. To our knowledge, long time scale stability (approx.10 micro-sec) and the correct electrophysiology have been shown so far for only one channel - the synthetic LS3 hexamer). In this presentation, this approach will be discussed in application to two viral channels - Vpu, encoded by the HIV-1 genome and p7 of hepatitis C.

Description: As interest in long duration effects of space habitation increases, understanding the behavior of model organisms living within the habitats engineered to fly them is vital for designing, validating, and interpreting future spaceflight studies. Only a handful of papers have previously reported behavior of mice and rats in the weightless environment of space (Andreev-Andrievskiy, et al., 2013; Cancedda et al., 2012; Ronca et al., 2008). The Rodent Research Hardware and Operations Validation Mission (Rodent Research-1; RR1) utilized the Rodent Habitat (RH) developed at NASA Ames Research Center to fly mice on the ISS. Ten adult (16-week-old) female C57BL6J mice were launched on September 21st, 2014 in an unmanned Dragon Capsule, and spent 37 days in flight. Here we report group behavioral phenotypes of the RR1 Flight (FLT) and environment-matched Ground Control (GC) mice in the RH during this long duration flight. Video was recorded for 34 days on the ISS, permitting daily assessments of overall health and well being of the mice, and providing a valuable repository for detailed behavioral analysis. As compared to GC mice, RR1 FLT mice exhibited the same range of behaviors, including eating, drinking, exploration, self- and allogrooming, and social interactions at similar or greater levels of occurrence. Overall activity was greater in FLT as compared to GC mice, with spontaneous ambulatory behavior, including organized circling or race-tracking behavior that emerged within the first few days of flight following a common developmental sequence, comprising the primary dark cycle activity of FLT mice. Circling participation by individual mice persisted throughout the mission. Analysis of group behavior over mission days revealed recruitment of mice into the group phenotype, coupled with decreasing numbers of collisions between circling mice. This analysis provides insights into the behavior of mice in microgravity, and clear evidence for the emergence of a distinctive, organized group behavior unique to the weightless space environment. Supported by the NASA Rodent Research Project, Space Biology Program, and Space Life Sciences Training Program.

Description: Recent studies of sensorimotor adaptation have found that individual differences in task-based functional brain activation are associated with the rate of adaptation and savings at subsequent sessions. However, few studies to date have investigated offline neural predictors of adaptation and multi-day savings. In the present study, we explore whether individual differences in the rate of visuomotor adaptation and multi-day savings are associated with differences in resting state functional connectivity and gray matter volume. Thirty-four participants performed a manual adaptation task during two separate test sessions, on average 9 days apart. We found that resting state functional connectivity strength between sensorimotor, anterior cingulate, and temporoparietal areas of the brain was a significant predictor of adaptation rate during the early, cognitive phase of practice. In contrast, default mode network functional connectivity strength was found to predict late adaptation rate and savings on day two, which suggests that these behaviors may rely on overlapping processes. We also found that gray matter volume in temporoparietal and occipital regions was a significant predictor of early learning, whereas gray matter volume in superior posterior regions of the cerebellum was a significant predictor of late adaptation. The results from this study suggest that offline neural predictors of early adaptation facilitate the cognitive mechanisms of sensorimotor adaptation, with support from by the involvement of temporoparietal and cingulate networks. In contrast, the neural predictors of late adaptation and savings, including the default mode network and the cerebellum, likely support the storage and modification of newly acquired sensorimotor representations. These findings provide novel insights into the neural processes associated with individual differences in sensorimotor adaptation.

Description: Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. Specifically CDKN1A/p-21 a cell senescence protein, was found to be upregulated in osteoprecursor cells of the femur during 15-day spaceflight, leading to the working hypothesis that CDKN1A/p-21 plays a role in inhibition of bone formation via mechanical regulation. To evaluate this hypothesis, utilizing a p-21 knockout mouse-line and relevant wildtype control, we cultured femoral bone marrow primary cells under unloaded (static) and cyclically stretched loading through a 30 day osteoblastogenesis protocol. Morphologic evaluation of the cultures demonstrated that mechanical stretching aligned the cells and increased the presence of defined focal adhesion expressing talin, integrin v3, and PTK2 protein tyrosine kinase 2, also known as focal adhesion kinase (FAK) in both mouse strains. In corroboration with previous investigations of cell survival signals relation to FAK, our study found that with greater concentration of focal adhesions via stretch stimulation the live cell percentage was significantly higher than the unloaded controls (p-21 knockout line: +49.70%, p*=0.009, wildtype control: +18.14%, p*=.01). Also evaluated was the mineralization and ECM secretion capability of the differentiating cells. Von Kossa staining has shown that in the p-21 knockout cells unloaded cells produce more matrix that the stretch stimulated, however the matrix is unorganized presenting in sporadic nodules covering approximately 30% of the culture area at day 14 (n=6 wells) while the stretch stimulated cultures have less mineralization content the surface area containing mineralized matrix is greater (~68% at day 14). Q-PCR evaluation of the p-21 knockout cells revealed that canonical (-catenin cascade) and non-canonical wnt11 and downstream planar cell polarity (wnt/PCP) pathway molecule RAC1 are prevalently upregulated with mechanical stimulation. Immunofluorescence for -catenin and RAC1 showed co-localization at the nuclear membrane of the p-21 knockout cells but not the wildtype (n=1) suggesting that molecular communication via the canonical and wnt/PCP pathway are initiated by mechanical loading and experience regulation along the signaling cascade by CDKN1A/p-21. Future investigations will further elucidate this relationship and provide causal data demonstrating mechanical loadings modulatory effect on p-21 expression change.

Description: Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice.

Description: Summary of temperature profiles of frozen tissues from various locations on ISS, transport, labs and the resultant quality of RNA obtained from those tissues.

Description: Cetacean (whales, dolphins and porpoises) mass strandings are a longstanding mystery in the field of marine biology that continue to be recorded in coastal environments around the world. For each of these events, anywhere from a few to several hundred otherwise healthy animals strand in onshore environments, often for no apparent reason. While the causes of these events remain unclear, anthropogenic and naturogenic mechanisms have been suggested. We present results of an inter-disciplinary study that draws expertise from space weather, marine mammal biology and ecology, and marine mammal stranding response. This study assessed 16 years of cetacean stranding events in the Cape Cod (Massachusetts, USA) area concurrently with a large dataset of meteorological, geophysical, biological, oceanographic and space weather data to produce inferences about possible causes for these unexplained events.

Description: Understanding how microbes impact an ecosystem has improved through advances of molecular and genetic tools, but creating complex systems that emulate natural biology goes beyond current technology. In fact, many chemical, biological, and metabolic pathways of even model organisms are still poorly characterized. Even then, standard laboratory techniques for testing microbial impact on environmental change can have many drawbacks; they are time-consuming, labor intensive, and are at risk of contamination. By having an automated process, many of these problems can be reduced or even eliminated. We are developing a benchtop system that can run for long periods of time without the need for human intervention, involve multiple environmental stressors at once, perform real-time adjustments of stressor exposure based on current state of the population, and minimize contamination risks. Our prototype device allows operators to generate an analogue of real world micro-scale ecosystems that can be used to model the effects of disruptive environmental change on microbial ecosystems. It comprises of electronics, mechatronics, and fluidics based systems to control, measure, and evaluate the before and after state of microbial cultures from exposure to environmental stressors. Currently, it uses four parallel growth chambers to perform tests on liquid cultures. To measure the population state, optical sensors (LED/photodiode) are used. Its primary selection pressure is UV-C radiation, a well-studied stressor known for its cell- and DNA-damaging effects and as a mutagen. Future work will involve improving the current growth chambers, as well as implementing additional sensors and environmental stressors into the system. Full integration of multiple culture testing will allow inter-culture comparisons. Besides the temperature and OD sensors, other types of sensors can be integrated such as conductivity, biomass, pH, and dissolved gasses such as CO and O. Additional environmental stressor systems like temperature (extreme heat or cold), metal toxicity, and other forms of radiation will increase the scale and testing range.

Description: No abstract available

Description: Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n22), normally loaded HU Cage Pair-Fed controls (CPF; n22), and Vivarium controls fed ad libitum (VIV; n10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, andor due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5 vs 3.0, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system.

Description: 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.

Description: Saccadic and pupillary responses are reliable indices of arousal decrement (e.g. fatigue), that might be exploited to improve work schedule guidelines. In this study, we tested the sensitivity of a short 30-s oculomotor test to detect diurnal arousal variations. Twelve participants (5 females, 7 males, 37.7+-11.9 years) volunteered to be assessed every hour (66+-20 min) for three consecutive working days, during their regular office-hours. We used a fully automated testing system, the FIT 2000 Fitness Impairment Tester (Pulse Medical Instruments Inc., Rockville, MD, USA), to measure and record saccadic peak velocity, pupil diameter, and latency and amplitude of the pupillary light reflex. In addition, we collected subjective levels of arousal using the Stanford Sleepiness Scale, and body core temperature. We analyzed the data using a linear mixed model approach for longitudinal data. Both saccadic velocity and subjective alertness decreased over the course of a day, while body core temperature increased (all p-values.05). The data also weakly suggested an increase of the pupil diameter (p 07). The findings support the use of oculomotor indices in the assessment of arousal and fatigue in applied settings.

Description: During weightlessness, the skeletal system of astronauts is negatively affected by decreased calcium absorption and bone mass loss. Therefore, it is necessary to counteract these changes for long-term skeletal health during space flights. Our long-term plan is to assess artificial gravity (AG) as a possible solution to mitigate these changes. In this study, we aim to determine the skeletal acclimation to chronic centrifugation. We hypothesize that a 2G hypergravity environment causes an anabolic response in growing male rats. Specifically, we predict chronic 2G to increase tissue mineral density, bone volume fraction of the cancellous tissue and to increase overall bone strength. Systemically, we predict that bone formation markers (i.e., osteocalcin) are elevated and resorption markers (i.e., tartrate resistant acid phosphatase) are decreased or unchanged from controls. The experiment has three groups, each with an n8: chronic 2g, cage control (housed on the centrifuge, but not spun), and a vivarium control (normal rat caging). Pre-pubescent, male Long-Evans rats were used to assess our hypothesis. This group was subject to 90 days of 2G via centrifugation performed at the Chronic Acceleration Research Unit (CARU) at University of California Davis. After 90 days, animals were euthanized and tissues collected. Blood was drawn via cardiac puncture and the right leg collected for structural (via microcomputed tomography) and strength quantification. Understanding how counteract these skeletal changes will have major impacts for both the space-faring astronauts and the people living on Earth.

Description: Growing plants in space will be an essential part of sustaining astronauts during long-range missions. To drive photosynthesis, light-emitting diodes (LEDs) are becoming superior because of their efficiency, longevity, small size, safety, and wavelength versatility. Isolating the effects of certain wavelengths on plant growth when combined with white light is attracting attention. To optimize crop production/quality in space, this study has aimed to configure novel light recipes for the Advanced Plant Habitat currently aboard the International Space Station (ISS). By using white light as a background to maintain normal growth, the addition of monochromatic wavelengths provides a clearer understanding of how each part of the visible spectrum affects plant growth. By growing Outredgeous lettuce under six treatments of White (W) LEDs, W + blue (B), W+ green (G), W + red (R), W + far red (FR), and RGB + FR LEDs with ratios similar to natural sunlight, this investigation has assessed differences in biomass, morphology, chlorophyll, and the synthesis of key phytonutrients. The potential for Outredgeous to produce anthocyanin, lutein, potassium, magnesium, and iron is paramount to maintaining astronaut health. The crop responses to each treatment have been evaluated and the optimal LED combination for both plant yield and nutrient content will be presented.

Description: Almost all modern proteins possess well-defined, relatively rigid scaffolds that provide structural preorganization for desired functions. Such scaffolds require the sufficient length of a polypeptide chain and extensive evolutionary optimization. How ancestral proteins attained functionality, even though they were most likely markedly smaller than their contemporary descendants, remains a major, unresolved question in the origin of life. On the basis of evidence from experiments and computer simulations, we argue that at least some of the earliest water-soluble and membrane proteins were markedly more flexible than their modern counterparts. As an example, we consider a small, evolved in vitro ligase, based on a novel architecture that may be the archetype of primordial enzymes. The protein does not contain a hydrophobic core or conventional elements of the secondary structure characteristic of modern water-soluble proteins, but instead is built of a flexible, catalytic loop supported by a small hydrophilic core containing zinc atoms. It appears that disorder in the polypeptide chain imparts robustness to mutations in the protein core. Simple ion channels, likely the earliest membrane protein assemblies, could also be quite flexible, but still retain their functionality, again in contrast to their modern descendants. This is demonstrated in the example of antiamoebin, which can serve as a useful model of small peptides forming ancestral ion channels. Common features of the earliest, functional protein architectures discussed here include not only their flexibility, but also a low level of evolutionary optimization and heterogeneity in amino acid composition and, possibly, the type of peptide bonds in the protein backbone.

Description: Space Biology current Rodent Research hardware and capabilities as of October 2017. These include the Life Sciences Glovebox, Rapid Freeze, sample cartridges and simulated carcass freezing, mass measurement device, habitat configuration with enrichment, and the non-Rodent capabilities they feature.