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NASA NTRS1992

Origin and early evolution of photosynthesis

Photosynthesis was well-established on the earth at least 3.5 thousand million years ago, and it is widely believed that these ancient organisms had similar metabolic capabilities to modern cyanobacteria. This requires that development of two photosystems and the oxygen evolution capability occurred very early in the earth's history, and that a presumed phase of evolution involving non-oxygen evolving photosynthetic organisms took place even earlier. The evolutionary relationships of the reaction center complexes found in all the classes of currently existing organisms have been analyzed using sequence analysis and biophysical measurements. The results indicate that all reaction centers fall into two basic groups, those with pheophytin and a pair of quinones as early acceptors, and those with iron sulfur clusters as early acceptors. No simple linear branching evolutionary scheme can account for the distribution patterns of reaction centers in existing photosynthetic organisms, and lateral transfer of genetic information is considered as a likely possibility. Possible scenarios for the development of primitive reaction centers into the heterodimeric protein structures found in existing reaction centers and for the development of organisms with two linked photosystems are presented.

NASA Program Exobiology↗

NASA NTRS2003

Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90)

We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

STS-90 Shuttle Project↗

NASA NTRSDate not supplied

Batch Effect Correction Methods for NASA GeneLab Transcriptomic Datasets

RNA sequencing (RNA-seq) data from space biology experiments promise to yield invaluable insights into the effects of spaceflight on terrestrial biology. However, sample numbers from each study are low due to limited crew availability, hardware, and space. To increase statistical power, spaceflight RNA-seq datasets from different missions are often aggregated together. However, this can introduce technical variation or "batch effects", often due to differences in sample handling, sample processing, and sequencing platforms. Several computational methods have been developed to correct for technical batch effects, thereby reducing their impact on true biological signals. In this study, we combined 7 mouse liver RNA-seq datasets from NASA GeneLab (part of the NASA Open Science Data Repository) to evaluate several common batch effect correction methods (ComBat and ComBat-seq from the sva R package, and Median Polish, Empirical Bayes, and ANOVA from the MBatch R package). We quantitatively evaluated the ability of these methods to correct for technical batch variables in space biology RNA-seq data using the following criteria: BatchQC, principal component analysis, dispersion separability criterion, log fold change correlation, and differential gene expression analysis. Each batch variable / correction method combination was then assessed using a custom scoring approach to identify the optimal correction method for the combined dataset, by geometrically probing the space of all allowable scoring functions to yield an aggregate volume-based scoring measure. Finally, we describe the way in which the GeneLab multi-study analysis and visualization portal will allow users to examine the presence or absence of batch effects using multiple metrics. If the user chooses to perform batch effect correction, the scoring approach described here can be implemented to identify the optimal correction method to use for their specific combined dataset prior to analysis.

Lauren M. Sanders↗

NASA NTRS2000

Molecular and biochemical evidence for the involvement of calcium/calmodulin in auxin action

The use of (35)S-labeled calmodulin (CaM) to screen a corn root cDNA expression library has led to the isolation of a CaM-binding protein, encoded by a cDNA with sequence similarity to small auxin up RNAs (SAURs), a class of early auxin-responsive genes. The cDNA designated as ZmSAUR1 (Zea mays SAURs) was expressed in Escherichia coli, and the recombinant protein was purified by CaM affinity chromatography. The CaM binding assay revealed that the recombinant protein binds to CaM in a calcium-dependent manner. Deletion analysis revealed that the CaM binding site was located at the NH(2)-terminal domain. A synthetic peptide of amino acids 20-45, corresponding to the potential CaM binding region, was used for calcium-dependent mobility shift assays. The synthetic peptide formed a stable complex with CaM only in the presence of calcium. The CaM affinity assay indicated that ZmSAUR1 binds to CaM with high affinity (K(d) approximately 15 nM) in a calcium-dependent manner. Comparison of the NH(2)-terminal portions of all of the characterized SAURs revealed that they all contain a stretch of the basic alpha-amphiphilic helix similar to the CaM binding region of ZmSAUR1. CaM binds to the two synthetic peptides from the NH(2)-terminal regions of Arabidopsis SAUR-AC1 and soybean 10A5, suggesting that this is a general phenomenon for all SAURs. Northern analysis was carried out using the total RNA isolated from auxin-treated corn coleoptile segments. ZmSAUR1 gene expression began within 10 min, increased rapidly between 10 and 60 min, and peaked around 60 min after 10 microM alpha-naphthaleneacetic acid treatment. These results indicate that ZmSAUR1 is an early auxin-responsive gene. The CaM antagonist N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride inhibited the auxin-induced cell elongation but not the auxin-induced expression of ZmSAUR1. This suggests that calcium/CaM do not regulate ZmSAUR1 at the transcriptional level. CaM binding to ZmSAUR1 in a calcium-dependent manner suggests that calcium/CaM regulate ZmSAUR1 at the post-translational level. Our data provide the first direct evidence for the involvement of calcium/CaM-mediated signaling in auxin-mediated signal transduction.

NASA Program Fundamental Space Biology↗

NASA NTRS1992

A phylogenetic analysis of Aquifex pyrophilus

The 16S rRNA of the bacterion Aquifex pyrophilus, a microaerophilic, oxygen-reducing hyperthermophile, has been sequenced directly from the the PCR amplified gene. Phylogenetic analyses show the Aq. pyrophilus lineage to be probably the deepest (earliest) in the (eu)bacterial tree. The addition of this deep branching to the bacterial tree further supports the argument that the Bacteria are of thermophilic ancestry.

Non-NASA Center↗

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 NTRSDate not supplied

Large Scale Transcriptional Analysis of Legacy Spaceflight Tissues From the Nasa Biological Institutional Science Collection

The NASA Biological Institutional Science Collection (NBISC) has amassed a collection of valuable space biology samples spanning from early Space Shuttle missions to recent missions on the International Space Station (ISS). However, the full potential of this archive has not been realized, with many samples having been stored for decades without being re-accessed. Given the pace of analytical advancement since NBISC began accumulating samples, we initiated a pilot study to reveal additional patterns that may have been missed during the original investigations and provide technical robustness and scientific value for a cost-effective sequencing approach. We selected 84 mouse and rat samples spanning four separate Space Shuttle, ISS and ground-analogue studies with a focus on muscle, spleen and thymus tissues to allow identification of important changes related to musculoskeletal unloading and immune function. High quality RNA from these tissues was extracted and used to generate transcriptional profiling data using a cost-effective Tag-seq approach and immediately released it on GeneLab. The released datasets included gastrocnemius tissues from rats during the 1991 Space Shuttle mission (GLDS-422) and a ground analogue study in 2019 (GLDS-418), and mice from the 2014 ISS mission (GLDS-419). Also released were two thymus datasets, one from rats flown on the 1993 Space Shuttle (GLDS-423) and another from mice flown on the 2017 ISS (GLDS-421) missions, along with one mouse spleen dataset from a 2017 ISS mission (GLDS-420). These data will serve as a pilot of a much larger and comprehensive study which would generate similar data from thousands of NBISC samples, enabling the discovery and validation of molecular networks influenced by space conditions.

Lovorka Degoricija↗

NASA NTRS2002

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Non-NASA Center↗

NASA NTRS1991

Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts

A major and too little recognized source of artifact in phylogenetic analysis of molecular sequence data is compositional difference among sequences. The problem becomes particularly acute when alignments contain ribosomal RNAs from both mesophilic and thermophilic species. Among prokaryotes the latter are considerably higher in G + C content than the former, which often results in artificial clustering of thermophilic lineages and their being placed artificially deep in phylogenetic trees. In this communication we review archaeal phylogeny in the light of this consideration, focusing in particular on the phylogenetic position of the sulfate reducing species Archaeoglobus fulgidus, using both 16S rRNA and 23S rRNA sequences. The analysis shows clearly that the previously reported deep branching of the A. fulgidus lineage (very near the base of the euryarchaeal side of the archaeal tree) is incorrect, and that the lineage actually groups with a previously recognized unit that comprises the Methanomicrobiales and extreme halophiles.

NASA Discipline Exobiology↗

NASA NTRSDate not supplied

A Pipeline for Assessing the Quality of Rna-Seq Datasets in GeneLab

Transcriptome profiling by RNA sequencing (RNA-seq) is a powerful approach to identify gene expression changes in organisms exposed to unique environments such as spaceflight. One of the challenges of evaluating RNA-seq data both within and across different space-relevant studies is the ability to control for technical differences, including the use of different library preparation kits, sequencing platforms, RNA yield, and person-to-person variation. To help address this issue, the National Institute of Standards and Technology (NIST, nist.gov) initiated a consortium, at the request of industry and academia, to develop a set of controls for gene expression measurements. The result was a set of 92 unlabeled, polyadenylated transcripts that range from 250 – 2,000 nucleotides in length to mimic natural eukaryotic mRNAs. These External RNA Controls Consortium (ERCC) genes can be used in any RNA-seq experiment, by adding known concentrations of the ERCC genes to samples after RNA extraction, to offer a standard measurement for data comparison. At NASA GeneLab, we employ these controls as part of our standard operating procedures for every in-house RNA-seq study to assess the limit of detection, dynamic range, and power of differential expression analysis both within and across experiments. Here we will discuss the use, benefits, and limitations of ERCC genes and other types of controls, such as universal RNA references, to generate quality control information for RNA-seq studies conducted at GeneLab.

GeneLab↗

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 NTRS1998

A natural view of microbial biodiversity within hot spring cyanobacterial mat communities

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Review↗

NASA NTRS1993

Evolution in vitro: analysis of a lineage of ribozymes

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

Non-NASA Center↗

NASA NTRSDate not supplied

Elevating the Quality of Space Omics Sequencing Data: Innovations and Methodologies from NASA GeneLab Sample Processing Laboratory

NASA’s GeneLab, part of the NASA Open Science Data Repository, is a space-related database that hosts a diverse range of transcriptomics, proteomics, epigenomics and genomics data. The NASA GeneLab Sample Processing Laboratory (SPL) generates omics data from biological experiments conducted aboard the International Space Station, Space Shuttle and space related ground experiments, this omics data then hosted on the GeneLab repository. Samples generated such experiments pose numerous technical challenges such as small experimental sample size, variance in dissection times, limited tissue preservation methods, prolonged storage time, and more. GeneLab SPL team had developed specialized expertise in nucleic acid extraction, library preparation and sequencing of such biological samples via extensive training and years of experience. In order to ensure data accuracy and consistency across experiments, SPL has developed standardized protocols for each species and tissue type. These protocols in conjunction with quality control metrics and data standards are crucial in generating of high-quality data. SPL protocols and standards have been developed in collaboration with the scientific community and had been made publicly available on the GeneLab portal, guaranteeing comparability of datasets across spaceflight experiments. To ensure reliability of data generation, SPL leverages cutting-edge innovations in laboratory automation for sample processing. By leveraging these state-of-the-art platforms, SPL achieves high levels of data reproducibility while significantly minimizing sources of bias and variability, especially across experiments with large numbers of samples. Over the past few years, the space biology investigator community has accessed SPL-generated data from the Open Science Data Repository for a myriad of data re-analysis and re-use studies. We observe a trend that in-house SPL-generated data consistently outperforms outsourced sequencing data in terms of technical standards, quality control metrics, timeliness of data delivery, and sequencing and reagent efficiency. Superior data generation has and will continue to enable discoveries in disease, diagnostic tools, and the biological effects of long duration spaceflight.

GeneLab↗

NASA NTRS1990

Immunological characterization of plant ornithine transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and Mr = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)+ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.

NASA Discipline Plant Biology↗

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 NTRS2002

Phylogeny and systematic position of Opiliones: a combined analysis of chelicerate relationships using morphological and molecular data

The ordinal level phylogeny of the Arachnida and the suprafamilial level phylogeny of the Opiliones were studied on the basis of a combined analysis of 253 morphological characters, the complete sequence of the 18S rRNA gene, and the D3 region of the 28S rRNA gene. Molecular data were collected for 63 terminal taxa. Morphological data were collected for 35 exemplar taxa of Opiliones, but groundplans were applied to some of the remaining chelicerate groups. Six extinct terminals, including Paleozoic scorpions, are scored for morphological characters. The data were analyzed using strict parsimony for the morphological data matrix and via direct optimization for the molecular and combined data matrices. A sensitivity analysis of 15 parameter sets was undertaken, and character congruence was used as the optimality criterion to choose among competing hypotheses. The results obtained are unstable for the high-level chelicerate relationships (except for Tetrapulmonata, Pedipalpi, and Camarostomata), and the sister group of the Opiliones is not clearly established, although the monophyly of Dromopoda is supported under many parameter sets. However, the internal phylogeny of the Opiliones is robust to parameter choice and allows the discarding of previous hypotheses of opilionid phylogeny such as the "Cyphopalpatores" or "Palpatores." The topology obtained is congruent with the previous hypothesis of "Palpatores" paraphyly as follows: (Cyphophthalmi (Eupnoi (Dyspnoi + Laniatores))). Resolution within the Eupnoi, Dyspnoi, and Laniatores (the latter two united as Dyspnolaniatores nov.) is also stable to the superfamily level, permitting a new classification system for the Opiliones. c2002 The Willi Hennig Society.

NASA Discipline Evolutionary Biology↗

NASA NTRS1998

Expression of extracellular calcium (Ca2 + o)-sensing receptor in the clonal osteoblast-like cell lines, UMR-106 and SAOS-2

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2 + o) homeostasis in parathyroid gland and kidney. More recent data have suggested the presence of this receptor in additional tissues, such as brain, intestine and skin. In this study, we examined the expression of the CaR in the rat and human osteosarcoma cell lines, UMR-106 and SAOS-2, respectively, which possess osteoblast-like characteristics. Both immunocytochemistry and Western blot analysis, using a polyclonal antiserum specific for the CaR, detected CaR protein in UMR-106 and SAOS-2 cells. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in each cell line. Therefore, taken together, our data strongly suggest that the osteoblast-like cell lines, UMR-106 and SAOS-2, possess both CaR protein and mRNA very similar if not identical to those in parathyroid and kidney.

NASA Discipline Musculoskeletal↗