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All sourcesNASA NTRSDOE OSTI
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 NTRS2019

Does Seed Sanitization Affect the Plant Rhizosphere Microbiome and Its Ability to Compete with the Human Associated Pathogen, E. coli on Salad Crops?

Cultivation of crops in controlled environmental agricultural systems may limit microbial colonization and reduce diversity of the microbial communities. Practices like seed and growth medium sanitization may further impact microbial communities in the mature plant and the plant’s capacity to limit the growth of pathogens through competition. As humans expand their travels to space, understanding plant growth, health, and development in closed environments will be critical to the success of producing a safe, supplemental food source for astronauts. To determine the persistence of a potential human pathogen in plant growth and development, sanitized and unsanitized seeds from, mizuna (Brassica rapa var japonica) and red romaine lettuce (Lactuca sativa cultivar ‘Outredgeous’), were inoculated with Escherichia coli, ATCC 21445, germinated under simulated International Space Station (ISS) environmental conditions and harvested every 7 days until maturity. The persistence of E. coli in the rhizosphere was determined by plating on selective media, real time PCR (Polymerase Chain Reaction) and community sequencing of the rhizosphere communities. E. coli was detected in the crops’ roots and leaves for several weeks post germination. At day 28, plants from sanitized seeds had significantly higher counts of E. coli on the roots than those from unsanitized seeds. E. coli was also detected on a few uninoculated plants indicating airborne cross contamination among plants in the same growth chamber and suggesting an influence of the natural microbiome on human pathogen survival and persistence in leafy greens. Sequencing analysis revealed variations in composition and diversity between the communities. Understanding the microbial community of the rhizospheric microbiome is only the first step in determining the relationships between plants. Additional studies to include genotypic and phenotypic variations in the plants should be considered to determine if the natural microbes in the rhizosphere may contribute to the health and therefore, safety of the edible plants.

Khodadad, Christina L. M.↗
NASA NTRS2005

Microbial assessment of cabin air quality on commercial airliners

The microbial burdens of 69 cabin air samples collected from commercial airliners were assessed via conventional culture-dependent, and molecular-based microbial enumeration assays. Cabin air samples from each of four separate flights aboard two different carriers were collected via air-impingement. Microbial enumeration techniques targeting DNA, ATP, and endotoxin were employed to estimate total microbial burden. The total viable microbial population ranged from 0 to 3.6 x10 4 cells per 100 liters of air, as assessed by the ATP-assay. When these same samples were plated on R2A minimal medium, anywhere from 2% to 80% of these viable populations were cultivable. Five of the 29 samples examined exhibited higher cultivable counts than ATP derived viable counts, perhaps a consequence of the dormant nature (and thus lower concentration of intracellular ATP) of cells inhabiting these air cabin samples. Ribosomal RNA gene sequence analysis showed these samples to consist of a moderately diverse group of bacteria, including human pathogens. Enumeration of ribosomal genes via quantitative-PCR indicated that population densities ranged from 5 x 10 1 ' to IO 7 cells per 100 liters of air. Each of the aforementioned strategies for assessing overall microbial burden has its strengths and weaknesses; this publication serves as a testament to the power of their use in concert.

microbe↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
NASA NTRS2005

Microbial Surveillance of Potable Water Sources of the International Space Station

To mitigate risk to the crew, the microbial surveillance of the quality of potable water sources of the International Space Station (ISS) has been ongoing since before the arrival of the first permanent crew. These water sources have included stored ground-supplied water, water produced by the shuttle fuel cells during flight, and ISS humidity condensate that is reclaimed and processed. Monitoring was accomplished using a self-contained filter designed to allow bacterial growth and enumeration during flight. Upon return to earth, microbial isolates were identified using 16S ribosomal gene sequencing. While the predominant isolates were common Gramnegative bacteria including Ralstonia eutropha, Methylobacterium fujisawaense, and Spingomonas paucimobilis, opportunistic pathogens such as Stenotrophomonas maltophilia and Pseudomonas aeruginosa were also isolated. Results of in-flight enumeration have indicated a fluctuation of bacterial counts above system design specifications. Additional in-flight monitoring capability for the specific detection of coliforms was added in 2004; no coliforms have been detected from any potable water source. Neither the bacterial concentrations nor the identification of the isolates recovered from these samples has suggested a threat to crew health.

Bruce, Rebekah J.↗
NASA NTRS2018

A Microbial Analysis of Space-Grown Produce

Before space crops become a permanent staple of the astronaut diet, we must first understand how plants and pathogenic microbes interact in microgravity. Crops such as red romaine lettuce and Chinese cabbage were grown on the International Space Station and sent back to Kennedy Space Center for microbial analysis. Aerobic plate counts and metagenomic sequencing were used to characterize bacterial and fungal communities for plants and their respective ''pillows''. These data will be used to create new guidelines for the microbial safety of space-grown produce, and will help us better protect astronauts from food-borne pathogens like E.coli, Staphylococcus, and Salmonella.

Barash, Eric L.↗
NASA NTRS1992

Microbiology operations and facilities aboard restructured Space Station Freedom

With the restructure and funding changes for Space Station Freedom, the Environmental Health System (EHS)/Microbiology Subsystem revised its scheduling and operational requirements for component hardware. The function of the Microbiology Subsystem is to monitor the environmental quality of air, water, and internal surfaces and, in part, crew health on board Space Station. Its critical role shall be the identification of microbial contaminants in the environment that may cause system degradation, produce unsanitary or pathogenic conditions, or reduce crew and mission effectiveness. EHS/Microbiology operations and equipment shall be introduced in concert with a phased assembly sequence, from Man Tended Capability (MTC) through Permanently Manned Capability (PMC). Effective Microbiology operations and subsystem components will assure a safe, habitable, and useful spacecraft environment for life sciences research and long-term manned exploration.

Cioletti, Louis A.↗
NASA NTRS2006

A Comprehensive Assessment of Biologicals Contained Within Commercial Airliner Cabin Air

Both culture-based and culture-independent, biomarker-targeted microbial enumeration and identification technologies were employed to estimate total microbial and viral burden and diversity within the cabin air of commercial airliners. Samples from each of twenty flights spanning three commercial carriers were collected via air-impingement. When the total viable microbial population was estimated by assaying relative concentrations of the universal energy carrier ATP, values ranged from below detection limits (BDL) to 4.1 x 106 cells/cubic m of air. The total viable microbial population was extremely low in both of Airline A (approximately 10% samples) and C (approximately 18% samples) compared to the samples collected aboard flights on Airline A and B (approximately 70% samples). When samples were collected as a function of time over the course of flights, a gradual accumulation of microbes was observed from the time of passenger boarding through mid-flight, followed by a sharp decline in microbial abundance and viability from the initiation of descent through landing. It is concluded in this study that only 10% of the viable microbes of the cabin air were cultivable and suggested a need to employ state-of-the art molecular assay that measures both cultivable and viable-but-non-cultivable microbes. Among the cultivable bacteria, colonies of Acinetobacter sp. were by far the most profuse in Phase I, and Gram-positive bacteria of the genera Staphylococcus and Bacillus were the most abundant during Phase II. The isolation of the human pathogens Acinetobacter johnsonii, A. calcoaceticus, Janibacter melonis, Microbacterium trichotecenolyticum, Massilia timonae, Staphylococcus saprophyticus, Corynebacterium lipophiloflavum is concerning, as these bacteria can cause meningitis, septicemia, and a handful of sometimes fatal diseases and infections. Molecular microbial community analyses exhibited presence of the alpha-, beta-, gamma-, and delta- proteobacteria, as well as Gram-positive bacteria, Fusobacteria, Cyanobacteria, Deinococci, Bacterioidetes, Spirochetes, and Planctomyces in varying abundance. Neisseria meningitidis rDNA sequences were retrieved in great abundance from Airline A followed by Streptococcus oralis/mitis sequences. Pseudomonas synxantha sequences dominated Airline B clone libraries, followed by those of N. meningitidis and S. oralis/mitis. In Phase II, Airline C, sequences representative of more than 113 species, enveloping 12 classes of bacteria, were retrieved. Proteobacterial sequences were retrieved in greatest frequency (58% of all clone sequences), followed in short order by those stemming from Gram-positives bacteria (31% of all clone sequences). As for overall phylogenetic breadth, Gram-positive and alpha-proteobacteria seem to have a higher affinity for international flights, whereas beta-and gamma-proteobacteria are far more common about domestic cabin air parcels in Airline C samples. Ultimately, the majority of microbial species circulating throughout the cabin airs of commercial airliners are commensal, infrequently pathogenic normal flora of the human nasopharynx and respiratory system. Many of these microbes likely originate from the oral and nasal cavities, and lungs of passengers and flight crew and are disseminated unknowingly via routine conversation, coughing, sneezing, and stochastic passing of fomites. The data documented in this study will be useful to generate a baseline microbial population database and can be utilized to develop biosensor instrumentation for monitoring microbial quality of cabin or urban air.

microbial diversity↗
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 NTRS1995

PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world.

Cassell, Gail H.↗
NASA NTRS1987

Nuclear morphometric analysis of osteoblast precursor cells in periodontal ligament, SL-3 rats

Five small (55 days old, 196 +/- 5 g) (mean +/- SE) and five large (83 days old, 382 +/- 4 g) Sprague-Dawley strain, specific pathogen-free rats were exposed to a 7-day spaceflight and 12-h postflight recovery period. As measured in 3-micron sections, periodontal ligament (PDL) fibroblastlike cells were classified according to nuclear size: A + A' (40-79), B (80-119), C (120-169), and D (greater than or equal to 170 microns 3). Since the histogenesis sequence is A----A'----C----D----osteoblast, the relative incidence of A + A' to C + D is an osteogenic index. No difference in A + A' or C + D cells in small rats may reflect partial recovery of preosteoblast formation (A----C) during the 12-h postflight period. Large flight rats demonstrated increased numbers of A + A', indicating an inhibition of preosteoblast formation (A----C). At least in the older group, a 7-day flight is adequate to reduce PDL osteogenic potential (inhibition in PDL osteoblast differentiation and/or specific attrition of C + D cells) that does not recover by 12-h postflight.

Flight Experiment↗
NASA NTRS2003

The Grand Assault

Plasmodium falciparum, the protozoan parasite responsible for most human malaria, is among the most studied pathogens of all time, probably only exceeded by the human immunodeficiency virus HTV and the bacterium Mycobacterium tuberculosis. The extent of human suffering and the devastating costs of malaria have long been recognized by world bodies, and numerous initiatives have been taken over the years in an effort to defeat this insidious microbe. Beginning in 1996, an international consortium of scientists from more than a dozen institutions set about to determine the sequence of the organism's 23-megabase genome. Their massive effort-which ended up going well beyond simple sequencing is reported in this special issue of Nature. The avowed goal of the project was to search for chinks in the parasite's armor so that new and effective drugs and vaccines might be developed.

Doolittle, Russell F.↗
NASA NTRS2009

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including the concentration and the characteristics of the infectious agent. Thus, a thorough knowledge of the microorganisms aboard spacecraft is essential in mitigating infectious disease risk to the crew. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The translation of 16S ribosomal DNA sequencing for the identification of bacteria from the SWAB experiment to nominal operations has increased bacterial speciation of environmental isolates from previous flights three fold compared to previous conventional methodology. The incorporation of molecular-based DNA fingerprinting using repetitive sequence-based polymerase chain reaction (rep-PCR) into the capabilities of the laboratory has provided a methodology to track microorganisms between crewmembers and their environment. Both 16S ribosomal DNA identification and bacterial fingerprinting have improved NASA s capability to better understand spacecraft environments and determine the source of contamination events. Preflight sampling has been completed for air, surface, and water samples. In-flight sample collection has been completed for a total of 8 air and surface sample collection sessions. In-flight hardware has performed well and the surface sampling device received positive feedback from the crew for its ease of use. While processing and analysis continue for these samples, early results have begun to provide information on the spacecraft environment. Using a method called Denaturing Gradient Gel Electrophoresis (DGGE), several air and samples were evaluated to determine the types of organisms that were present. Using only molecular techniques, DGGE does not depend on any microbial growth on culture media, allowing a more comprehensive assessment of the spacecraft interior. Preliminary results have identified several microorganisms that would not have been isolated using current technology, though none of these organisms would be considered medically significant. Interestingly, the isolation of Gram negative organisms is greater using DGGE than conventional media based isolation. The cause of this finding is unclear, though it may be the result of the technique s ability to isolate both viable and non-viable bacteria. The next phase of the SWAB sample analysis is the use of quantitative polymerase chain reaction (QPCR) to look for specific medically significant organisms. While not as broad as DGGE, QPCR is much more sensitive and may reveal findings that were not seen during the initial evaluation. Together, this information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Castro, V.A.↗
NASA NTRS2007

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

The determination of risk from infectious disease during long-duration missions is composed of several factors including (1) the host#s susceptibility, (2) the host#s exposure to the infectious disease agent, and (3) the concentration of the infectious agent, and (4) the characteristics of the infectious agent. While stringent steps are taken to minimize the transfer of potential pathogens to spacecraft, several medically significant organisms have been isolated from both the Mir and International Space Station (ISS). Historically, the method for isolation and identification of microorganisms from spacecraft environmental samples depended upon their growth on culture media. Unfortunately, only a fraction of the organisms may grow on a culture medium, potentially omitting those microorganisms whose nutritional and physical requirements for growth are not met. Thus, several pathogens may not have been detected, such as Legionella pneumophila, the etiological agent of Legionnaire#s disease. We hypothesize that environmental analysis using non-culture-based technologies will reveal microorganisms, allergens, and microbial toxins not previously reported in spacecraft, allowing for a more complete health assessment. The development of techniques for this flight experiment, operationally named SWAB, has already provided advances in NASA laboratory processes and beneficial information toward human health risk assessment. The first accomplishment of the SWAB experiment was the incorporation of 16S ribosomal DNA sequencing for the identification of bacteria. The use of this molecular technique has increased bacterial speciation of environmental isolates from previous flights three fold compared to conventional methodology. This increased efficiency in bacterial speciation provides a better understanding of the microbial ecology and the potential risk to the crew. Additional SWAB studies focused on the use of molecular-based DNA fingerprinting using repetitive sequencebased polymerase chain reaction (rep-PCR). This technology has allowed contamination tracking of microorganisms between crewmembers and their environment. This study not only demonstrated that ISS has a greater diversity of organisms than originally expected, but also provided insight into possible routes of infection to the crew. Additional ground-based studies used rep-PCR and protein based assays to determine the potential of methicillin resistant Staphylococcus aureus (MRSA) aboard ISS. MRSA has become increasingly common on Earth and pose a treatment problem for infections during flight. While no MRSA have been isolated from ISS to date, the mecA gene product that is responsible for methicillin resistance was isolated in other Staphylococcus species aboard ISS suggesting a potential of MRSA through gene transfer. Using improved sample collection technologies, flight sampling for SWAB was initiated in August 2006 and should continue through spring of 2007. The focus of these flight samples is the collection of DNA for evaluation by Denaturing Gradient Gel Electrophoresis (DGGE). Unlike other techniques, DGGE does not depend on any microbial growth on culture media allowing a more comprehensive assessment of the spacecraft interior. This study should provide insight into the true microbial ecology that is experienced by the crew during flight. This information will lead toward an accurate microbial risk assessment to help set flight requirements to protect the safety, health, and performance of the crew.

Ott, C. M.↗
NASA NTRS2011

Automated Miniaturized Instrument for Space Biology Applications and the Monitoring of the Astronauts Health Onboard the ISS

Human space travelers experience a unique environment that affects homeostasis and physiologic adaptation. The spacecraft environment subjects the traveler to noise, chemical and microbiological contaminants, increased radiation, and variable gravity forces. As humans prepare for long-duration missions to the International Space Station (ISS) and beyond, effective measures must be developed, verified and implemented to ensure mission success. Limited biomedical quantitative capabilities are currently available onboard the ISS. Therefore, the development of versatile instruments to perform space biological analysis and to monitor astronauts' health is needed. We are developing a fully automated, miniaturized system for measuring gene expression on small spacecraft in order to better understand the influence of the space environment on biological systems. This low-cost, low-power, multi-purpose instrument represents a major scientific and technological advancement by providing data on cellular metabolism and regulation. The current system will support growth of microorganisms, extract and purify the RNA, hybridize it to the array, read the expression levels of a large number of genes by microarray analysis, and transmit the measurements to Earth. The system will help discover how bacteria develop resistance to antibiotics and how pathogenic bacteria sometimes increase their virulence in space, facilitating the development of adequate countermeasures to decrease risks associated with human spaceflight. The current stand-alone technology could be used as an integrated platform onboard the ISS to perform similar genetic analyses on any biological systems from the tree of life. Additionally, with some modification the system could be implemented to perform real-time in-situ microbial monitoring of the ISS environment (air, surface and water samples) and the astronaut's microbiome using 16SrRNA microarray technology. Furthermore, the current system can be enhanced substantially by combining it with other technologies for automated, miniaturized, high-throughput biological measurements, such as fast sequencing, protein identification (proteomics) and metabolite profiling (metabolomics). Thus, the system can be integrated with other biomedical instruments in order to support and enhance telemedicine capability onboard ISS. NASA's mission includes sustained investment in critical research leading to effective countermeasures to minimize the risks associated with human spaceflight, and the use of appropriate technology to sustain space exploration at reasonable cost. Our integrated microarray technology is expected to fulfill these two critical requirements and to enable the scientific community to better understand and monitor the effects of the space environment on microorganisms and on the astronaut, in the process leveraging current capabilities and overcoming present limitations.

Human space travelers↗
NASA NTRS2021

Decoding the effects of synonymous variants

Synonymous single nucleotide variants (sSNVs) are common in the human genome but are often overlooked. However, sSNVs can have significant biological impact and may lead to disease. Existing computational methods for evaluating the effect of sSNVs suffer from the lack of gold-standard training/evaluation data and exhibit over-reliance on sequence conservation signals. We developed synVep (synonymous Variant effect predictor), a machine learning-based method that overcomes both of these limitations. Our training data was a combination of variants reported by gnomAD (observed) and those unreported, but possible in the human genome (generated). We used positive-unlabeled learning to purify the generated variant set of any likely unobservable variants. We then trained two sequential extreme gradient boosting models to identify subsets of the remaining variants putatively enriched and depleted in effect. Our method attained 90% precision/recall on a previously unseen set of variants. Furthermore, although synVep does not explicitly use conservation, its scores correlated with evolutionary distances between orthologs in cross-species variation analysis. synVep was also able to differentiate pathogenic vs. benign variants, as well as splice-site disrupting variants (SDV) vs. non-SDVs. Thus, synVep provides an important improvement in annotation of sSNVs, allowing users to focus on variants that most likely harbor effects.

Zishuo Zeng↗
NASA NTRS2001

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

NASA Discipline Plant Biology↗
NASA NTRS2002

Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

Case Reports↗