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Surface-slip equations for multicomponent, nonequilibrium air flow

Equations are presented for the surface slip (or jump) values of species concentration, pressure, velocity, and temperature in the low-Reynolds-number, high-altitude flight regime of a space vehicle. These are obtained from closed-form solutions of the mass, momentum, and energy flux equations using the Chapman-Enskog velocity distribution function. This function represents a solution of the Boltzmann equation in the Navier-Stokes approximation. The analysis, obtained for nonequilibrium multicomponent air flow, includes the finite-rate surface catalytic recombination and changes in the internal energy during reflection from the surface. Expressions for the various slip quantities have been obtained in a form which can readily be employed in flow-field computations. A consistent set of equations is provided for multicomponent, binary, and single species mixtures. Expression is also provided for the finite-rate species-concentration boundary condition for a multicomponent mixture in absence of slip.

Gupta, Roop N.↗

Calbindins decreased after space flight

Exposure of the body to microgravity during space flight causes a series of well-documented changes in Ca2+ metabolism, yet the cellular and molecular mechanisms leading to these changes are poorly understood. Calbindins, vitamin D-dependent Ca2+ binding proteins, are believed to have a significant role in maintaining cellular Ca2+ homeostasis. In this study, we used biochemical and immunocytochemical approaches to analyze the expression of calbindin-D28k and calbindin-D9k in kidneys, small intestine, and pancreas of rats flown for 9 d aboard the space shuttle. The effects of microgravity on calbindins in rats from space were compared with synchronous Animal Enclosure Module controls, modeled weightlessness animals (tail suspension), and their controls. Exposure to microgravity resulted in a significant and sustained decrease in calbindin-D28k content in the kidney and calbindin-D9k in the small intestine of flight animals, as measured by enzyme-linked immunosorbent assay (ELISA). Modeled weightlessness animals exhibited a similar decrease in calbindins by ELISA. Immunocytochemistry (ICC) in combination with quantitative computer image analysis was used to measure in situ the expression of calbindins in the kidney and the small intestine, and the expression of insulin in pancreas. There was a large decrease of immunoreactivity in renal distal tubular cell-associated calbindin-D28k and in intestinal absorptive cell-associated calbindin-D9k of space flight and modeled weightlessness animals compared with matched controls. No consistent difference in pancreatic insulin immunoreactivity between space flight, modeled weightlessness, and controls was observed. Regression analysis of results obtained by quantitative ICC and ELISA for space flight, modeled weightlessness animals, and their controls demonstrated a significant correlation. These findings after a short-term exposure to microgravity or modeled weightlessness suggest that a decreased expression of calbindins may contribute to the disorders of Ca2+ metabolism induced by space flight.

Non-NASA Center↗

Characterization of the DNA binding properties of polyomavirus capsid protein

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

NASA Discipline Cell Biology↗

Systemic Microgravity Response: Utilizing GeneLab to Develop Hypotheses for Spaceflight Risks

Biological risks associated with microgravity is a major concern for space travel. Although determination of risk has been a focus for NASA research, data examining systemic (i.e., multi- or pan-tissue) responses to space flight are sparse. The overall goal of our work is to identify potential master regulators responsible for such responses to microgravity conditions. To do this we utilized the NASA GeneLab database which contains a wide array of omics experiments, including data from: 1) different flight conditions (space shuttle (STS) missions vs. International Space Station (ISS); 2) different tissues; and 3) different types of assays that measure epigenetic, transcriptional, and protein expression changes. We have performed meta-analysis identifying potential master regulators involved with systemic responses to microgravity. The analysis used 7 different murine and rat data sets, examining the following tissues: liver, kidney, adrenal gland, thymus, mammary gland, skin, and skeletal muscle (soleus, extensor digitorum longus, tibialis anterior, quadriceps, and gastrocnemius). Using a systems biology approach, we were able to determine that p53 and immune related pathways appear central to pan-tissue microgravity responses. Evidence for a universal response in the form of consistency of change across tissues in regulatory pathways was observed in both STS and ISS experiments with varying durations; while degree of change in expression of these master regulators varied across species and strain, some change in these master regulators was universally observed. Interestingly, certain skeletal muscle (gastrocnemius and soleus) show an overall down-regulation in these genes, while in other types (extensor digitorum longus, tibialis anterior and quadriceps) they are up-regulated, suggesting certain muscle tissues may be compensating for atrophy responses caused by microgravity. Studying these organtissue-specific perturbations in molecular signaling networks, we demonstrate the value of GeneLab in characterizing potential master regulators associated with biological risks for spaceflight.

Microgravity↗

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗

An active role of the amyloplasts and nuclei of root statocytes in graviperception

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception. c 2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

short duration↗

Molecular Signaling in Muscle Plasticity

Extended spaceflight under microgravity conditions leads to significant atrophy of weight-bearing muscles. Atrophy and hypertrophy are the extreme outcomes of the high degree of plasticity exhibited by skeletal muscle. Stimuli which control muscle plasticity include neuronal, hormonal, nutritional, and mechanical inputs. The mechanical stimulus for muscle is directly related to the work or exercise against a load performed. Little or no work is performed by weight-bearing muscles under microgravity conditions. A major hypothesis is that focal adhesion kinase (FAK) which is associated with integrin at the adherens junctions and costa meres of all skeletal muscles is an integral part of the major mechanism for molecular signaling upon mechanical stimulation in all muscle fibers. Additionally, we propose that myotonic protein kinase (DMPK) and dystrophin (DYSTR) also participate in distinct mechanically stimulated molecular signaling pathways that are most critical in type I and type II muscle fibers, respectively. To test these hypotheses, we will use the paradigms of hindlimb unloading and overloading in mice as models for microgravity conditions and a potential exercise countermeasure, respectively, in mice. We expect that FAK loss-of-function will impair hypertrophy and enhance atrophy in all skeletal muscle fibers whereas DYSTR and DMPK loss-of-function will have similar but more selective effects on Type IT and Type I fibers, respectively. Gene expression will be monitored by muscle-specific creatine kinase M promoter-reporter construct activity and specific MRNA and protein accumulation in the soleus (type I primarily) and plantaris (type 11 primarily) muscles. With these paradigms and assays, the following Specific Project Aims will be tested in genetically altered mice: 1) identify the roles of DYSTR and its pathway; 2) evaluate the roles of the DMPK and its pathway; 3) characterize the roles of FAK and its pathway and 4) genetically analyze the mechanisms and interactions between the FAK, DYSTR, and DMPK-associated pathways in single and specific combinations of mutants. The identification of potential signaling mechanisms may permit future development of pharmacological countermeasures for amelioration and prevention of the microgravity-induced atrophy in extended spaceflight, and the analysis of both overloading and unloading paradigms may provide further support for development of exercise-based countermeasures. Understanding the basic mechanisms of molecular signaling in muscle plasticity may aid our understanding and treatment of skeletal muscle atrophy not only in spaceflight but in similar problems of the aging population, in prolonged bed rest, and in cachexia associated with chronic disease.

Epstein, Henry F.↗

SPACEFLIGHT-INDUCED CHANGES IN MICROBIAL VIRULENCE AND THE IMPACT TO THE HOST IMMUNE RESPONSE

INTRODUCTION Over the past 50 years, microorganisms have displayed unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, and virulence. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system; consisting of altered leukocyte distribution, reductions in T and NK cell function, altered cytokine profiles, and reactivation of latent herpesviruses. Further, astronauts have some degree of clinical incidence, primarily infectious disease episodes and atopic dermatitis. The impact of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. SPECIFIC AIMS In the Host-Microorganism, Microbial Response aspect of this study, the following Specific Aims are being investigated, using the microbial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. Aim 1: Characterize the effect of spaceflight analogue culture on microbial pathogenesis-related stress responses and in vitro host-pathogen interactions. Analysis will include microbial stress responses as well as colonization and viability following pathogen challenge of three-dimensional (3-D) tissue co-culture models containing immune cells. We specifically selected obligate and opportunistic pathogens that are medically important and have been or are likely to be found aboard spacecraft. Aim 2: Characterize the effect of spaceflight analogue culture on the virulence potential of pathogenic microorganisms. Changes in virulence will be assessed using a mouse model of infection. SIGNIFICANCE The goal of this study is to use spaceflight analogue conditions to gain insight into the breadth of medically-significant International Space Station microorganisms that have altered virulence and the impact of those changes on the immune response of the host, including astronaut immune cells. This information will provide critical understanding into the impact of microgravity on potential alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C. M. Ott↗

Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development

BACKGROUND: The identification of the function of all genes that contribute to specific biological processes and complex traits is one of the major challenges in the postgenomic era. One approach is to employ forward genetic screens in genetically tractable model organisms. In Drosophila melanogaster, P element-mediated insertional mutagenesis is a versatile tool for the dissection of molecular pathways, and there is an ongoing effort to tag every gene with a P element insertion. However, the vast majority of P element insertion lines are viable and fertile as homozygotes and do not exhibit obvious phenotypic defects, perhaps because of the tendency for P elements to insert 5' of transcription units. Quantitative genetic analysis of subtle effects of P element mutations that have been induced in an isogenic background may be a highly efficient method for functional genome annotation. RESULTS: Here, we have tested the efficacy of this strategy by assessing the extent to which screening for quantitative effects of P elements on sensory bristle number can identify genes affecting neural development. We find that such quantitative screens uncover an unusually large number of genes that are known to function in neural development, as well as genes with yet uncharacterized effects on neural development, and novel loci. CONCLUSIONS: Our findings establish the use of quantitative trait analysis for functional genome annotation through forward genetics. Similar analyses of quantitative effects of P element insertions will facilitate our understanding of the genes affecting many other complex traits in Drosophila.

Non-NASA Center↗

GeneLab: A Systems Biology Platform for Spaceflight Omics Data

NASA's mission includes expanding our understanding of biological systems to improve life on Earth and to enable long-duration human exploration of space. Resources to support large numbers of spaceflight investigations are limited. NASA's GeneLab project is maximizing the science output from these experiments by: (1) developing a unique public bioinformatics database that includes space bioscience relevant "omics" data (genomics, transcriptomics, proteomics, and metabolomics) and experimental metadata; (2) partnering with NASA-funded flight experiments through bio-sample sharing or sample augmentation to expedite omics data input to the GeneLab database; and (3) developing community-driven reference flight experiments. The first database, GeneLab Data System Version 1.0, went online in April 2015. V1.0 contains numerous flight datasets and has search and download capabilities. Version 2.0 will be released in 2016 and will link to analytic tools. In 2015 Genelab partnered with two Biological Research in Canisters experiments (BBRIC-19 and BRIC-20) which examine responses of Arabidopsis thaliana to spaceflight. GeneLab also partnered with Rodent Research-1 (RR1), the maiden flight to test the newly developed rodent habitat. GeneLab developed protocols for maxiumum yield of RNA, DNA and protein from precious RR-1 tissues harvested and preserved during the SpaceX-4 mission, as well as from tissues from mice that were frozen intact during spaceflight and later dissected. GeneLab is establishing partnerships with at least three planned flights for 2016. Organism-specific nationwide Science Definition Teams (SDTs) will define future GeneLab dedicated missions and ensure the broader scientific impact of the GeneLab missions. GeneLab ensures prompt release and open access to all high-throughput omics data from spaceflight and ground-based simulations of microgravity and radiation. Overall, GeneLab will facilitate the generation and query of parallel multi-omics data, and deep curation of metadata for integrative analysis, allowing researchers to uncover cellular networks as observed in systems biology platforms. Consequently, the scientific community will have access to a more complete picture of functional and regulatory networks responsive to the spaceflight environment.. Analysis of GeneLab data will contribute fundamental knowledge of how the space environment affects biological systems, and enable emerging terrestrial benefits resulting from mitigation strategies to prevent effects observed during exposure to space. As a result, open access to the data will foster new hypothesis-driven research for future spaceflight studies spanning basic science to translational science.

proteomics↗

Multiple melanocortin receptors are expressed in bone cells

Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

NASA Discipline Musculoskeletal↗

Antisense and sense expression of cDNA coding for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco

A number of plant species contain the class II of genes encoding the cytochrome P450, CYP73, the cognate protein of which cinnamic acid 4-hydroxylase, is the second enzyme of the phenylpropanoid pathway. In order to begin to determine possible functionality, tobacco has been transformed with a truncated French bean class II cinnamate hydroxylase (CYP73A15) in the sense and antisense orientations. Signals for C4H protein could be detected in vascular tissue from wild-type plants using heterologous probes. The transformed plants showed a normal phenotype, even though detectable C4H protein was much reduced in tissue prints. Young propagated transformants displayed a range of reduced C4H activities, as well as either reduced or no phloroglucinol-stainable lignin. However, all mature tobacco plants showed the accumulation of lignin, even though its deposition was apparently delayed. This was not due to induction of tyrosine ammonia-lyase activity, which was not detected, but instead it is presumed due to sufficient C4H residual activity. Analysis of the lignin content of the plants showed reductions of up to 30% with a slightly reduced syringyl to guaiacyl ratio as compared to wild type. This reduction level was favourable in comparison with some other targets in the lignification pathway that have been manipulated including that of class I cinnamate 4-hydroxylase. It is proposed that the class II cinnamate 4-hydroxylase might also function in lignification in a number of species including French bean and tobacco, based on these data.

NASA Discipline Plant Biology↗

Advances in Rodent Research Missions on the International Space Station

A Research platform for rodent experiment on the ISS is an essential tool for advancing biomedical research in space. The Rodent Research allows for experiments of much longer duration that experiments on the Shuttle. NASA’s Rodent Research (RR)-1 mission was successfully completed, including post-flight analysis and achieved a number of objectives including validation of flight hardware, on-orbit operations, and science capabilities that were developed at the NASA Ames Research Center. Briefly, twenty C57BL/6J adult female mice were launched on the SpX4 Dragon vehicle, which thrived for up to 37 days in microgravity. Daily health checks of the mice were performed during the mission via downlinked video; all animals were healthy and displayed normal behavior without any significant signs of stress. Behavioral analysis demonstrated that Flight and Ground Control mice exhibited the same range of behaviors, including eating, drinking, exploratory behavior, self- and allo-grooming, and social interactions indicative of healthy animals. The animals were euthanized and select tissues were collected from some of the mice on orbit to assess the long-term sample storage capabilities of the ISS. The data obtained from the flight mice were comparable to those from the 3 groups of control mice (baseline, vivarium and ground controls), suggesting that the ISS has adequate capability to support long-duration rodent experimentations. We recovered over 35 tissues from 40 RR1 frozen carcasses, yielded over 3200 aliquots of tissues, and distributed to the scientific community, including NASA’s GeneLab and scientists in the U.S. through Biospecimen Sharing Program via Ames Life Science Data Archive. Tissues were also distributed to Russian research colleagues at the Institute for Biomedical Problems. The expression levels of select genes including albumin, catalase, GAPDH, HMGCoA Reductase, and IGF1 were determined using RNA isolated from the livers by qPCR and no significant differences by one factor ANOVA were found between flight and ground control groups. In addition, some of the liver samples were subject to transcriptomics, epigenomics and proteomics. The data are now available to the scientific community through GeneLab’s open science data website. Since the RR1 mission, another long duration mission (Rodent Research-2) was completed on the ISS in 2015 in which 20 female C57 BL/6J mice were successfully maintained on the ISS for varying time points, with the last group of 5 animals being on-orbit for 54 days. This second Rodent Research flight expanded the program’s capabilities with the introduction of new technologies including blood collection and separation and bone densitometry scanning. Furthermore, we have continued to expand the ISS’s capabilities by running a series of ground-based verification testing using male mice. Our next step is to fly male mice for Rodent Research-4 on SpaceX-10 to study the effects of microgravity on bone healing and regeneration. It will be the first long-duration mission using male mice using Rodent Hardware. In addition, the number of mice will increase from 20 mice (on RR-1 and RR-2) to 40 for RR-4. When samples return to Earth, a number of tissues will be dissected from the frozen carcasses and select tissue samples will become available to the scientific community via BSP. Altogether, we have continued to expand our capabilities for performing long-duration missions on the ISS as emphasized in the National Research Council’s Decadal Survey released in 2011 and to maximize science return from each mission.

Choi, S.↗

Radiation Quality Effects on Transcriptome Profiles in 3-D Cultures After Charged Particle Irradiation

In this work, we evaluated the differential effects of low- and high-LET radiation on 3-D organotypic cultures in order to investigate radiation quality impacts on gene expression and cellular responses. Current risk models for assessment of space radiation-induced cancer have large uncertainties because the models for adverse health effects following radiation exposure are founded on epidemiological analyses of human populations exposed to low-LET radiation. Reducing these uncertainties requires new knowledge on the fundamental differences in biological responses (the so-called radiation quality effects) triggered by heavy ion particle radiation versus low-LET radiation associated with Earth-based exposures. In order to better quantify these radiation quality effects in biological systems, we are utilizing novel 3-D organotypic human tissue models for space radiation research. These models hold promise for risk assessment as they provide a format for study of human cells within a realistic tissue framework, thereby bridging the gap between 2-D monolayer culture and animal models for risk extrapolation to humans. To identify biological pathway signatures unique to heavy ion particle exposure, functional gene set enrichment analysis (GSEA) was used with whole transcriptome profiling. GSEA has been used extensively as a method to garner biological information in a variety of model systems but has not been commonly used to analyze radiation effects. It is a powerful approach for assessing the functional significance of radiation quality-dependent changes from datasets where the changes are subtle but broad, and where single gene based analysis using rankings of fold-change may not reveal important biological information.

Patel, Zarana S.↗

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗

Advances in Rodent Research Missions on the International Space Station

A research platform for rodent experiment on the ISS is a valuable tool for advancing biomedical research in space. Capabilities offered by the Rodent Research project developed at NASA Ames Research Center can support experiments of much longer duration on the ISS than previous experiments performed on the Space Shuttle. NASAs Rodent Research (RR)-1 mission was completed successfully and achieved a number of objectives, including validation of flight hardware, on-orbit operations, and science capabilities as well as support of a CASIS-sponsored experiment (Novartis) on muscle atrophy. Twenty C57BL6J adult female mice were launched on the Space-X (SpX) 4 Dragon vehicle, and thrived for up to 37 days in microgravity. Daily health checks of the mice were performed during the mission via downlinked video; all flight animals were healthy and displayed normal behavior, and higher levels of physical activity compared to ground controls. Behavioral analysis demonstrated that Flight and Ground Control mice exhibited the same range of behaviors, including eating, drinking, exploratory behavior, self- and allo-grooming, and social interactions indicative of healthy animals. The animals were euthanized on-orbit and select tissues were collected from some of the mice on orbit to assess the long-term sample storage capabilities of the ISS. In general, the data obtained from the flight mice were comparable to those from the three groups of control mice (baseline, vivarium and ground controls, which were housed in flight hardware), showing that the ISS has adequate capability to support long-duration rodent experiments. The team recovered 35 tissues from 40 RR-1 frozen carcasses, yielding 3300 aliquots of tissues to distribute to the scientific community in the U.S., including NASAs GeneLab project and scientists via Space Biology's Biospecimen Sharing Program Ames Life Science Data Archive. Tissues also were distributed to Russian research colleagues at the Institute for Biomedical Problems. The expression levels of select genes including albumin, catalase, GAPDH, HMGCoA Reductase, and IGF1 were determined using RNA isolated from the livers by qPCR and no significant differences by one factor ANOVA were found between flight and ground control groups. In addition, some of the liver samples were analyzed for transcriptomic, epigenomic and proteomic profiles; some of the data sets are now available to the scientific community through GeneLabs open science data website. A second long duration mission, Rodent Research-2 (RR-2) was completed on the ISS in 2015; 20 female C57BL6J mice were successfully maintained on the ISS for various durations, with the last group of 5 animals living on-orbit for 54 days. Furthermore, we continue to expand the ISSs capabilities by introducing new on-orbit technologies including blood collection and separation, bone densitometry scanning, muscle grip strength and anesthesia with recovery. In addition, series of ground-based verification testing to fly male mice and increase the total number of mice on-orbit from 20 to 40. Subsequent missions will provide the capability to return live mice from the ISS animals to evaluate recovery on Earth, further expanding operational and science capabilities of the RR project on the ISS.

Choi, S. Y.↗

Nasa GeneLab Computomics Reveal Horizontal Gene Transfer on International Space Station Environmental Metagenomes

Prokaryotic lifeforms can be observed to demonstrate many keen adaptive advantages, perhaps facilitated by a nature simplistic relative to divergent domains of life. In particular, decompartmentalized gene expression facilitates adaptation by allowing free exchange of genetic material, albeit at the cost of increased susceptibility to genetic damage. Thus, these lifeforms must compensate by embracing diverse investment strategies in an attempt to “brute force” the evolvability equation through precipitous genesis, lean metabolic efficiency, and sheer population. This prokaryotic archetype also enables symbiotic relationships with secondary mobile genetic elements known as plasmids, which have been shown to drive evolution on rapid temporal scales through processes such as conjugation and transformation. This study attempts to decipher whether these mechanisms of horizontal gene transfer (HGT) are major factors in determining prokaryote fitness within a unique isolated environment, the International Space Station (ISS). The ISS Microbial Tracking (MT) project has generated a wealth of data concerning the successive reigns of microbial genera that appear to thrive amidst harsh conditions for life. Despite relatively higher doses of ionizing radiation as compared to Earth, complications associated with microgravity, and the anti-microbial mélange deployed, microbial life still persists in this environment. The NASA GeneLab serves as a data repository and analysis platform to enable researchers to access space flight factor related data. With the use of GeneLab’s modern computational suites (computomics), phylogenetic and functional genomic investigations of HGT events were conducted on the data generated from the MT-1 project. The putative data concerning the plasmid population (plasmidome) of the ISS was algorithmically derived and compared to those of habitats with similar environmental dynamics- such as living quarters and hospitals- to investigate whether these HGT elements may play crucial role(s) in shaping the microbiome of this closed habitat that serves as the only inhabited structure in space.

Bense, Nicholas↗