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At least 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
NASA NTRS2001

Complex expression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate unique lymphoid tissues in generating an immune repertoire

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.

Non-NASA Center↗
NASA NTRS2011

Stress-Driven Selection of Novel Phenotypes

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Fox, George E.↗
NASA NTRS1980

How many theories for the origin of /proto/life

The sequence of primordial chemical events leading to contemporary metabolism is considered, taking into account primordial reactants, amino acids, proteinoid, protocells, ATP, polynucleotides, and protein. The right kind of matter, thermal copolyamino acids, can organize itself into cell-like structures, in the absence of discrete lipid, when triggered to do so by water. Another unpredicted result of examination of the microsystems formed was the step-by-step realization that the component processes of a primitive form of replication were latent in the proteinoid microsystems. At the present time, four modes of primitive replication of proteinoid microsystems have been identified, plus one that has the appearance of protosexual reproduction. Two main conceptual pathways have received attention. One is the proteinoid theory, derived from experiments. The other is the DNA-first theory, for which attempts at conceptual construction and experimental support continue to be sought.

Fox, S. W.↗
NASA NTRS1993

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Wu, Liu-Lai↗
NASA NTRS1998

Transposon tagging and the study of root development in Arabidopsis

The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

NASA Discipline Plant Biology↗
NASA NTRS1999

Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences

Relatively low levels of expression from naturally occurring promoters have limited the use of muscle as a gene therapy target. Myogenic restricted gene promoters display complex organization usually involving combinations of several myogenic regulatory elements. By random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries, and screening of hundreds of individual clones for transcriptional activity in vitro and in vivo, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.

Non-NASA Center↗
NASA NTRS2001

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Review↗
NASA NTRS1993

Molecular Basis of the Increase in Invertase Activity Elicited by Gravistimulation of Oat-Shoot Pulvini

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oatshoot pulvini starting within 3h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly(A)(+)RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the Polymerase Chain Reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52 % deduced amino-acid sequence homology to that of carrot beta-fructosi- dase and a 48 % homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

Wu, Liu-Lai↗
NASA NTRSDate not supplied

Differential Responses to Mechanostimulation in Embryonic Stem Cells Versus the Embryoid Body Model of Development Assessed at Single Cell RNA-Seq Resolution

Mechanicalforces generated by gravity have shaped life on Earth and impact gene expression and morphogenesis during early development. In contrast disuse canreduce normal mechanical loading, resulting in altered cell and tissue function. Although loading in adult mammals is known to promote increased cell proliferation and differentiation, little is known about how cells respondto this stimulusduring early development. In this study we sought to understand, with single cell RNA-sequencing resolution, how a 60-minute pulse of 50xg hypergravity-generated 5kPa hydrostatic pressure, influences transcriptomic regulation of developmental processes in the Embryoid Body (EB) model. Our study included both day-9 EBs and progenitor mouse embryonic stem cells (ESCs) with or without the hydrostatic pressurepulse. Single cell tSNE mapping shows limited transcriptome shifts in response to thispulse in either ESCs or EBs; this pulse,however, induces greater positional shifts in EB mapping compared to ESCs, indicating the influence of mechanotransduction is more pronounced in later states of cell commitment within the developmental program.We assessed ESCs and EBs for differentially expressed (DE) genes with hydrostatic pressurepulse and found approximately 1/3 DE genes were shared. However, gene ontology (GO) pathway analysis show that EBs have choreographed responses associated with upregulation ofpathways formulticellular development, mechanical signal transduction, and DNA damage repair. Cluster transcriptome analysis of the EBs showsmechanostimulationpromotes maintenance of transitory cell phenotypes in early development,including EB cluster co-expression of markers for progenitor, post-implant epiblast and primitive endoderm phenotypes versus expression exclusivity in the non-pulsed clusters. Pseudotime analysisidentified three branching cell types susceptible tohydrostatic pressureinduction of cell fate decisions. In summary, this study provides novel evidence that ESC maintenance and EB development can be regulated by mechanostimulation,and that stem cells committed to a differentiation program are more sensitive to force-induced changes to their transcriptome.

Cassandra Juran↗
NASA NTRS1999

Cell and molecular biology of simian virus 40: implications for human infections and disease

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963. SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin. This virus has been a favored laboratory model for mechanistic studies of molecular processes in eukaryotic cells and of cellular transformation. The viral replication protein, named large T antigen (T-ag), is also the viral oncoprotein. There is a single serotype of SV40, but multiple strains of virus exist that are distinguishable by nucleotide differences in the regulatory region of the viral genome and in the part of the T-ag gene that encodes the protein's carboxyl terminus. Natural infections in monkeys by SV40 are usually benign but may become pathogenic in immunocompromised animals, and multiple tissues can be infected. SV40 can replicate in certain types of simian and human cells. SV40-neutralizing antibodies have been detected in individuals not exposed to contaminated polio vaccines. SV40 DNA has been identified in some normal human tissues, and there are accumulating reports of detection of SV40 DNA and/or T-ag in a variety of human tumors. This review presents aspects of replication and cell transformation by SV40 and considers their implications for human infections and disease pathogenesis by the virus. Critical assessment of virologic and epidemiologic data suggests a probable causative role for SV40 in certain human cancers, but additional studies are necessary to prove etiology.

Review↗
NASA NTRS2017

NASA's Space Launch System: Deep-Space Delivery for Smallsats

Designed for human exploration missions into deep space, NASA's Space Launch System (SLS) represents a new spaceflight infrastructure asset, enabling a wide variety of unique utilization opportunities. While primarily focused on launching the large systems needed for crewed spaceflight beyond Earth orbit, SLS also offers a game-changing capability for the deployment of small satellites to deep-space destinations, beginning with its first flight. Currently, SLS is making rapid progress toward readiness for its first launch in two years, using the initial configuration of the vehicle, which is capable of delivering 70 metric tons (t) to Low Earth Orbit (LEO). On its first flight test of the Orion spacecraft around the moon, accompanying Orion on SLS will be small-satellite secondary payloads, which will deploy in cislunar space. The deployment berths are sized for "6U" CubeSats, and on EM-1 the spacecraft will be deployed into cislunar space following Orion separate from the SLS Interim Cryogenic Propulsion Stage. Payloads in 6U class will be limited to 14 kg maximum mass. Secondary payloads on EM-1 will be launched in the Orion Stage Adapter (OSA). Payload dispensers will be mounted on specially designed brackets, each attached to the interior wall of the OSA. For the EM-1 mission, a total of fourteen brackets will be installed, allowing for thirteen payload locations. The final location will be used for mounting an avionics unit, which will include a battery and sequencer for executing the mission deployment sequence. Following the launch of EM-1, deployments of the secondary payloads will commence after sufficient separation of the Orion spacecraft to the upper stage vehicle to minimize any possible contact of the deployed CubeSats to Orion. Currently this is estimated to require approximately 4 hours. The allowed deployment window for the CubeSats will be from the time the upper stage disposal maneuvers are complete to up to 10 days after launch. The upper stage will fly past the moon at a perigee of approximately 100km, and this closest approach will occur about 5 days after launch. The limiting factor for the latest deployment time is the available power in the sequencer system. Several NASA Mission Directorates were involved in the development of programs for the competition, selection, and development of EM-1 payloads that support directorate priorities. CubeSat payloads on EM-1 will include both NASA research experiments and spacecraft developed by industry, international and potentially academia partners. The Human Exploration and Operations Mission Directorate (HEOMD) Advanced Exploration Systems (AES) Division was allocated five payload opportunities on the EM-1 mission. Near Earth Asteroid (NEA) Scout is designed to rendezvous with and characterize a candidate NEA. A solar sail, an innovation the spacecraft will demonstrated for the CubeSat class, will provide propulsion. Lunar Flashlight will use a green propellant system and will search for potential ice deposits in the moon's permanently shadowed craters. BioSentinel is a yeast radiation biosensor, planned to measure the effects of space radiation on deoxyribonucleic acid (DNA). Lunar Icecube, a collaboration with Morehead State University, will prospect for water in ice, liquid, and vapor forms as well as other lunar volatiles from a low-perigee, highly inclined lunar orbit using a compact Infrared spectrometer. Skyfire, a partnership with Lockheed Martin, is a technology demonstration mission that will perform a lunar flyby, collecting spectroscopy, and thermography data to address questions related to surface characterization, remote sensing, and site selection. NASA's Space Technology Mission Directorate (STMD) was allocated three payload opportunities on the EM-1 mission. These slots will be filled via the Centennial Challenges Program, NASA's flagship program for technology prize competitions, which directly engages the public, academia, and industry in open prize competitions to stimulate innovation. The NASA Science Mission Directorate (SMD) was allocated two payload opportunities on the EM-1 mission. The CubeSat Mission to Study Solar Particles (CuSP) payload will study the sources and acceleration mechanisms of solar and interplanetary particles in near-Earth orbit, support space weather research by determining proton radiation levels during Solar Energetic Particle (SEP) events and identifying suprathermal properties that could help predict geomagnetic storms. The LunaH-Map payload will help scientists understand the quantity of H-bearing materials in lunar cold traps (~10 km), determine the concentration of H-bearing materials with 1m depth, and constrain the vertical distribution of H-bearing materials. The final three payload opportunities for the EM-1 mission were allocated for NASA's international space agency counterparts. The flight opportunities are intended to benefit the international space agency and NASA as well as further the collective space exploration goals. ArgoMoon is sponsored by ESA/ASI and will fly along with the ICPS on its disposal trajectory to perform proximity operations with the ICPS post-disposal, take external imagery of engineering and historical significance, and perform an optical communications demonstration. EQUULEUS, sponsored by JAXA, will fly to a libration orbit around the Earth-Moon L2 point and demonstrate trajectory control techniques within the Sun-Earth- Moon region for the first time by a nano spacecraft. The mission will also contribute to the future human exploration scenario by understanding the radiation environment in geospace and deep space, characterizing the flux of impacting meteors on the far side of the moon, and demonstrating the future deep space exploration scenario using the "deep space port" at Lagrange points. OMOTENASHI, also sponsored by JAXA, will land the smallest lunar lander to date on the lunar surface to demonstrate the feasibility of the hardware for distributed cooperative exploration system. Small landers will enable multi-point exploration, which is complimentary with large-scale human exploration. Once on the lunar surface, the OMOTENASHI spacecraft will observe the radiation and soil environments of the lunar surface by active radiation measurements and soil shear measurements. Following EM-1, Space Launch System will evolve to the more-powerful Block 1B configuration, which uses a new Exploration Upper Stage to increase the vehicle's LEO payload capability from 70 t to 105 t. With that transition, the Orion Stage Adapter, which will carry the secondary payloads on EM-1, will be phased out, and a new Universal Stage Adapter will be introduced, creating opportunities for flying larger secondary payloads. This paper will provide a brief status of SLS progress toward first launch; an overview of smallsat accommodations, integration, and operations on EM-1; information about the specific payloads flying on that launch; and a discussion of future accommodations and opportunities for secondary payloads on SLS for Exploration Mission-2 and beyond.

Robinson, Kimberly F.↗
NASA NTRS2017

NASA's Space Launch System: Deep-Space Opportunities for SmallSats

Designed for human exploration missions into deep space, NASA's Space Launch System (SLS) represents a new spaceflight infrastructure asset, enabling a wide variety of unique utilization opportunities. While primarily focused on launching the large systems needed for crewed spaceflight beyond Earth orbit, SLS also offers a game-changing capability for the deployment of small satellites to deep-space destinations, beginning with its first flight. Currently, SLS is making rapid progress toward readiness for its first launch in two years, using the initial configuration of the vehicle, which is capable of delivering 70 metric tons (t) to Low Earth Orbit (LEO). On its first flight test of the Orion spacecraft around the moon, accompanying Orion on SLS will be small-satellite secondary payloads, which will deploy in cislunar space. The deployment berths are sized for "6U" CubeSats, and on EM-1 the spacecraft will be deployed into cislunar space following Orion separate from the SLS Interim Cryogenic Propulsion Stage. Payloads in 6U class will be limited to 14 kg maximum mass. Secondary payloads on EM-1 will be launched in the Orion Stage Adapter (OSA). Payload dispensers will be mounted on specially designed brackets, each attached to the interior wall of the OSA. For the EM-1 mission, a total of fourteen brackets will be installed, allowing for thirteen payload locations. The final location will be used for mounting an avionics unit, which will include a battery and sequencer for executing the mission deployment sequence. Following the launch of EM-1, deployments of the secondary payloads will commence after sufficient separation of the Orion spacecraft to the upper stage vehicle to minimize any possible contact of the deployed cubesats to Orion. Currently this is estimated to require approximately 4 hours. The allowed deployment window for the cubesats will be from the time the upper stage disposal maneuvers are complete to up to 10 days after launch. The upper stage will fly past the moon at a perigee of approximately 100km, and this closest approach will occur about 5 days after launch. The limiting factor for the latest deployment time is the available power in the sequencer system. Several NASA Mission Directorates were involved in the development of programs for the competition, selection, and development of EM-1 payloads that support directorate priorities. CubeSat payloads on EM-1 will include both NASA research experiments and spacecraft developed by industry, international and potentially academia partners. The Human Exploration and Operations Mission Directorate (HEOMD) Advanced Exploration Systems (AES) Division was allocated five payload opportunities on the EM-1 mission. Near Earth Asteroid (NEA) Scout is designed to rendezvous with and characterize a candidate NEA. A solar sail, an innovation the spacecraft will demonstrated for the CubeSat class, will provide propulsion. Lunar Flashlight will use a green propellant system and will search for potential ice deposits in the moon's permanently shadowed craters. BioSentinel is a yeast radiation biosensor, planned to measure the effects of space radiation on deoxyribonucleic acid (DNA). Lunar Icecube, a collaboration with Morehead State University, will prospect for water in ice, liquid, and vapor forms as well as other lunar volatiles from a low-perigee, highly inclined lunar orbit using a compact Infrared spectrometer. Skyfire, a partnership with Lockheed Martin, is a technology demonstration mission that will perform a lunar flyby, collecting spectroscopy, and thermography data to address questions related to surface characterization, remote sensing, and site selection. NASA's Space Technology Mission Directorate (STMD) was allocated three payload opportunities on the EM-1 mission. These slots will be filled via the 2 Centennial Challenges Program, NASA's flagship program for technology prize competitions, which directly engages the public, academia, and industry in open prize competitions to stimulate innovation. The NASA Science Mission Directorate (SMD) was allocated two payload opportunities on the EM-1 mission. The CubeSat Mission to Study Solar Particles (CuSP) payload will study the sources and acceleration mechanisms of solar and interplanetary particles in near-Earth orbit, support space weather research by determining proton radiation levels during Solar Energetic Particle (SEP) events and identifying suprathermal properties that could help predict geomagnetic storms. The LunaH-Map payload will help scientists understand the quantity of H-bearing materials in lunar cold traps (~10 km), determine the concentration of H-bearing materials with 1m depth, and constrain the vertical distribution of H-bearing materials. The final three payload opportunities for the EM-1 mission were allocated for NASA's international space agency counterparts. The flight opportunities are intended to benefit the international space agency and NASA as well as further the collective space exploration goals. ArgoMoon is sponsored by ESA/ASI and will fly along with the ICPS on its disposal trajectory to perform proximity operations with the ICPS post-disposal, take external imagery of engineering and historical significance, and perform an optical communications demonstration. EQUULEUS, sponsored by JAXA, will fly to a libration orbit around the Earth-Moon L2 point and demonstrate trajectory control techniques within the Sun-Earth-Moon region for the first time by a nano spacecraft. The mission will also contribute to the future human exploration scenario by understanding the radiation environment in geospace and deep space, characterizing the flux of impacting meteors on the far side of the moon, and demonstrating the future deep space exploration scenario using the "deep space port" at Lagrange points. OMOTENASHI, also sponsored by JAXA, will land the smallest lunar lander to date on the lunar surface to demonstrate the feasibility of the hardware for distributed cooperative exploration system. Small landers will enable multi-point exploration, which is complimentary with large-scale human exploration. Once on the lunar surface, the OMOTENASHI spacecraft will observe the radiation and soil environments of the lunar surface by active radiation measurements and soil shear measurements. Following EM-1, Space Launch System will evolve to the more-powerful Block 1B configuration, which uses a new Exploration Upper Stage to increase the vehicle's LEO payload capability from 70 t to 105 t. With that transition, the Orion Stage Adapter, which will carry the secondary payloads on EM-1, will be phased out, and a new Universal Stage Adapter will be introduced, creating opportunities for flying larger secondary payloads. This paper will provide a brief status of SLS progress toward first launch; an overview of smallsat accommodations, integration, and operations on EM-1; information about the specific payloads flying on that launch; and a discussion of future accommodations and opportunities for secondary payloads on SLS for Exploration Mission-2 and beyond.

Robinson, Kimberly F.↗
NASA NTRS1995

Asymmetric structure of five and six membered DNA hairpin loops

The tertiary structure of nucleic acid hairpins was elucidated by means of the accessibility of the single-strand-specific nuclease from mung bean. This molecular probe has proven especially useful in determining details of the structural arrangement of the nucleotides within a loop. In this study 3'-labeling is introduced to complement previously used 5'-labeling in order to assess and to exclude possible artifacts of the method. Both labeling procedures result in mutually consistent cleavage patterns. Therefore, methodological artifacts can be excluded and the potential of the nuclease as structural probe is increased. DNA hairpins with five and six membered loops reveal an asymmetric loop structure with a sharp bend of the phosphate-ribose backbone between the second and third nucleotide on the 3'-side of a loop. These hairpin structures differ from smaller loops with 3 or 4 members, which reveal this type of bend between the first and second 3' nucleotide, and resemble with respect to the asymmetry anticodon loops of tRNA.

NASA Discipline Exobiology↗
NASA NTRS2023

ChIPPS: Charged Information-storage Polymer Preparation System

Technological advances are required to support principal science objectives of missions to the solar system’s icy worlds to seek biosignatures of past/extant life. Sensitivity and reliability are key concerns due to small sample sizes (µL – mL) and the extraordinary import of the results. The preparation and processing of small samples can constrain limits of detection (LoDs); therefore, the Charged Information-storage Polymer Preparation System (ChIPPS) project is advancing the technologies of autonomous sample preparation and processing to add a new class of reliably detectable biosignatures: charged polymers and particles, which can be the information storage-and-transmission means for life. Specifically, we are developing an integrated microfluidic sample-processing unit to prepare icy-world samples to support complementary solid-state nanopore-based analyses: 1) charged-polymer analysis, to characterize variations in polymer chain size, shape, and charge vs. position along the chain; 2) polymer and nanoparticle sizing-and-counting, to characterize the relative abundance of polymer chains, as well as small (virus-sized) particles, by their dimensions and charge. Although no such autonomous system presently exists, such measurements can reveal the nature and abundance of charged polymers that could be used by biological systems to store and transfer information—as DNA and RNA are used terrestrially—without limitation to terrestrial nucleic acids, given that life elsewhere may utilize different information store-and-transfer moieties. Key system components include (a) a lysis unit for mechanical sample disruption; b) an ion-exchange column for charged macromolecule/nanoparticle purification; c) dialyzer to remove excess salt; d) concentrator to enhance signal; e) supporting pumps, valves, bubble traps, connectors, filters, etc.; f) interface to nanopore detection instruments.

Space Biology↗
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

Oligodeoxynucleotide Probes for Detecting Intact Cells

A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of target bacteria in samples are usually amplified by overnight pre-enrichment growth. These tests are usually performed in laboratories by skilled technicians. The present test was designed to overcome the shortcomings of the commercial hybridization tests. The figure summarizes the major steps of the test. It is important to emphasize that the hybridization process used in this test differs from that of other hybridization tests in that it does not extract target nucleic acids. This process is based on intact-cell hybridization (so-called in situ hybridization ), wherein the intact cells act as immobilizing agents. The cells are identified and enumerated by measuring the chemiluminescence emitted from alkaline phosphatase-probe (AP-probe) hybridization; the chemiluminescence is detected or measured by use of photographic film or a luminometer, respectively.

Rosson, Reinhardt A.↗
NASA NTRSDate not supplied

Differential Gene Expression in A Cross-Feeding Two-Species Model Microbial Community Under Simulated Microgravity and Deep-Space Radiation

A long-term goal of space biology is to understand interspecies microbial interactions in space. Presently, little is known about the combined effect of microgravity and ionizing radiation on bacterial community response when species are interdependent through exchange of metabolites in fluid medium (cross-feeding). Microgravity is expected to slow interspecies mass transfer and growth in cross-feeding communities in the low-shear, diffusion-limited environment, while ionizing radiation may influence stress response to direct (DNA damage) and indirect damage (ROS). Using a well-understood, two-species (Escherichia coli and Salmonella enterica) microbial community engineered to be a model for studying cross-feeding, we simulated galactic cosmic rays (GCRsim) and microgravity to test the hypothesis: exposure to ionizing radiation causes cell damage or stress, altering transcriptomic community responses in metabolically interdependent cells, which is exacerbated by microgravity. We expect to see differential gene expression between cross-feeding and non-cross-feeding communities. We measured GCRsim effects on growth and gene expression in well-mixed versus simulated-microgravity conditions and in cross-feeding and non-cross-feeding medium. Microbial cultures were inoculated into liquid medium in rotating wall vessels (RWV) with different rotation rates: 5 RPM (simulated microgravity) and 50 RPM (well-mixed). The E. coli-S. enterica consortium, under simulated microgravity, were exposed to 500 mGy of Simplified 5-ion Galactic Cosmic Ray Simulation for 2 hours at Brookhaven National Lab. We harvested samples 40 minutes after irradiation for extraction and sequencing (NASA GeneLab). Here we present the differential gene expression analysis results, which reveal altered transcriptomic community responses, even where growth rate differences are not observed. Gene expression of these actively metabolizing microbial communities in GCRsim may illuminate molecular mechanisms of microbial interactions in space. Understanding how microbial community gene expression, metabolism, and other cellular processes are influenced by spaceflight stressors can inform the use of microbes in human life support for low Earth orbit missions and beyond.

microgravity↗
NASA NTRSDate not supplied

Developing Open-Source Training Materials for AI/ML and Space Biological Sciences Using NASA Cloud-Based Data

Artificial Intelligence (AI) and Machine Learning (ML) has gained significant traction in the biological and biomedical research fields, in part due to a culture of open data sharing and reuse. AI/ML methodology is well-suited to recognize and predict biological patterns from high-dimensional next-generation sequencing data (e.g. whole genome sequencing, transcriptomic sequencing), as well as from biological or medical imaging data (e.g. microscopy, computed tomography, ultrasound, magnetic resonance imaging, radiography). These methodologies hold particular promise for space biosciences research and automated space health monitoring systems. However, there are key considerations for properly training, validating, and testing a machine learning model in biological research or clinical application. Inexperienced researchers can produce models that perform poorly outside of the training dataset. Open Science principles such as data sharing and open-source code must go hand-in-hand with publicly available, high-quality training curricula in best practices, with modules centered on real-life scientific use cases and data so future AI/ML practitioners gain experience on real problems. Here we present the development of open-source training materials for AI/ML and space biosciences, as part of the NASA Transform to Open Science Training (TOPST) initiative. We develop 4 independent training programs, focused on the following topics: 1) Fundamentals of Machine Learning and Space Biosciences Domain, 2) Open Science, Artificial Intelligence, and Ethical Best Practices for Data Sharing and Analysis, 3) Using AI/ML Classification to Identify Gene Networks Affected By Space Exposure in Mouse Liver, and 4) Using Neural Networks to Find DNA Damage Patterns in Immune Cells after Radiation. All programs leverage cloud-based NASA biological datasets. The curriculum we present will enable worldwide access to training in AI/ML and scientific analysis.

James Casaletto↗