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All sourcesNASA NTRSDOE OSTI
NASA NTRS2018

Detection of Antimicrobial Resistance Genes Associated With the International Space Station Environmental Surfaces

Antimicrobial resistance (AMR) is a global health issue. In an effort to minimize this threat to astronauts, who may be immunocompromised and thus at a greater risk of infection from antimicrobial resistant pathogens, a comprehensive study of the ISS “resistome’ was conducted. Using whole genome sequencing (WGS) and disc diffusion antibiotic resistance assays, 9 biosafety level 2 organisms isolated from the ISS were assessed for their antibiotic resistance. Molecular analysis of AMR genes from 24 surface samples collected from the ISS during 3 different sampling events over a span of a year were analyzed with Ion AmpliSeq™ and metagenomics. Disc diffusion assays showed that Enterobacter bugandensis strains were resistant to all 9 antibiotics tested and Staphylococcus haemolyticus being resistant to none. Ion AmpliSeq™ revealed that 123 AMR genes were found, with those responsible for beta-lactam and trimethoprim resistance being the most abundant and widespread. Using a variety of methods, the genes involved in antimicrobial resistance have been examined for the first time from the ISS. This information could lead to mitigation strategies to maintain astronaut health during long duration space missions when return to Earth for treatment is not possible.

Antimicrobial resistance↗
NASA NTRSDate not supplied

Improving Efficacy and Safety of Pharmacological Treatment Through Precision Medicine and Pharmacogenomics for Human Deep Space Exploration

INTRODUCTION: Future spaceflight will require increased crew medical autonomy as exploration class missions expanding duration and distance from Earth, especially for Mars missions. As mission duration increases, it will be even more essential to have appropriate amounts of effective medication to ensure the maintenance of crew health and performance. Conversely, mass and volume constraints will become more severe as future spaceflight expands beyond low Earth orbit, where resupply is difficult or becomes impossible. These constraints thus convey an urgency to further tailor medications included in the spacecraft formulary and increased examination of appropriate dosing regimens. BACKGROUND: Precision Health is an exciting area of cutting-edge research and medicine focused on maintaining an individual’s health and performance through in-depth understanding of an individual’s unique factors and molecular profiles. This approach can be adapted to better predict, monitor, and address physiological responses to the spaceflight environment. One example is the field of pharmacogenomics (PGX),the study of how the expressed genome impacts drug responses with the goal of prescribing the right dose of the right drug at the right time. Specifically, PGX testing provides valuable information on an individual’s precise allelic variations to guide physicians in making informed decisions on pharmaceutical choice and dosing to avoid adverse drug events and maximize pharmacological efficacy. The goal of this study was to evaluate which drugs in the current space pharmacy could be evaluated using PGX testing and to understand the potential impact on the health and wellness of the astronaut population. Additionally, we sought to evaluate clinically available FDA-approved PGX testing solutions to better understand its applicability. METHODS: A complete list of drugs onboard the International Space Station (ISS) was analyzed for risk and likelihood of drug failure and PGX actionability. This analysis encompassed both personal astronaut medications, including supplements and over the counter drugs (n=151) and ISS MedKit formulary medications (n=95). Duplicate medications and different formulations were removed, which resulted in 157 total drugs used in the subsequent analysis. A 5x5 risk assessment table was produced by examining the likelihood of drug failure compared to the consequence of drug failure. Likelihood of individual drug failure was defined by whether existing processes are sufficient to prevent adverse events, as ranked from 1 (very low, can easily be prevented) to 5 (very high, cannot be prevented) during a Mars mission. In contrast, the consequence of drug failure was defined by impact to safety, schedule, cost or technical and ranked from 1 (very low) to 5 (very high).A comprehensive assessment of commercially available PGX solutions is currently underway to evaluate specimen requirements, cost/benefit analysis (cost vs. number of alleles assessed), utility of variant analysis, relevance to inflight medication usage, quality of reporting in enabling clinical application, and ease of integration into electronic medical records. RESULTS: Risk assessments(LxC 5x5 table) indicated29medicationswere in the yellow or red zone driven predominantly by drug failure or safety concerns, with the remainder(n=128)of the medications in the green zone where risk is acceptable. We found that current PGX testing results could impact 21% of the total medications in the ISS MedKit and IMAK; of these, 9 medications currently have direct clinically actionable guidance available. Results of the clinical PGX solution evaluations as related to these medications will be presented. CONCLUSION: PGX testing has demonstrated clear benefits in terrestrial medicine and clinical environments for the selection of proper medications, avoiding adverse drug reactions, and maximizing drug efficacy. We propose that similar benefits would be bestowed on the astronaut and commercial spaceflight passenger population by performing preemptive pre-flight PGX testing to reduce risk of mission failure due to ineffective or toxic medications, improve targeting drug efficacy and safety, and further open the door to countermeasure research exploring PGX-related allelic variants. For example, PGX results could allow tailoring of specific medications at optimal doses more precisely to each individual astronaut, particularly in areas of space motion sickness, sleep aids, and analgesics. An additional benefit is that PGX results could provide information for better planning of the components of a space pharmacy for deep space missions to be more cost effective and more efficient in the utilization of limited pharmaceutical resources. Finally, while PGX testing of the astronaut corps is not currently conducted, this approach could provide immediate impact in support of mission success by reducing risks, optimizing astronaut performance, and providing valuable insights into long-term astronaut health. Such advancements in clinical decision making are important next steps in building dynamic individual risk profiles for astronauts, increasing crew autonomy and providing tailored countermeasures

Alice R W Tang↗
NASA NTRSDate not supplied

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

The CubeSat platform has proven successful in obtaining meaningful life science information when biological payloads are incorporated. Examples include: 1) the first-ever CubeSat with a biological payload, GeneSat-1, which demonstrated decreased growth rates for flight samples of Escherichia coli in low Earth orbit (Parra et al. 2008); 2) PharmaSat, demonstrated that Saccharomyces cerevisiae in the microgravity environment exhibits a significant level of metabolic activity even at high doses of applied antifungal (Ricco et al. 2011); 3) O/OREOS, which used Bacillus subtilis(bacteria) to demonstrate for the first time that microorganisms can be loaded in a dried, dormant form and then rehydrated and grown in orbit months after launch (Nicholson et al. 2011; Ehrenfreund et al. 2014; 4) the SporeSat payload, which investigated Ceratopteris richardii(fern spores) using lab-on-a-chip devices (BioCDs) and minicentrifuges to produce artificial gravitational forces in ground studies (Park et al. 2017), with demonstration of the BioCD and minicentrifuge in space; 5) EcAMSat, the first CubeSat to be directly deployed from the ISS for an experiment assessing antibiotic resistance of E. coli in the microgravity environment (Padgen et al. 2020); 6) BioSentinel, exposed a culture of yeast to galactic cosmic radiation (GCR) and solar particle events while in heliocentric orbit to measure the rate of double-strand-break repair using DNA-repair-deficient mutants. This effort measures the metabolic parameters of yeast in a deep-space environment compared to Earth ambient conditions using a 3-color LED detection system (Ricco et al. 2020; Padgen et al. 2021). We aim to expand this list to include a Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS). SAMMS will allow for the genome level understanding of changes in growth and metabolic activity for any organism. While microbes are suitable for early studies in our proposed platform because of their small size, small and relatively less-complicated genomes, fast generation times, and relevance to life support systems; multicellular organisms can similarly be evaluated for their genetic response to the spaceflight environment. The Spaceflight Autonomous Multigenerational Microbial Sequencer (SAMMS) will enable autonomous sequencing of biological samples in plant production units, cislunar orbit and on the lunar surface to examine spaceflight effects (ie. radiation, altered gravity, reduced pressures) on plant and microbial genomes.On this team a Kennedy Space Center (KSC) space crop production and water systems microbiologist/molecular biologist works with a Johnson Space Center (JSC) International Space Station (ISS) microbial sequencing expert and an Ames Research Center (ARC) CubeSat Engineering team to convert an automated Oxford Nanopore librarypreparation and sequencing method to a fluidic CubeSat payload system. The Oxford Nanopore MinION sequencing platform has proven successful in the spaceflight environment onboard the ISS (Stahl-Rommel et al. 2021). Further long-duration spaceflight and exposure to high levels of radiation will cause genotypic effects in biological organisms that may affect their function. Monitoring the adaption of a population to the spaceflight environment and any subsequent beneficial mutations will allow for the harnessing of organisms best suited for use in life support systems. This will ensure that the selected life support-essential microorganisms maintain their intended specified function over generations of culturing in the relevant spaceflight environment without becoming hazardous to crew or spacecraft systems.

Aubrie E Orourke↗
NASA NTRS2015

The Twins Study: NASA's First Foray into 21st Century Omics Research

The full array of 21st century omics-based research methods should be intelligently employed to reduce the health and performance risks that astronauts will be exposed to during exploration missions beyond low Earth Orbit. In March of 2015, US Astronaut Scott Kelly will launch to the International Space Station for a one year mission while his twin brother, Mark Kelly, a retired US Astronaut, remains on the ground. This situation presents an extremely rare flight opportunity to perform an integrated omics-based demonstration pilot study involving identical twin astronauts. A group of 10 principal investigators has been competitively selected, funded, and teamed together to form the Twins Study. A very broad range of biological function are being examined including the genome, epigenome, transcriptome, proteome, metabolome, gut microbiome, immunological response to vaccinations, indicators of atherosclerosis, physiological fluid shifts, and cognition. The plans for the Twins Study and an overview of initial results will be described as well as the technological and ethical issues raised for such spaceflight studies. An anticipated outcome of the Twins Study is that it will place NASA on a trajectory of using omics-based information to develop precision countermeasures for individual astronauts.

Kundrot, C. E.↗
NASA NTRS2006

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗
NASA NTRS2015

Materials Genome Initiative

The Materials Genome Initiative (MGI) project element is a cross-Center effort that is focused on the integration of computational tools to simulate manufacturing processes and materials behavior. These computational simulations will be utilized to gain understanding of processes and materials behavior to accelerate process development and certification to more efficiently integrate new materials in existing NASA projects and to lead to the design of new materials for improved performance. This NASA effort looks to collaborate with efforts at other government agencies and universities working under the national MGI. MGI plans to develop integrated computational/experimental/ processing methodologies for accelerating discovery and insertion of materials to satisfy NASA's unique mission demands. The challenges include validated design tools that incorporate materials properties, processes, and design requirements; and materials process control to rapidly mature emerging manufacturing methods and develop certified manufacturing processes

Vickers, John↗
NASA NTRS1993

Stabilization of pH in solid-matrix hydroponic systems

2-[N-morpholino]ethanesulfonic acid (MES) buffer or Amberlite DP-1 (cation-exchange resin beads) were used to stabilize substrate pH of passive-wicking, solid-matrix hydroponic systems in which small canopies of Brassica napus L. (CrGC 5-2, genome : ACaacc) were grown to maturity. Two concentrations of MES (5 or 10 mM) were included in Hoagland 1 nutrient solution. Alternatively, resin beads were incorporated into the 2 vermiculite : 1 perlite (v/v) growth medium at 6% or 12% of total substrate volume. Both strategies stabilized pH without toxic side effects on plants. Average seed yield rates for all four pH stabilization treatments (13.3 to 16.9 g m-2 day-1) were about double that of the control (8.2 g m-2 day-1), for which there was no attempt to buffer substrate pH. Both the highest canopy seed yield rate (16.9 g m-2 day-1) and the highest shoot harvest index (19.5%) occurred with the 6% resin bead treatment, even though the 10 mM MES and 12% bead treatments maintained pH within the narrowest limits. The pH stabilization methods tested did not significantly affect seed oil and protein contents.

NASA Discipline Number 93-10↗
NASA NTRS2012

PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

LaDuc, Myron T.↗
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

Variational Asymptotic Homogenization of Finitely Deformed Viscoelastic-Viscoplastic Composites

The objective of this paper is to develop a constitutive model for finitely deformed viscoelastic-viscoplastic materials and a micromechanics approach to homogenizing composites consisting of such materials. The development of the constitutive model involves establishing a thermodynamic framework based on finite strain theory, developing a viscoelasticity and a viscoplasticity model based on the thermodynamic framework, developing a radial return algorithm based on a classic framework, and deriving a closed-from incremental constitutive relation in the spatial configuration. The development of the micromechanics approach involves pulling-back the above constitutive relation to the material configuration, formulating a variational statement with the resulting constitutive relation, discretizing variational statement in a finite-dimensional space, and solving the discretized variational statement using an Euler–Newton predictor–corrector method. The constitutive model is calibrated via monotonic uniaxial compression tests on a polymer, and the calibrated model is validated by comparing its predictions with the cyclic test data. It is shown capable of characterizing viscoelasticity, viscoplasticity, and complex loading paths. The micromechanics approach’s capabilities are demonstrated through homogenizing a unidirectional fiber-reinforced composite, subjected to uniaxial, biaxial, and shear loading, at different strain rates. It is demonstrated to be capable of handling rate dependence and complex loading paths. The present framework can be further improved by implementing more sophisticated viscoelasticity and viscoplasticity models in future work.

Finite element analysis↗
NASA NTRS2026

Does the International Space Station Leak DNA? Preliminary Results from the ISS External Microorganisms Payload

Existing crewed spacecraft like the ISS (International Space Station) leak by design. The ISS routinely releases gas to maintain life support systems and when astronauts exit the station to perform space walks. The chemical component of this leakage is well characterized, but the biological components are not. The ISS is not subject to planetary protection requirements, but planned missions to Mars will use similar systems and will be subject to planetary protection requirements. If detectable microorganisms are escaping through vents and or airlocks we may need to redesign our crewed habitats to minimize this type of contamination. To test the hypothesis that microorganisms from inside ISS are detectable on exterior surfaces an astronaut used the ISS External Microorganisms sampling kit (Rucker et al. 2018) to sample exterior surfaces of the ISS during an EVA (Extra Vehicular Activity) in January of 2025. These samples were returned to Earth for DNA extraction and sequencing. We successfully, extracted and sequenced bacterial, fungal and viral DNA from these samples that was not present in the negative controls. These results should help NASA refine the planetary protection requirements for crewed missions. Methods: The samples were collected using sterile, DNA free, buccal swabs (23 mm. diameter) housed in custom canisters. Each canister uses a 0.2 μm Teflon filter to maintain sterility as the caddy, holding 8 swabs moves in and out of vacuum. The astronaut sampled the: 1) airlock vestibule, 2) airlock thermal cover, 3) a gap in the micrometeorite shielding near the airlock, 4) a handrail near the airlock, 5) the Carbon Dioxide Removal Assembly vent, and 6) the Vacuum Exhaust System vent. The seventh swab was exposed to vacuum during the EVA without touching it to a surface. The eighth swab, a negative control, was not opened until the caddy returned to Earth. DNA was extracted from the swabs using a QIamp UCP Pathogen kit and prepared for sequencing on an Aviti (Element Biosciences) sequencer (Arslan et al. 2024). The resulting sequences were analyzed using the EDGE Bioinformatics platform (Li et al. 2017). The sequences were analyzed individually using tools like BLAST, GOTTCHA2, Kraken2, and PanGIA. The data were also assembled into metagenome assembled genomes) using tools like CONCOCT, MaxBin2 and MetaBAT2. Results: We successfully extracted and sequenced bacterial, archaeal, fungal and viral DNA from all seven samples. The handrail swab had the lowest number of reads (768,651) and the airlock thermal cover had the highest number of reads (8,819,230). These samples contain DNA from human associated bacteria (e.g. Crynebacterium riegelii ), fungi (.e.g. Penicillium rubens ), and viruses (e.g Alphapapillomavirus ). Conclusion: Preliminary interpretation suggest that the airlock and the space suits themselves are the largest sources of contaminant DNA. Most if not all of the DNA is from organisms known to be present inside the ISS. Vents attached to life support systems may be a lesser source of biological contamination. Further analysis should help NASA address planetary protection knowledge gaps for crewed missions.

Aaron B Regberg↗
NASA NTRS2017

Small Ion Channel Linking Molecular Simulations and Electrophysiology

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.

Pohorille, Andrzej↗
NASA NTRSDate not supplied

Cleanroom Microbes Survive Drying, Vacuum, and Proton Irradiation

Introduction : The goal of planetary protection at NASA is to mitigate the risk of contaminating sensitive target bodies with biological life. While many cleaning procedures have been put in place to reduce bioburden on spacecraft, microbes are experts at evolving to survive harsh conditions. Specifically, the dry, low-nutrient environment of a cleanroom (commonly used for assembly of spacecraft) can represent an environment where extremophiles can survive. Methods : Scientists at NASA MSFC wished to gather a snapshot of the microbial population within a variety of cleanrooms on site. A study was undertaken to collect air, surface, and floor samples from clean-rooms and isolate unique morphologies. From this study, 95 isolates were collected and saved in a microbial library. About 86% of these were identified at least to a genus level. Following identification, 24 microbes were selected, based on a literature review, as potential extremophiles. These were grown in liquid cultures, diluted to a set optical density, washed with water, and then applied to a sterilized Kapton coupon. Droplets were allowed to dry overnight in a biosafety cabinet. Coupons were then installed in a pelletron and pumped down to high vacuum (~1E-6 Torr). Samples were then subjected 100 keV protons at a fluence of 2x10 15 p+/cm 2 up to 4x10 15 p+/cm 2 . Following exposure, samples were returned to the microbiology lab where they were pro-cessed by submerging in water, vortexing, and then plating either droplets or spread plates. Recovery data collected was qualitative with a ranking or +, minor, or – for growth. Some selected radiotolerant strains were sequenced using the Illumina sequencing platform. The resulting genomes were annotated with the Rapid Annotations using Subsystems Technology (RAST) server and analyzed for conserved and unique stress response relevant genomic signatures to identify clues related to specific tolerances. Results and Discussion : After five rounds of proton radiation, we narrowed our isolates to five, non-spore forming bacteria that demonstrated survival: Arthrobacter koreensis, Paenarthrobacter nitroguajacolicus, Mycetocola manganoxydans , and an Erwinia sp. Furthermore, we exposed these four microbes to 254 nm wavelength light at an intensity of 80 W/m 2 at a distance of ~18 cm for 10 minutes. Only A. koreensis demonstrated survival following UV exposure. Finally, we performed whole genome sequencing on the four strains to look for genetic markers of stress resistance. When we compared the genomes of the four strains, we found that genes coding for GGDEF and EAL domains with PAS/PAC sensors were only found in A. koreensis . These domains, modulated by PAS/PAC sensors, are hypothesized to facilitate survival under drying, desiccation, and proton irradiation. Drying and Desiccation : PAS domains sense hydration changes and modulate GGDEF and EAL domain activity to adjust c-di-GMP levels, enhancing resistance to desiccation. For instance, in Pseudomonas aeruginosa , the PAS domain of RbdA modulates activity under varying hydration conditions, affecting stress responses [1]. Proton Irradiation : Proton irradiation causes oxidative stress, leading to ROS generation. PAS domains detect this stress and modulate GGDEF and EAL domains to manage oxidative stress responses. In Shewanella , EAL domain proteins modulated by PAS sensors help bacteria adapt to extreme conditions [2]. These genes upregulate other stress response genes, protecting membrane function, protein stability, DNA repair, and antioxidant defenses. The modulation of c-di-GMP by PAS domains is crucial for bacterial adaptation to stress conditions, enabling dynamic physio-logical adjustments [3]. Understanding these mechanisms provides insights into bacterial stress responses and strategies for controlling bacterial growth [4]. Conclusions : These findings indicate that clean-rooms harbor extremophile microbes that may be able to survive conditions in deep space. Furthermore, while we identified certain stress-response genes that may be at least partly responsible for the phenotypes observed in this study, there are likely unidentified genes or characteristics about A. koreensis , and other bacteria, that may allow them to survive in harsh environments. Future studies will focus on identifying these unknown genes and characteristics, further elucidating the mechanisms of extremophile survival and potentially informing the development of new biotechnologies for space exploration and other extreme environments.

Chelsi Cassilly↗
NASA NTRS2004

Characterization of Two Microbial Isolates from Andean Lakes in Bolivia

We are currently investigating the biological population present in the highest and least explored perennial lakes on earth in the Bolivian and Chilean Andes, including several volcanic crater lakes of more than 6000 m elevation, in combination of microbiological and molecular biological methods. Our samples were collected in saline lakes of the Laguna Blanca Laguna Verde area in the Bolivian Altiplano and in the Licancabur volcano crater (27 deg. 47 min S/67 deg. 47 min. W) in the ongoing project studying high altitude lakes. The main goal of the project is to look for analogies with Martian paleolakes. These Bolivian lakes can be described as Andean lakes following the classification of Chong. We have attempted to isolate pure cultures and phylogenetically characterize prokaryotes that grew under laboratory conditions. Sediment samples taken from the Licancabur crater lake (LC), Laguna Verde (LV), and Laguna Blanca (LB) were analyzed and cultured using enriched liquid media under both aerobic and anaerobic conditions. All cultures were incubated at room temperature (15 to 20 C) and under light exposure. For the reported isolates, 36 hours incubation were necessary for reaching optimal optical densities to consider them viable cultures. Ten serial dilutions starting from 1% inoculum were required to obtain a suitable enriched cell culture to transfer into solid media. Cultures on solid medium were necessary to verify the formation of colonies in order to isolate pure cultures. Different solid media were prepared using several combinations of both trace minerals and carbohydrates sources in order to fit their nutrient requirements. The microorganisms formed individual colonies on solid media enriched with tryptone, yeast extract and sodium chloride. Cells morphology was studied by optical and electronic microscopy. Rodshape morphologies were observed in most cases. Total bacterial genomic DNA was isolated from 50 ml late-exponential phase culture by using the CTAB miniprep protocol. The 16S rRNA genes were amplified by PCR using both Bacteria- and Archaeauniversal primer sets: 27f and 1492r, 21f and 1492r respectively. Sequences of 16S rRNA gene were determined and initially compared with reference sequences contained in the EMBL nucleotide sequence database by using the BLAST program and were subsequently aligned with 16S rRNA reference sequences in the ARB package (http://www.mikro.biologie.tu-muenchen.de). Aligned sequences were inserted within a stable phylogenetic tree by using the ARB parsimony tool. In this work we report the morphology and phylogenetic characterization of two isolates belonged to Laguna Blanca sediments.

Demergasso, C.↗
NASA NTRS2017

Network Analysis of Rodent Transcriptomes in Spaceflight

Network analysis methods leverage prior knowledge of cellular systems and the statistical and conceptual relationships between analyte measurements to determine gene connectivity. Correlation and conditional metrics are used to infer a network topology and provide a systems-level context for cellular responses. Integration across multiple experimental conditions and omics domains can reveal the regulatory mechanisms that underlie gene expression. GeneLab has assembled rich multi-omic (transcriptomics, proteomics, epigenomics, and epitranscriptomics) datasets for multiple murine tissues from the Rodent Research 1 (RR-1) experiment. RR-1 assesses the impact of 37 days of spaceflight on gene expression across a variety of tissue types, such as adrenal glands, quadriceps, gastrocnemius, tibalius anterior, extensor digitorum longus, soleus, eye, and kidney. Network analysis is particularly useful for RR-1 -omics datasets because it reinforces subtle relationships that may be overlooked in isolated analyses and subdues confounding factors. Our objective is to use network analysis to determine potential target nodes for therapeutic intervention and identify similarities with existing disease models. Multiple network algorithms are used for a higher confidence consensus.

genomics↗
NASA NTRS2021

Characterization of Spacesuit Associated Microbial Communities and their Implications for NASA Missions

Crewed National Aeronautics and Space Administration (NASA) missions to other solar system bodies are currently being planned. One high-profile scientific focus during such expeditions would be life detection, specifically the discovery of past or present microbial life, if they exist. However, both humans and associated objects typically carry a high microbial burden. Thus, it is essential to distinguish between microbes brought with the expedition and those present on the exploring planets. Modern spacesuits are unique, customized spacecraft which provide protection, mobility and life support to crew during spacewalks, yet they vent continuously, and the mobility of microbes through spacesuits has not been studied. To evaluate the microbial colonization of spacesuits, NASA used an Extravehicular Activity swab kit to examine viable microbial populations of 48 samples from spacesuits using both traditional microbiological methods and molecular sequencing methods. The cultivable microbial population ranged from below the detection limit to 9 x 102 colony forming units per 25 cm2 of sample and also significantly varied by the location. The cultivable microbial diversity was dominated by members of Bacillus, Arthrobacter, and Ascomycota. However, 16S rRNA-based viable bacterial burden ranged from 105 to 106 copies per 25 cm2 of sample. Shotgun metagenome sequencing revealed the presence of a diverse microbial population on the spacesuit surfaces, including Curtobacterium and Methylobacterium from across all sets of spacesuits in high abundance. Among bacterial species identified, higher abundance of Cutibacterium acnes, Methylobacterium oryzae, and M. phyllosphaerae reads were documented. The results of this study provide evidence that identical microbial strains may live on the wrist joint, inner gauntlet, and outer gauntlet of spacesuits. This raises the possibility, but does not confirm, that viable microbes are able to migrate between the interior and exterior of spacesuits. Overall, these data provide the first estimate of microbial dispersion from internal to external spacesuit surfaces, which will help future mission planners develop effective planetary protection strategies.

spacesuit↗
NASA NTRS2001

Monte Carlo predictions of DNA fragment-size distributions for large sizes after HZE particle irradiation

DSBs (double-strand breaks) produced by densely ionizing space radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. DSB clustering at large scales, from >100 Mbp down to approximately 2 kbp, is modeled using a Monte-Carlo algorithm. A random-walk model of chromatin is combined with a track model, that predicts the radial distribution of energy from an ion, and the RLC (randomly-located-clusters) formalism, in software called DNAbreak. This model generalizes the random-breakage model, whose broken-stick fragment-size distribution is applicable to low-LET radiation. DSB induction due to track interaction with the DNA volume depends on the radiation quality parameter Q. This dose-independent parameter depends only weakly on LET. Multi-track, high-dose effects depend on the cluster intensity parameter lambda, proportional to fluence as defined by the RLC formalism. After lambda is determined by a numerical experiment, the model reduces to one adjustable parameter Q. The best numerical fits to the experimental data, determining Q, are obtained. The knowledge of lambda and Q allows us to give biophysically based extrapolations of high-dose DNA fragment-size data to low doses or to high LETs.

NASA Center JSC↗
NASA NTRS2018

Variability in Galactic Cosmic Radiation- Induced DNA Damage Response in Inbred Mice Is Modulated by Genetics

In radiation biology, the ability to predict cancer risk associated with exposure to low doses of high-LET (Linear Energy Transfer) ionizing radiation remains a challenge. Epidemiological methods lack the sensitivity and power to provide detailed risk estimates for cancer and ignore individual sensitivity. We have hypothesized that DNA repair capacity is the primary factor differentiating peoples radiation sensitivity. We previously showed in immortalized human cell lines that characterizing the dose and time dependence of p53-binding protein 1 (53BP1) foci formation in the nucleus following X-rays exposure is sufficient to predict DNA repair response to any other LET in the same cell line. We now tested this hypothesis across a population of mice with different genetic background. Fibroblast cells were extracted and cultivated from 76 individual mice from 15 different strains and exposed to HZE (high (H) atomic number (Z) and energy (E) galactic cosmic ray particles) particles and X-rays. Individual radiation sensitivities were investigated by high throughput measurement of DNA repair kinetics that evaluated 53bp1 foci numbers as a surrogate for DNA double-strand breaks at various times post-irradiation. Instead of just counting foci which can be hard to distinguish for high-LET or high doses, we also took into account the track structure of high-LET particles to compute the remaining number of unrepaired tracks as a function of time post-irradiation. As expected, the percentage of unrepaired track over a 48 hours follow-up period increased with LET. In addition, repair rate was modulated by genetics, with animals from the same strain showing small variance while large rate differences were observed between strains. Radiation strain sensitivity ranking was estimated based on repair rates from exposure to each LET evaluated in this work, and ranking for high-LET correlated better with ranking from high dose of X-ray, not low dose. At the in-vivo level, drops in T-cells and B-cells number measured 24 hours after 0.1 Gy (Gray) X-ray exposure, correlated with slower DNA repair kinetic in fibroblast cells of the same strains of mice. At the genomic level, mouse genome wide association (GWA) analysis identified seven significant genetic loci on chromosomes 2, 3, 7, 10, 11, 13 and 19 with different significance depending on the LET. Interestingly, for the two highest LET, a common locus on Chromosome 10 was identified with high enrichment for DNA repair associated genes.Overall, this work suggests that repair kinetics of primary skin fibroblasts is a good surrogate marker for in-vivo radiation sensitivities in other tissues and that this response is modulated by genetics. Our study also confirms that DNA repair kinetics following high doses of X-ray can be used to predict radiation sensitivity to high-LET.

High-LET Radiation↗