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NASA NTRS2017

Microbial Characteristics of ISS Environmental Surfaces

The microbiome of environmental surfaces from the International Space Station were characterized in order to examine the relationship to crew and hardware maintenance. The Microbial Observatory (ISS-MO) experiment generated a microbial census of ISS environments using advanced molecular microbial community analyses along with traditional culture-based methods. Since the “omics” methodologies generated an extensive microbial census, significant insights into spaceflight-induced changes in the populations of beneficial and/or potentially harmful microbes were gained. Surface samples were collected from several ISS surface locations from three flight opportunities, and were returned to Earth via the Soyuz TMA-14M or the Space X Dragon capsule. In addition to cultivation methods, viable microbial burden, iTag-based sequencing, and metagenome analyses were carried out. The cultivable microbial bioburden differed by location and sampling event. Exploring the ISS environmental microbiome revealed presence of opportunistic pathogens and antibiotic resistant microbes. Genes involved in ATP binding cassette transporters, two component systems, and beta-lactam resistance were among a diverse set of metabolic and genetic information processing pathways. Whole genome sequencing (WGS) of 50 ISS strains exhibiting resistance to various antibiotics was carried out. The antibiotic resistant genes deduced from the WGS were compared with the resistomes generated directly from the gene pool of the environmental samples. Two unique Aspergillus fumigatus strains isolated from the ISS were characterized and compared to the experimentally established clinical isolates Af293 and CEA10. A virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis indicated that both ISSFT-021 and IF1SW-F4 were significantly more lethal compared to Af293 and CEA10. The findings from this Environmental “Omics” project should be exploited to enhance human health and well-being of a closed system. In other words, the ISS-MO research aims to "translate" findings in fundamental research into medical practice (pathogen detection) and meaningful health outcomes (countermeasure development).

Perry, Jay↗

NASA NTRS2016

The Biomolecule Sequencer Project: Nanopore Sequencing as a Dual-Use Tool for Crew Health and Astrobiology Investigations

Human missions to Mars will fundamentally transform how the planet is explored, enabling new scientific discoveries through more sophisticated sample acquisition and processing than can currently be implemented in robotic exploration. The presence of humans also poses new challenges, including ensuring astronaut safety and health and monitoring contamination. Because the capability to transfer materials to Earth will be extremely limited, there is a strong need for in situ diagnostic capabilities. Nucleotide sequencing is a particularly powerful tool because it can be used to: (1) mitigate microbial risks to crew by allowing identification of microbes in water, in air, and on surfaces; (2) identify optimal treatment strategies for infections that arise in crew members; and (3) track how crew members, microbes, and mission-relevant organisms (e.g., farmed plants) respond to conditions on Mars through transcriptomic and genomic changes. Sequencing would also offer benefits for science investigations occurring on the surface of Mars by permitting identification of Earth-derived contamination in samples. If Mars contains indigenous life, and that life is based on nucleic acids or other closely related molecules, sequencing would serve as a critical tool for the characterization of those molecules. Therefore, spaceflight-compatible nucleic acid sequencing would be an important capability for both crew health and astrobiology exploration. Advances in sequencing technology on Earth have been driven largely by needs for higher throughput and read accuracy. Although some reduction in size has been achieved, nearly all commercially available sequencers are not compatible with spaceflight due to size, power, and operational requirements. Exceptions are nanopore-based sequencers that measure changes in current caused by DNA passing through pores; these devices are inherently much smaller and require significantly less power than sequencers using other detection methods. Consequently, nanopore-based sequencers could be made flight-ready with only minimal modifications.

John, K. K.↗

NASA NTRSDate not supplied

Spaceflight Biospecimen and Data Sharing in Support of Science Discovery and Exploration

For decades, NASA and international partners have conducted biological experiments in space to understand effects of spaceflight and address potential hazards. To enable spaceflight back to the Moon, and then to Mars and beyond, it is imperative to further understand basic science and health risks associated with spaceflight, along with developing countermeasures. The sending of experiments and organisms into space is a costly endeavor. To maximize scientific return, sharing with the scientific community both space-flown biospecimens and data from completed experiments is essential. New fundamental, applied, and bioinformatic science insights can be gained from specimen and data sharing efforts. Data reuse enables spaceflight health risk modeling, analyzing adverse outcomes across spaceflight hazards, and deep space autonomous support for the flight medical officer. Space-flown biospecimens not required by mission Principal Investigators are regularly archived and made available for scientific request. The largest biorepository of these samples are found within NASA’s Institutional Scientific Collection at Ames Research Center (ISC-ARC), which stores over 32,000 specimens mostly from Shuttle and International Space Station (ISS) missions, but also some ground-based analog samples. The Ames Life Sciences Data Archive manages the ISC-ARC. Tissues are predominantly from mice and rats, though samples are also available from bacteria and quail. Only a handful of other similar collections exist worldwide. Rodent biospecimens exposed to simulated space radiation at Brookhaven National Laboratory are archived under the purview of NASA HRP Space Radiation Element. Microbial collection and analyses from 20 years of routine environmental monitoring of air, surfaces, and water systems of the ISS were performed to ensure a safe environment for astronauts. Samples from the ISC-ARC, space radiation and microbial collections are searchable and requestable through the NASA Life Sciences Data Archive (LSDA). Decades of planetary protection microbial isolates derived from spacecraft bioburden are archived in JPL’s microbial collection. Rodent biospecimens from spaceflight investigations conducted by the Japan Aerospace Exploration Agency (JAXA) are archived and available at the JAXA Biorepository at Tsukuba Space Center. The Russian Institute of Biomedical Problems also has a collection of animal, microbial, cellular, and fungi available for research from ground analog experiments. Several data repositories exist for scientists to utilize. The LSDA is the primary NASA source of life sciences research data and information. It contains decades of spaceflight and ground-analog research involving human, microbial, cellular, plant, and animal subjects. Data is collected from NASA-funded investigations through the Human Research Program and the Space Biology Program. The NASA Lifetime Surveillance of Astronaut Health collects and grants access to clinical and occupational health monitoring data from astronauts, with a list and description of data collected available for request through the LSDA. NASA GeneLab at ARC collects genomic, transcriptomic, proteomic, and metabolomic data from any species. It is a repository and platform for collaborative open-science bioinformatic approaches. JAXA is establishing an ‘omics-based repository in collaboration with the Tohoku Medical Megabank (ToMMo), called the JAXA-ToMMo Integrated Biobank for Space Life Science. Overall, the sharing of these biospecimen and data resources can assist researchers worldwide in understanding spaceflight effects on biology, along with enabling next generation data science applications for space exploration platforms. Websites: https://lsda.jsc.nasa.gov/ ; https://www.nasa.gov/ames/research/space-biosciences/isc-bsp ; https://www.nasa.gov/ames/research/space-biosciences/alsda

Ryan T. Scott↗

NASA NTRS2017

The Transcriptional Response of Diverse Saccharomyces Cerevisiae Strains to Simulated Microgravity

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.

yeast↗

NASA NTRS2017

The Transcriptional Response of Diverse Saccharomyces Cerevisiae Strains to Simulated Microgravity

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.

genetic diversity↗

NASA NTRS2018

The Transcriptional Response of Diverse Saccharomyces cerevisiae Strains to Simulated Microgravity

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 a typical 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.

spaceflight↗

NASA NTRS2023

Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS) in Support of Plant-Growth Systems

As the National Aeronautics and Space Association (NASA) begins to pursue long-duration space flights, they will need to be able to provide astronauts with a nutritious and reliable food source. To meet the administration’s goal of traveling to the Moon and Mars, astronauts will need to begin to grow their own food in space. To protect their food source, extensive monitoring will occur to test for the effects of a space flight environment (e.g., radiation) as well as for early pathogen and disease detection. Genomic sequencing allows for both concerns to be tested on a regular basis. However, NASA’s current sequencer is unable to process plant tissues. Therefore, a novel method for plant DNA extraction using microneedle (MN) patches that will be able to feed into NASA’s existing system, but also require minimal human input is proposed. To support this, the design was broken down into four components (1) MN patch fabrication (2) MN patch extraction, (3) automated sampling motion control, and (4) a processing module. The MN patch is fabricated using a custom mold with conically shaped needles. The mold is filled with Polyvinyl alcohol (PVA) solution and placed in a vacuum desiccator. The mold is left in the vacuum overnight until the patch is dry and ready for use. The protocol was tested with varying pressures, drying times, volume amounts, and preparation methods to determine if highquality needles can be produced. A MN is a method of DNA extraction where the patch is applied to a leaf, the needles penetrate the leaf, breaking the rigid plant cell wall to isolate the DNA. A protocol for this method of extraction was tested to ensure the patch could produce the needed yield and purity. The tests varied by the number of patches, number of applications, and plant type. To automate the MN extraction method, motion control will utilize two separate axis tables which move in the x and y directions. The y-axis table will have an end effector that fits a MN patch and will have the ability to apply the patch to the leaf sample. This end effector will also act as a lid for a downstream processing module. The other axis will position the leaf sample and processing container so that the patch can be applied accurately. The Joint Comprehensive Sequencing System (JCSS) module integrates all the components together. The output of this module feeds into the NASA Charged Information-Storage Polymer Preparation System (CHIPPS) for genomic sequencing. The extraction module operates using a series of syringes and tubing to pump the varying reagents needed for the extraction protocol. The results of the study proved that MN patches are a viable method of DNA extraction. While fabrication of high-quality needles was unsuccessful, the protocol was able to be further developed using centrifugation. The integrated design between the motion control and the JCSS enabled the potential for automation with a complete conceptual design and prototype. Future research and development for this study would include (1) further testing for fabrication (2) expanding the range of plant species compatible with the MN patch, and (3) building a working prototype for the integrated system.

Peter Ling↗

NASA NTRS2002

Lipid Biomarkers for Methanogens in Hypersaline Cyanobacterial Mats for Guerrero Negro, Baja California Sur

Analyses of sediments from the vicinity of active methane seeps have uncovered a particular suite of lipid biomarker patterns that characterize methane consuming archaea and their syntrophic, sulfate reducing partners. These isoprenoid biomarkers, largely identified by their anomalously light carbon isotopic signatures, have been a topic of intense research activity and are recorded in numerous methane-rich environments from Holocene to Cenozoic. This phenomenon has implications for depleted kerogens at 2.7 Ga on early Earth (Hinrichs 2002). In contrast, the lipid biosignatures of methane producing archaea are not readily identified through distinct isotopic labels and have received comparably little attention in analyses of archaea in environmental samples. Indeed, environmental analyses generally detect only free archaeal lipids, not the intact, polar molecules found in the membrane of living organisms. As part of the Ames NAI, the 'Early Microbial Ecosystem Research Group' (EMERG) is working to understand microbial processes in the hypersaline cyanobacterial mats growing in the salt evaporation ponds of the Exportadora de Sal at Guerrero Negro, Baja California Sur, Mexico. The aim of this study was to develop methods by which we could identify the organisms responsible for methane generation in this environment. While the ester-bound fatty acids, hopanoids and wax esters provide a means to identify most of the bacterial components of these mats, the archaea which Ere evidently present through genomic assays and the fact of intense methane production (Hoehler et al. 200l), have not been identified through their corresponding lipid signatures. Archaeal core lipids present a number of analytical challenges. The core lipids of methanogens comprise C20, C40 and sometimes C25 isoprenoid chains, linked through ether bonds to glycerol. As well as archaeal (C20), sn-2- and sn-3-hydroxyarchaeol are associated particularly with methylotrophic methanogens. Recently, we have also identified a dihydroxyarchaeol in a hyperthermophilic methanogen (Summons et al. 2002). Additional structural diversity is encoded into the polar head groups that are attached to the glycerol ether cores. The C20 core lipids are readily analyzed by GC-MS as their volatile trimethylsilyl derivatives while compounds with intact polar head groups can only be detected using LC-MS approaches. Our approach was to utilize the alternative of an ether cleavage reagent (BBr3 vs. HI) and a hydride reducing agent to convert all ether lipids to hydrocarbon in order to provide a vertical profile of quantitative information that might be matched to methane fluxes. We have found that while conventional acid hydrolysis and HI treatment will destroy hydroxyarchaeols, molecular information remains intact through use of BBr3 for ether cleavage. This method revealed the presence of traces of biphytane and various ether alkyls associated with some sulfate reducing bacteria within the mat structure. An interesting, and potentially valuable, byproduct of the method utilizing HI was the identification of abundant homohopanoids after superhydride reduction. Evidently present as sulfur-bound diagenetic products these hopanoids are likely cyanobacterial biomarkers in the early stages of diagenetic preservation.

Jahnke, Linda L.↗

NASA NTRS2013

Conducting On-orbit Gene Expression Analysis on ISS: WetLab-2

WetLab-2 will enable expanded genomic research on orbit by developing tools that support in situ sample collection, processing, and analysis on ISS. This capability will reduce the time-to-results for investigators and define new pathways for discovery on the ISS National Lab. The primary objective is to develop a research platform on ISS that will facilitate real-time quantitative gene expression analysis of biological samples collected on orbit. WetLab-2 will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on orbit. WetLab-2 will significantly expand the analytical capabilities onboard ISS and enhance science return from ISS.

quantitative PCR↗

NASA NTRSDate not supplied

Enabling Open and Interoperable Science: Multi-Omics Data Processing Platform with NASA GeneLab Standardized Bioinformatics Workflows for Space and Earth Research

Multi-omics biological data continues to be generated at an astounding pace. Genomics, transcriptomics, metabolomics, and proteomics, or collectively known as multi-omics data, are used to assess biological functions, and provide invaluable insights into human, animal, plant, and environmental health both on Earth and in Space. Despite the abundance of these valuable data, the need for bioinformatics expertise, particularly as it relates to the niche filed of space biology, and a lack of accessible resources for processing these data limit their usefulness in deriving biological insights. The NASA Open Science Data Repository (OSDR) provides access to omics data from various spaceflight and analog studies. To enhance the accessibility and reusability of these data, GeneLab (part of OSDR) designs and implements standardized, community-driven, open-source bioinformatics workflows to transform raw omics data into standardized processed data. Currently, GeneLab-processed data from hundreds of space studies have been reused for meta-analyses. This has led to new insights and scientific publications that extend beyond the initial research, thereby enriching our understanding of molecular-scale biological responses to the space environment. To make these bioinformatics workflows open and accessible, GeneLab teamed up with DOE-funded initiatives, including the National Microbiome Data Collaborative (NMDC), to create the NASA EDGE [Empowering the Development of Genomics Expertise] Bioinformatics web-based platform. NASA EDGE utilizes shared compute resources to run the GeneLab standardized bioinformatics workflows, which eliminates the need for researchers to have their own high performance computing cluster. The web-based platform makes complicated biological analyses incredibly easy to perform, thus expanding the reach of these analyses to bioinformatics novices, students, and even citizen scientists enabling them to contribute to scientific discoveries and progress. The authors will demonstrate how the NASA EDGE platform can be used to process microbial omics data hosted on OSDR as well as user-generated omics datasets using GeneLab’s standard workflows.

Amanda M. Saravia-Butler↗

NASA NTRS2000

Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

NASA Discipline Exobiology↗

NASA NTRS2007

Effect Of Spaceflight On Microbial Gene Expression And Virulence: Preliminary Results From Microbe Payload Flown On-Board STS-115

Human presence in space, whether permanent or temporary, is accompanied by the presence of microbes. However, the extent of microbial changes in response to spaceflight conditions and the corresponding changes to infectious disease risk is unclear. Previous studies have indicated that spaceflight weakens the immune system in humans and animals. In addition, preflight and in-flight monitoring of the International Space Station (ISS) and other spacecraft indicates the presence of opportunistic pathogens and the potential of obligate pathogens. Altered antibiotic resistance of microbes in flight has also been shown. As astronauts and cosmonauts live for longer periods in a closed environment, especially one using recycled water and air, there is an increased risk to crewmembers of infectious disease events occurring in-flight. Therefore, understanding how the space environment affects microorganisms and their disease potential is critically important for spaceflight missions and requires further study. The goal of this flight experiment, operationally called MICROBE, is to utilize three model microbial pathogens, Salmonella typhimurium, Pseudomonas aeruginosa, and Candida albicans to examine the global effects of spaceflight on microbial gene expression and virulence attributes. Specifically, the aims are (1) to perform microarray-mediated gene expression profiling of S. typhimurium, P. aeruginosa, and C. albicans, in response to spaceflight in comparison to ground controls and (2) to determine the effect of spaceflight on the virulence potential of these microorganisms immediately following their return from spaceflight using murine models. The model microorganisms were selected as they have been isolated from preflight or in-flight monitoring, represent different degrees of pathogenic behavior, are well characterized, and have sequenced genomes with available microarrays. In particular, extensive studies of S. typhimurium by the Principal Investigator, Dr. Nickerson, using ground-based analog systems demonstrate important changes in the genotypic, phenotypic, and virulence characteristics of this pathogen resulting from exposure to a flight-like environment (i.e. modeled microgravity).

Wilson, J. W.↗

NASA NTRSDate not supplied

Enterococci in Space: Adaptation, Antibiotic Resistance, and Clinical Implications

Enterococci are gram-positive bacteria that originated when our ancient animal ancestors emerged from the oceans to live on land, and brought their gut flora with them. Enterococcus faecalis (EF) and Enterococcus faecium, are common human commensals and can harbor multidrug resistance. Both have been previously isolated from the International Space Station (ISS). Likely as a consequence of their evolutionary origins, enterococci show remarkable stress resistance within, but also outside, their human hosts. Their antibiotic resistance, coupled with tolerance to desiccation, starvation, and disinfection, make some EF strains potent pathogens in the built environment (e.g., hospitals), and a potential risk to crew health during space missions. Here we describe our planned flight studies, currently in development. Genomic Enumeration of Antibiotic Resistance in Space (GEARS) will characterize the frequency and genomic identity of antibiotic resistant organisms, including enterococci, on the ISS, and expand our future in-space sequencing-based diagnostic capabilities. Enterococcus Growth Advantage on ISS via Tn-seq (EnteroGAIT) will assess the evolutionary selective pressure of the space environment (microgravity, space radiation) using EF as a model system during a long-duration culture and persistence experiment. Adaptation & Evolution of Resilient Enterococcus in Space (AERES) will study existing and newly identified isolates to characterize the “natural” evolutionary history of EF on Earth and in space to reveal mechanisms of microbial adaption including, possibly, natural selection. Our work to date has identified that EF ISS isolates share virulence factors found in clinical strains and commensals, and revealed limitations of current pathogenicity assessment tools. While ISS isolates were, in some cases, multi-drug resistant, current data suggests these are largely commensal strains. Our work will further refine potential crew health risks and improve understanding of EF adaptation to the built environment, of great relevance on Earth where EF is the second leading cause of hospital acquired infection.

Jordan McKaig↗

NASA NTRS2016

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

The WetLab-2 system was developed by NASA Ames Research Center to offer new capabilities to researchers. The system can lyse cells and extract RNA (Ribonucleic Acid) on-orbit from different sample types ranging from microbial cultures to animal tissues. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (Polymerase Chain Reaction) (qRT-PCR) analysis without the need for sample return. The qRT-PCR results can be downlinked to the ground a few hours after the completion of the run. The validation flight of the WetLab-2 system launched on SpaceX-8 on April 8, 2016. On orbit operations started on April 15th with system setup and was followed by three quantitative PCR runs using an E. coli genomic DNA template pre-loaded at three different concentrations. These runs were designed to discern if quantitative PCR functions correctly in microgravity and if the data is comparable to that from the ground control runs. The flight data showed no significant differences compared to the ground data though there was more variability in the values, this was likely due to the numerous small bubbles observed. The capability of the system to process samples and purify RNA was then validated using frozen samples prepared on the ground. The flight data for both E. coli and mouse liver clearly shows that RNA was successfully purified by our system. The E. coli qRT-PCR run showed successful singleplex, duplex and triplex capability. Data showed high variability in the resulting Cts (Cycle Thresholds [for the PCR]) likely due to bubble formation and insufficient mixing during the procedure run. The mouse liver qRT-PCR run had successful singleplex and duplex reactions and the variability was slightly better as the mixing operation was improved. The ability to purify and stabilize RNA and to conduct qRT-PCR on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. The ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

gene expression↗

NASA NTRS2016

Successful Validation of Sample Processing and Quantitative Real-Time PCR Capabilities on the International Space Station

quantitative PCR↗

NASA NTRSDate not supplied

NASA Omics Archive Project

The space environment consists of a complex set of hazards including altered gravity, radiation, psychological/physiological stress, isolation, and confinement leading to complex biological responses. Advances in biotechnology capabilities offer considerable potential to provide novel insight into those responses as well as innovative diagnostic, treatment, and countermeasure solutions for astronauts as NASA begins to travel beyond low Earth orbit. Omics data (genomics, transcriptomics, proteomics, etc.) is one example that can provide NASA with critical knowledge of how a crewmember’s genetics, environment, and lifestyle can be used to develop individualized approaches for disease prevention, advance diagnostics, and improve treatment strategies. NASA ventured into the field of omics on human subjects with the successful completion of the NASA Twins Study which was the first step in mapping the multi-omic profile of astronauts to understand and mitigate the health consequences of spaceflight. The Human Research Program aims to build upon the success of the Twins Study with the NASA Omics Archive flight study, establishing a longitudinal biospecimen archive and efficiently generating a comprehensive high-quality multi-omic dataset from astronauts for the purpose of studying molecular, metabolic, and microbial changes associated with longduration spaceflight missions. The goal is to facilitate scientific and medical research community efforts to characterize and mitigate spaceflight health and performance risks. In this presentation, we will review details regarding the biospecimen and data archive to be generated by the NASA Omics Archive flight study. Data generated as part of this project will be archived in the NASA Life Sciences Portal (NLSP) and be made available for future hypothesis-driven research efforts or occupational surveillance through Institutional Review Board-approved data sharing and retrospective data requests submitted to the Life Sciences Data Archive (LSDA) team. We will also present results of a ground study performed to evaluate in-house procedures, new sample collection hardware, and vendor capabilities. The data repository generated and the samples to be archived by this study will enable future research efforts to assess an astronauts’ unique molecular and genetic profile with respect to individual spaceflight responses. Results of which will be instrumental in enabling precision health capabilities to better assess and mitigate spaceflight risks, detect disease states earlier, and actively monitor countermeasure treatments, ultimately improving clinical outcomes during future exploration class missions.

C. A. Theriot↗

NASA NTRS2011

Post-Fragmentation Whole Genome Amplification-Based Method

This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

Benardini, James↗

NASA NTRS2023

Molecular Identification of Microbial Contaminants

Microorganisms can have significant impacts on the success of NASA’s missions, including the integrity of materials, the reliability of scientific results, and maintenance of crew health. Robust cleaning and sterilization protocols are currently in place in NASA facilities, but agency experts agree that microbial contamination is unavoidable and its impact on NASA’s missions and science must be minimized. Therefore, it is critical to understand: 1) what specific microorganisms are present, 2) how they may impact scientific objectives, and 3) how to select appropriate mitigation strategies. The Marshall Space Flight Center (MSFC) Planetary Protection (PP) microbiology lab historically relied solely upon enumeration of culturable microbial contamination associated with spacecraft materials or cleanrooms. However, this process is time consuming, many microbes cannot be cultured, and very few can be identified with any fidelity using NASA standard microbiological methods. The work described in this white paper includes the establishment of molecular identification capabilities at MSFC, including DNA isolation, amplification, purification, and Sanger sequencing. This capability will not only improve planetary protection efforts at MSFC (i.e. by identifying contaminating microorganisms in cleanrooms or on spacecraft) but also offers a service center-wide for the identification of contaminants that arise in other projects, processing locations, or during set up and roll out of spacecraft. This work also lays the foundation for higher throughput efforts to identify large populations of microbes across the lifetime of a project and serves as the starting point for future work into whole genome sequencing, non-culture based methods, or additional characterization studies. Ultimately, accurate identification informs appropriate mitigation strategies, increasing the chances of success for NASA’s missions and objectives.

C. D. Cassilly↗