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All sources NASA NTRS DOE OSTI

NASA NTRS2003

Macromolecular Expression and Function: A New Paradigm for NASA Risk Assessment

Predicting risks in humans of either acute effects such as bone loss or muscle wasting, or late effects such as cancer, is challenging. To an approximation, this is because uncertainties of exposure to stress factors or toxic agents and the uniformity of processing subsequent damage at the cellular level within a complex set of biological variables degrade the confidence of predicting pathologic outcome. A cellular biodosimeter that simultaneously reports 1) the type of damage due to that exposure, 2) the quantity of damage incurred by that exposure, and 3) the dataset used to assess risk of developing pathologic outcome caused by that exposure would therefore be useful for predicting ultimate risks faced by an individual, such as an astronaut. It is suggested that such a biodosimeter can be based upon analyses of gene-expression and protein expression whereby large datasets of cellular response to damage are obtained and analyzed for expression-profiles correlated with established end points and molecular markers predictive for risks being assessed. The usefulness of multiparametric cellular biodosimeters could be realized by quantitatively profiling these datasets using techniques of bioinformatics. Such an approach contributes to the foundation of molecular epidemiology as a new scientific discipline, and represents a new paradigm of risk assessment.

Richmond, Robert↗

NASA NTRS1998

Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness

Shortly after the discovery of polonium and radium by Marie Curie and her husband and colleague, Pierre Curie, it was learned that exposure to these alpha-particle emitters produced deleterious biological effects. The mechanisms underlying the increased biological effectiveness of densely ionizing radiations, including alpha particles, neutrons and highly energetic heavy charged particles, remain an active area of investigation. In this paper, we review recent advances in several areas of the radiobiology of these densely ionizing radiations, also known as heavy ions. Advances are described in the areas of DNA damage and repair, chromosome aberrations, mutagenesis, neoplastic transformation in vitro, genomic instability, normal tissue radiobiology and carcinogenesis in vivo. We focus on technical innovations, including novel applications of pulsed-field gel electrophoresis, fluorescence in situ hybridization (FISH), linkage analysis, and studies of gene expression and protein expression. We also highlight the use of new cellular and animal systems, including those with defined DNA repair deficiencies, as well as epithelial cell model systems to assess neoplastic transformation both in vitro and in vivo. The studies reviewed herein have had a substantial impact on our understanding of the genotoxic effects of heavy ions as well as their distinct effects on tissue homeostasis. The use of these radiations in cancer therapy is also discussed. The use of both heavy-ion and proton therapy is on the upswing in several centers around the world, due to their unique energy deposition characteristics that enhance the therapeutic effect and help reduce damage to normal tissue.

Review, Academic↗

NASA NTRS2003

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

Atherosclerosis is now viewed as an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions, including oscillatory shear stress (OS), in branched arteries. In contrast, the arterial regions exposed to laminar shear (LS) are relatively lesion-free. The mechanisms underlying the opposite effects of OS and LS on the inflammatory and atherogenic processes are not clearly understood. Here, through DNA microarrays, protein expression, and functional studies, we identify bone morphogenic protein 4 (BMP4) as a mechanosensitive and pro-inflammatory gene product. Exposing endothelial cells to OS increased BMP4 protein expression, whereas LS decreased it. In addition, we found BMP4 expression only in the selective patches of endothelial cells overlying foam cell lesions in human coronary arteries. The same endothelial patches also expressed higher levels of intercellular cell adhesion molecule-1 (ICAM-1) protein compared with those of non-diseased areas. Functionally, we show that OS and BMP4 induced ICAM-1 expression and monocyte adhesion by a NFkappaB-dependent mechanism. We suggest that BMP4 is a mechanosensitive, inflammatory factor playing a critical role in early steps of atherogenesis in the lesion-prone areas.

NASA Program Fundamental Space Biology↗

NASA NTRS2001

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

NASA Discipline Musculoskeletal↗

DOE OSTI2024

Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical β-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Spatial Proteomics towards cellular Resolution

Introduction: Spatial biology is an emerging interdisciplinary field facilitating biological discoveries through the use of spatial omics technologies. Recent advancements in spatial transcriptomics, spatial genomics (e.g. genetic mutations and epigenetic marks), multiplexed immunofluorescence, and spatial metabolomics/lipidomics have enabled high-resolution spatial profiling of gene expression, genetic variation, protein expression, and metabolites/lipids profiles in tissue. These developments contribute to a deeper understanding of the spatial organization within tissue microenvironments at the molecular level. Areas covered: This report provides an overview of the untargeted, bottom-up mass spectrometry (MS)-based spatial proteomics workflow. It highlights recent progress in tissue dissection, sample processing, bioinformatics, and liquid chromatography (LC)-MS technologies that are advancing spatial proteomics toward cellular resolution. Expert opinion: The field of untargeted MS-based spatial proteomics is rapidly evolving and holds great promise. To fully realize the potential of spatial proteomics, it is critical to advance data analysis and develop automated and intelligent tissue dissection at the cellular or subcellular level, along with high-throughput LC-MS analyses of thousands of samples. In conclusion, achieving these goals will necessitate significant advancements in tissue dissection technologies, LC-MS instrumentation, and computational tools.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2005

Resolving Risks in Individual Astronauts: A New Paradigm for Critical Path Exposures

The limited number of astronauts available for risk-assessment prevents classic epidemiologic study, and thereby requires alternative approach to assessing risks within individual astronauts exposed to toxic agents identified within the Bioastronautics Critical Path Roadmap (BCPR). Developing a system of noninvasive real-time biodosimetry that provides large datasets for analyses before, during, and after missions for simultaneously determining 1) the kinds of toxic insult, 2) the degree of that insult, both within tissues absorbing that insult, would be usehl for resolving statistically significant risk-assessment in individual astronauts. Therefore, a currently achievable multiparametric paradigm is presented for use in analyzing gene-expression and protein-expression so as to establish predictive outcomes.

Richmond, Robert C.↗

NASA NTRS2010

Osteocytes Mechanosensing in NASA Rotating Wall Bioreactor

Osteocyte cells are the most abundant (90%) yet least understood bone cell type in the human body. Osteocytes are theorized to be the mechanosensors and transducers of mechanical load for bones, yet the biological mechanism of this action remains elusive. However, recent discoveries in osteocyte cell biology have shed light on their importance as key mechanosensing cells regulating bone remodeling and phosphate homeostasis. The aim of this project was to characterize gene expression patterns and protein levels following exposure of MLO-Y4, a very well characterized murine osteocyte-like cell line, to simulated microgravity using the NASA Rotating Wall Vessel (RWV) Bioreactor. To determine mechanistic pathways of the osteocyte's gravity sensing ability, we evaluated in vitro gene and protein expression of osteocytes exposed to simulated microgravity. Improved understanding of the fundamental mechanisms of mechano transduction at the osteocyte cellular level may lead to revolutionary treatment otions to mitigate the effects of bone loss encountered by astronauts on long duration space missions and provide tailored treatment options for maintaining bone strength of immobilized/partially paralyzed patients here on Earth.

Spatz, Jordan↗

NASA NTRS2014

MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

Wu, Honglu↗

DOE OSTI2024

Corn stover variability drives differences in bisabolene production by engineered Rhodotorula toruloides

Microbial conversion of lignocellulosic biomass represents an alternative route for production of biofuels and bioproducts. While researchers have mostly focused on engineering strains such as Rhodotorula toruloides for better bisabolene production as a sustainable aviation fuel, less is known about the impact of the feedstock heterogeneity on bisabolene production. Critical material attributes like feedstock composition, nutritional content, and inhibitory compounds can all influence bioconversion. Further, the given feedstocks can have a marked influence on selection of suitable pretreatment and hydrolysis technologies, optimizing the fermentation conditions, and possibly even modifying the microorganism's metabolic pathways, to better utilize the available feedstock. Here, this work aimed to examine and understand how variations in corn stover batches, anatomical fractions, and storage conditions impact the efficiency of bisabolene production by R. toruloides. All of these represent different facets of feedstock heterogeneity. Deacetylation, mechanical refining, and enzymatic hydrolysis of these variable feedstocks served as the basis of this research. The resulting hydrolysates were converted to bisabolene via fermentation, a sustainable aviation fuel precursor, using an engineered R. toruloides strain. This study showed that different sources of feedstock heterogeneity can influence microbial growth and product titer in counterintuitive ways, as revealed through global analysis of protein expression. The maximum bisabolene produced by R. toruloides was on the stalk fraction of corn stover hydrolysate (8.89 ± 0.47 g/L). Further, proteomics analysis comparing the protein expression between the anatomic fractions showed that proteins relating to carbohydrate metabolism, energy production, and conversion as well as inorganic ion transport metabolism were either significantly upregulated or downregulated. Specifically, downregulation of proteins related to the iron–sulfur cluster in stalk fraction suggests a coordinated response by R. toruloides to maintain overall metabolic balance, and this was corroborated by the concentration of iron in the feedstocks.

09 BIOMASS FUELS↗

NASA NTRS1997

The Structure and Function of Non-Collagenous Bone Proteins

The research done under the cooperative research agreement for the project titled 'The structure and function of non-collagenous bone proteins' represented the first phase of an ongoing program to define the structural and functional relationships of the principal noncollagenous proteins in bone. An ultimate goal of this research is to enable design and execution of useful pharmacological compounds that will have a beneficial effect in treatment of osteoporosis, both land-based and induced by long-duration space travel. The goals of the now complete first phase were as follows: 1. Establish and/or develop powerful recombinant protein expression systems; 2. Develop and refine isolation and purification of recombinant proteins; 3. Express wild-type non-collagenous bone proteins; 4. Express site-specific mutant proteins and domains of wild-type proteins to enhance likelihood of crystal formation for subsequent solution of structure.

Hook, Magnus↗

NASA NTRS1996

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.

Non-NASA Center↗

NASA NTRS2002

Computational Model of the Modulation of Gene Expression Following DNA Damage

High linear energy transfer (LET) radiation, such as heavy ions or neutrons, has an increased biological effectiveness compared to X rays for gene mutation, genomic instability, and carcinogenesis. In the traditional paradigm, mutations or chromosomal aberrations are causative of late effects. However, in recent years experimental evidence has demonstrated the important role of the description of the modification of gene expression by radiation in understanding the mechanisms of radiation action. In this report, approaches are discussed to the mathematical description of mRNA and protein expression kinetics following DNA damage. Several hypotheses for models of radiation modulation of protein expression are discussed including possible non-linear processes that evolve from the linear dose responses that follow the initial DNA damage produced by radiation.

NASA Center JSC↗

NASA NTRS1992

Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore

The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.

Non-NASA Center↗

NASA NTRS2005

Analysis of Pdeudomonas aeruginosa Growth and Virulence in Modelled Microgravity

Stress, radiation and microgravity cause astronauts to experience secondary immunosuppression. Spaceflight conditions enhance bacterial growth and alter antimicrobial susceptibility. Clinostats are used to model microgravity effects at lxg. In controls rotated on the vertical axis, the g-vector acts on cells as in static cultures. Salmonella enterica serovar Typhimurium virulence genes are up-regulated in modelled microgravity (MMG); a MMG regulon has been postulated. We hypothesize that the virulence of P. aeruginosa (PA) may be affected similarly by microgravity, which could be observed in MMG. This study focused on regulation of the ETA protein by PA during growth in MMG. PA103 was grown in an ETA production medium at 37 C. One series of media was inoculated with frozen cultures and grown using horizontal (MMG) or static incubation. Another series inoculated with refrigerated cultures included vertical rotating controls. Analyses included optical density (OD), agar plate counts (PC) on R2A, ETA ELISA, and protein expression by 2-D gel analyses. Growth and ETA results differed depending on inoculum, with minor effects of MMG. Proteomic analysis of 2-D gels indicate differences in protein expression with MMG. Growth and ETA results show that consistent methodology is critical when studying environmental effects. This study provides information on the relationships between environmental changes and virulence regulation, especially for flight experiments, when ground experiments are used to predict potential spaceflight effects.

Guadarrama, Seratna↗

NASA NTRS2002

Estrogen has opposing effects on vascular reactivity in obese, insulin-resistant male Zucker rats

We hypothesized that estradiol treatment would improve vascular dysfunction commonly associated with obesity, hyperlipidemia, and insulin resistance. A sham operation or 17beta-estradiol pellet implantation was performed in male lean and obese Zucker rats. Maximal vasoconstriction (VC) to phenylephrine (PE) and potassium chloride was exaggerated in control obese rats compared with lean rats, but estradiol significantly attenuated VC in the obese rats. Estradiol reduced the PE EC50 in all groups. This effect was cyclooxygenase independent, because preincubation with indomethacin reduced VC response to PE similarly in a subset of control and estrogen-treated lean rats. Endothelium-independent vasodilation (VD) to sodium nitroprusside was similar among groups, but endothelium-dependent VD to ACh was significantly impaired in obese compared with lean rats. Estradiol improved VD in lean and obese rats by decreasing EC50 but impaired function by decreasing maximal VD. The shift in EC50 corresponded to an upregulation in nitric oxide synthase III protein expression in the aorta of the estrogen-treated obese rats. In summary, estrogen treatment improves vascular function in male insulin-resistant, obese rats, partially via an upregulation of nitric oxide synthase III protein expression. These effects are counteracted by adverse factors, such as hyperlipidemia and, potentially, a release of an endothelium-derived contractile agent.

Non-NASA Center↗

NASA NTRS2004

Biodosimetry as a New Paradigm for Determination of Radiation Risks and Risk-Mitigation in Astronauts Exposed to Space Radiation

Predicting risk of cancer in astronauts exposed to space radiation is challenging partly because uncertainties of absorption of dose and the processing of dose-related damage at the cellular level degrade the confidence of predicting the expression of cancer. Cellular biodosimeters that simultaneously report: 1) the quantity of absorbed dose after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the macromolecular profiles related to malignant transformation in cells absorbing that dose would therefore be useful. An approach to such a multiparametric biodosimeter will be reported, This is the demonstration of two dose-responsive field-effects of enhanced protein-expression. In one case, expression of keratin 18 (K18) in cultures of human mammary epithelial cells (HMEC) irradiated with cesium-137 gamma-rays is enhanced following exposure of log phase cells to relatively low doses of 30 to 90 cGy. K18 has been reported by a marker for tumor staging and for apoptosis. In the second case, expression of connexin 43 (Cx43) is increased in irradiated stationary phase cultures of HMEC, indicating enhanced formation of gap junctions. Gap junctions have been reported to be involved in bystander effects following irradiation. It is a biodosimeter for assessing radiogenic damage. It is suggested further that such biomolecular dosimetry may introduce a new paradigm for assessing cancer risk and risk-mitigation in individuals, a requirement for managing radiation health in astronauts during extended missions in space. This new paradigm is built upon the statistical power provided by the use of functional genomics and proteomics represented in combined gene- and protein-expression assays.

Richmond, Robert↗

DOE OSTI2024

Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli

YcjN is a putative substrate binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 Å, revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein. Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and nonlipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and nonlipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

Escherichia coli↗