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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

Non-NASA Center↗
NASA NTRS2000

Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

Non-NASA Center↗
NASA NTRS1999

A molecular description of the evolution of resistance

BACKGROUND: In vitro evolution has been used to obtain nucleic acid molecules with interesting functional properties. The evolution process usually is carried out in a stepwise manner, involving successive rounds of selection, amplification and mutation. Recently, a continuous in vitro evolution system was devised for RNAs that catalyze the ligation of oligonucleotide substrates, allowing the evolution of catalytic function to be studied in real time. RESULTS: Continuous in vitro evolution of an RNA ligase ribozyme was carried out in the presence of a DNA enzyme that was capable of cleaving, and thereby inactivating, the ribozyme. The DNA concentration was increased steadily over 33.5 hours of evolution, reaching a final concentration that would have been sufficient to inactivate the starting population in one second. The evolved population of ribozymes developed resistance to the DNA enzyme, reducing their vulnerability to cleavage by 2000-fold but retaining their own catalytic function. Based on sequencing and kinetic analysis of the ribozymes, two mechanisms are proposed for this resistance. One involves three nucleotide substitutions, together with two compensatory mutations, that alter the site at which the DNA enzyme binds the ribozyme. The other involves enhancement of the ribozyme's ability to bind its own substrate in a way that protects it from cleavage by the DNA enzyme. CONCLUSIONS: The ability to direct the evolution of an enzyme's biochemical properties in response to the behavior of another macromolecule provides insight into the evolution of resistance and may be useful in developing enzymes with novel or enhanced function.

Non-NASA Center↗
NASA NTRS1999

Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region

Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.

NASA Discipline Regulatory Physiology↗
NASA NTRS1999

Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

Non-NASA Center↗
NASA NTRS1999

Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1-34) (1 h/day at 8 microg/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5-fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle-treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH-induced factor. We hypothesize that this factor is insulin-like growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF-I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF-I.

NASA Discipline Musculoskeletal↗
NASA NTRS1997

Neonatal rat heart cells cultured in simulated microgravity

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

Non-NASA Center↗
NASA NTRS2007

Cooperative Effects of Corticosteroids and Catecholamines upon Immune Deviation of the Type-1/Type-2 Cytokine Balance in Favor of Type-2 Expression in Human Peripheral Blood Mononuclear Cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-gamma) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS+CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

Salicru, A. N.↗
NASA NTRS2005

Evaluation of Late Effects of Heavy-Ion Radiation on Mesenchymal Stem Cells

The overall objective of this recently funded study is to utilize well-characterized model test systems to assess the impact of pluripotent stem cell differentiation on biological effects associated with high-energy charged particle radiation. These stem cells, specifically mesenchymal stem cells (MSCs), have the potential for differentiation into bone, cartilage, fat, tendons, and other tissue types. The characterization of the regulation mechanisms of MSC differentiation to the osteoblastic lineage by transcription factors, such as Runx2/Cbfa1 and Osterix, and osteoinductive proteins such as members of the bone morphogenic protein family are well established. More importantly, for late biological effects, MSCs have been shown to contribute to tissue restructuring and repair after tissue injury. The complex regulation of and interactions between inflammation and repair determine the eventual outcome of the responses to tissue injury, for which MSCs play a crucial role. Additionally, MSCs have been shown to respond to reactive oxygen species, a secondary effector of radiation, by differentiating. With this, we hypothesized that differentiation of MSCs can alter or exacerbate the damage initiated by radiation, which can ultimately lead to late biological effects of misrepair/fibrosis which may ultimately lead to carcinogenesis. Currently, studies are underway to examine high-energy X-ray radiation at low and high doses, approximately 20 and 200 Rad, respectively, on cytogenetic damage and gene modulation of isolated MSCs. These cells, positive for MSC surface markers, were obtained from three persons. In vitro cell samples were harvested during cellular proliferation and after both cellular recovery and differentiation. Future work will use established in vitro models of increasing complexity to examine the value of traditional 2D tissue-culture techniques, and utilize 3D in vitro tissue culture techniques that can better assess late effects associated with radiation.

Gonda, S.R.↗
NASA NTRS2010

Simulated Microgravity Induces SOST/Sclerostin Upregulation in Osteocytes

Osteocytes are theorized to be the mechanosensors and transducers of mechanical forces in bone, yet the biological mechanism of this action remains elusive. Recent evidence suggests that SOST/Sclerostin is an important regulator of mechano-transduction. To investigate the molecular mechanisms of SOST/Sclerostin regulation under in-vitro and ex-vivo unloading we used the NASA Rotating Wall Vessel(RWV) Bioreactor. For in-vitro experiments, MLOY-4 osteocytic cells were seeded at a concentration of 250,000 cells onto 3D collagen scaffold (BD). Scaffolds (4 per condition) were either rotated in a vertical 50ml NASA/bioreactor vessel at 18 rpm (unloaded), cultured in a horizontal 50 ml NASA bioreactor vessel at 18 rpm (control for the sheared environment of vertical rotating vessel), or cultured in a static T-75 cm dish (static condition ) for 7days. For ex-vivo experiments, calvaria bones were harvested from 12-week old C57/Bl6 mice and sequentially digested with type I/II collagenase to remove periosteal osteoblasts. Calvaria halves (10 per condition) were then exposed to the same set of culture conditions described above. Simulated unloading, as achieved in the NASA RWV, resulted in enlarged, round osteocytes, as assessed by H&E staining, that was reminiscent of prior reports of unloading causing loss of osteocyte morphology and dendritic network connectivity. Semiquantitative realtime qPCR and immunohistochemistry from both in-vitro and ex-vivo RWV experiments demonstrated a four-fold up-regulation of SOST/Sclerostin. Furthermore, mRNA of the transcriptional SOST enhancer Mef2C was upregulated 1.4 fold in ex-vivo calvaria subjected to unloading conditions of the NASA RWV, suggesting that Mef2C might be an important regulator of mechano-sensation. These findings are consistent with results from seven day hindlimb unloading experiments, C57/B6 females, conducted in our laboratory and validate the use of the NASA RWV as a tool to study osteocyte mechanotransduction

Spatz, Jordan↗
NASA NTRS2012

Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

Respiratory syncytial virus and parainfluenza virus cause severe respiratory disease, especially in infants, children and the elderly. An in vitro model that accurately mimics infection of the human respiratory epithelium (HRE) would facilitate vaccine development greatly. Monolayer cultures traditionally used to study these viruses do not accurately and precisely differentiate the replication efficiencies of wild type and attenuated viruses. Therefore, we engineered novel three-dimensional (3D) tissue-like assemblies (TLAs) of human broncho-epithelial (HBE) cells to produce a more physiologically relevant in vitro model of the HRE. TLAs resemble HRE structurally and by expression of differentiated epithelial cell markers. Most significantly, wild type viruses exhibited a clear growth advantage over attenuated strains in TLAs unlike monolayer cultures. In addition, the TLAs responded to virus infection by secreting pro-inflammatory mediators similar to the respiratory epithelia of infected children. These characteristics make the TLA model a valuable platform technology to develop and evaluate live, attenuated respiratory virus vaccine candidates for human use. Respiratory virus diseases, the most frequent and least preventable of all infectious diseases, range in severity from the common cold to severe bronchiolitis and pneumonia . Two paramyxoviruses, respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3), are responsible for a majority of the most severe respiratory diseases of infants and young children. RSV causes 70% of all bronchiolitis cases and is a major cause of morbidity and mortality worldwide, especially in infants. PIV3 causes 10-15% of bronchiolitis and pneumonia during infancy, second only to RSV, and 40% of croup in infants To date, licensed vaccines are not available to prevent these respiratory diseases. At present, traditional monkey kidney (Vero and LLC-MK2) and human (HEp-2) tissue culture cells and small animal models (mouse, cotton rat, guinea pig, ferret, and hamster) fail to accurately imitate viral replication and human disease states (8). Lacking an authentic model has impeded the development and evaluation of live, attenuated vaccine candidates. Development of a physiologically relevant in vitro tissue culture model that reproduces characteristics of the HRE, the primary target of RSV and PIV3, would aid in predicting clinical attenuation and safety of vaccine candidates. Successful tissue engineering of a 3D human intestinal model using novel NASA technology inspired the development of a tri-culture 3D model for the HRE. Sequential layering of primary mesenchymal cells (comprised of normal human fibroblasts and endothelial cells) followed by BEAS-2B epithelial cells derived from human bronchi and tracheae were recapitulated on Cultisphere and/or cytodex3 microcarriers in cylindrical vessels that rotate horizontally creating an organized epithelial structure. Horizontal rotation randomizes the gravity vector modeling aspects of microgravity. Mesenchymal and epithelial cells grown under these conditions reproduce the structural organization, multi-cellular complexity, and differentiation state of the HRE. The opportunity to study respiratory viruses in a nasal epithelium model is invaluable because the most promising respiratory virus vaccine candidates are live attenuated viruses for intranasal administration. Here we characterize the interactions of respiratory viruses and epithelial cells grown under modeled microgravity in comparison to gravity-ladened monolayers. 3D HBE TLAs and traditional monolayers (2D) are infected at 35 C, the upper temperature of the upper HRE, to simulate in vivo infection conditions. Growth kinetics of wild type (wt) RSV and PIV3 viruses were compared in 2D and 3D cells to that of strains attenuated in humans or rhesus macaques. This novel 3D HBE model also offers an opportunity to study whether the epithelial cell function, especially in host defenses recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity mediators. Therefore, 3D HBE TLAs offer advantages for the study of respiratory viruses and the development of viral vaccine candidates.

Deatly, Anne M.↗
NASA NTRS2012

Alterations of Cellular Immune Reactions in Crew Members Overwintering in the Antarctic Research Station Concordia

Background: Concordia Station is located inside Antarctica about 1000km from the coast at an altitude of 3200m (Dome C). Hence, individuals living in this harsh environment are exposed to two major conditions: 1.) hypobaric hypoxia and 2.) confinement and extreme isolation. Both hypoxia and confinement can affect human immunity and health, and are likely to be present during exploration class space missions. This study focused on immune alterations measured by a new global immunity test assay, similar to the phased out delayed type hypersensitivity (DTH) skin test. Methods: After informed written consent 14 healthy male subjects were included to the CHOICE-study (Consequences-of-longterm-Confinement-and-Hypobaric-HypOxia-on-Immunity-in-the Antarctic-Concordia-Environment). Data collection occurred during two winter-over periods lasting each one year. During the first campaign 6 healthy male were enrolled followed by a second campaign with 8 healthy males. Blood was drawn monthly and incubated for 48h with various bacterial, viral and fungal antigens followed by an analysis of plasma cytokine levels (TNF-alpha, IL2, IFN-gamma, IL10). As a control, blood was incubated without stimulation ("resting condition"). Goals: The scope of this study was to assess the consequences of hypoxia and confinement on cellular immunity as assessed by a new in vitro DTH-like test. Results: Initial results indicate that under resting conditions the in vitro DTH-like test showed low cytokine levels which remained almost unchanged during the entire observation period. However, cytokine responses to viral, bacterial and fungal antigens were remarkably reduced at the first month after arrival at Concordia when compared to levels measured in Europe prior to departure for Antarctica. With incrementing months of confinement this depressed DTH-like response tended to reverse, and in fact to show an "overshooting" immune reaction after stimulation. Conclusion: The reduced in vitro DTH-like test response in the early phase of Antarctic wintering over con rms distinct immune suppressive effects seen after (sub-)acute hypobaric hypoxia. The reversal and overshooting reaction of cellular immune responses upon stimulation, but not the resting state, indicate either a) priming of immune answers and/or b) an uncoupled or disregulated control of cellular immune answers by auto-, para- and endocrine pathways. Further analyses and correlations are warranted. Acknowledgement: Supported by the European Space Agency (ESA), the French (IPEV) and Italian (PNRA) polar institutes, the German National Space Program (DLR, 50WB0719/WB0919), by BELSPO/PROEDEX/ESA (C90-380/-391), NASA and by the Concordia crews who have participated with great enthusiasm.

Crucian, Brian↗
NASA NTRS2020

Circulating miRNA Signature Predicts Health Risks Associated with Cancer and Spaceflight

Biological risks associated with space radiation and microgravity are major concerns for long-term space travel. Through a Systems Biology approach, our previous NASA work has shown both TGF signaling pathways and miRNAs have a critical impact on defining health risks with and without space irradiation. We hypothesize that circulating microRNA (miRNA) signatures are driving microvascular disease and muscle degeneration associated with accelerating aging and will be enhanced by exposure to the space environment (radiation and microgravity). We are investigating this hypothesis with both in vivo and in vitro models to test novel antagonist therapies to these miRNA signatures as countermeasures to reduce space radiation-induced health risks. A comprehensive Systems Biology approach is utilized to examine the influence by high atomic number by high (H) atomic number (Z) and energy (E) (HZE) irradiation. To simulate low-dose exposure due to galactic cosmic rays (GCR), we used ions, energy, and doses determined by a NASA consensus formula of 7 different ions to represent GCR (referred to as GCR sim model). To similate high-dose radiation exposure due to solar particle events (SPE), we used a solar particle event (SPE) sim model which gave a total dose of 1Gy protons with energy ranges from 50MeV to 150MeV. C57BL/6 wild-type female mice were utilized for the irradiations with our established simulated microgravity model (hindlimb suspension model) and an in vitro 3D microvasculature tissue model under simulated microgravity (clinostat) conditions was also irradiated. To expand on the circulating miRNA signature determined from our preliminary data, we determined a group of conserved miRNAs which are commonly being regulated in the majority of the organs and tissues throughout the host using our established techniques. MiRNA-sequencing was done on serum (at time of sacrifice), liver, heart, and muscle (soleus muscle) tissue for all radiation groups. Additional validation of the key miRNAs was performed by droplet digital PCR (ddPCR). This revealed a key circulating miRNA signature (consisting of multiple miRNAs) impacting cardiovascular and muscular disease risk. Further in vitro experiments with CRISPR/Cas9 system to knockout the key miRNA signatures, novel self-delivering antagomirs, overexpression of the miRNAs test the functional impact of the miRNA signatures on both microvascular disease and muscle degeneration due to space irradiation. The current work has started to allow the possible development of a novel minimally invasive miRNA based radioprotector to be used as a countermeasure for space radiation. Collectively, understanding of how whole body space radiation impacts microvascular and tissue degeneration through circulating miRNAs will greatly enhance health risk prognostication and provide possible new mechanisms for protection against space radiation. This work is supported by the Translational Research Institute through NASA Cooperative Agreement NNX16AO69A (T-0404) awarded to AB.

Beheshti, Afshin↗
NASA NTRSDate not supplied

Single-Cell mRNA Sequencing of Bone Marrow Mesenchymal Stem Cell Lineages Shows that Gravity Mechanical Loading Regulates Regenerative Osteogenesis in a CDKN1A-Dependent Manner

Weight-bearing at 1g is increasingly recognized as an important stimulus driving stem cell-based tissue regenerative homeostasis and health. In the bone marrow compartment, mechanical stimuli from gravity can drive osteogenesis from mesenchymal progenitors and hematopoiesis from hematopoietic progenitors. Conversely, hematopoietic monocyte/macrophage progenitors can also be driven into osteoclastogenesis by mechanical unloading in microgravity. Although the cell cycle inhibitor gene Cdkn1a is known to be upregulated in microgravity, the great diversity of stem cell lineages and stages of differentiation in bone marrow, make it difficult to attribute gene expression changes to specific cell types. To overcome this limitation, we used single-cell mRNAseq analyses with both in-vitro and in-vivo strategies, including in-vitro stretch of marrow osteogenic cultures, and in-vivo hindlimb unloading, running wheels, treadmills, and exposure to real microgravity. For in-vitro studies marrow osteoprogenitors from WT and Cdkn1a-null mice were subjected to substrate stretch during osteogenesis, with static cultures simulating unloading. From those experiments we determined Cdkn1a-null cells transition more readily from progenitors to early osteoblasts and further to mineralizing osteoblasts. Additionally, single-cell mRNAseq resolution identified Cdkn1a suppression in response to mechanical loading predominantly in early proliferative osteoblasts rather than progenitor or mineralizing osteoblasts. These findings support the hypothesis that CDKN1A plays a mechano-reversible, anti-proliferative role during bone regenerative osteogenesis. To investigate in-vivo mechanoregulation of osteogenesis, mice were flown in microgravity during the Rodent Research-10 experiment comparing wildtype and Cdkn1a-null mice kept on ISS for 30 days. The mice were euthanized and dissected on-orbit in microgravity and femur bone marrow was collected using a custom sample isolation and live-cell cryo-preservation technique allowing for single cell transcriptomics post-flight processing. Ground control experiments with hindlimb-unloading, running wheels, and treadmills, were also conducted for a comprehensive analysis of mechanical loading effects on the regenerative health of bone tissue and its marrow stem cell progenitor lineages.

single cell sequencing↗
NASA NTRS2002

Regulation of period 1 expression in cultured rat pineal

The aim of the present study was to investigate the in vitro expression of Period 1 (Per1), Period 2 (Per2) and arylalkylamine N-acetyltransferase (AA-NAT) genes in the rat pineal gland to understand the mechanism(s) regulating the expression of these genes in this organ. Pineals, when maintained in vitro for 5 days, did not show circadian rhythmicity in the expression of any of the three genes monitored. Norepinephrine (NE) induced AA-NAT and Per1, whereas its effect on Per2 was negligible. Contrary to what was observed in other systems, NE stimulation did not induce circadian expression of Per1. The effect of NE on Per1 level was dose- and receptor subtype-dependent, and both cAMP and cGMP induced Per1. Per1 was not induced by repeated NE - or forskolin - stimulation. Protein synthesis was not necessary for NE-induced Per1, but it was for reduction of Per1 following NE stimulation. Per1 transcription in pinealocytes was activated by BMAL1/CLOCK. Our results indicate that important differences are present in the regulation of these genes in the mammalian pineal. Copyright 2002 S. Karger AG, Basel.

NASA Discipline Space Human Factors↗
NASA NTRS1987

Thermal dependence of neural activity in the hamster hippocampal slice preparation

1. Neural activity was recorded in an in vitro hamster hippocampal slice preparation while the temperature of the Ringer's solution bathing in the slice was controlled at selected levels. 2. The amplitude of the population spike (action potentials from a group of pyramidal cells) was measured as bath temperature was lowered from 35 degrees C to temperatures where a response could not be evoked. 3. Plots of population spike amplitude versus temperature have bell-shaped curves. The population spikes increased in amplitude as temperature was lowered from 35 degrees C, reached a peak amplitude between 25 and 20 degrees C, and then decreased until a response could not be evoked when temperature was further lowered. 4. These in vitro results obtained in the slice preparation are related to in vivo hippocampal studies. Results are interpreted as consistent with the proposal reviewed here that neural activity in the hippocampus plays a role at specific stages of entrance into and arousal from hibernation.

Non-NASA Center↗
NASA NTRS2000

Effects of 2-deoxy-D-glucose administration on cytokine production in BDF1 mice

Physical exercise and diet changes have been shown to affect immune parameters, and similar effects are also induced by the administration of a nonmetabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The present study was designed to characterize the effects of glucoprivation induced by 2-DG administration on concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the blood and interferon-gamma (IFN-gamma), IL-2, and IL-4 in vitro production by partially purified T splenocytes in BDF1 mice. Mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, or 1000 mg/kg of 2-DG, and blood and spleens were collected 2 h after the last injection. Partially purified T splenocytes were cultured 24 h in the presence of concanavalin A (ConA). A significant increase in the corticosterone levels with the amount of 2-DG injected was observed after one or three injections (p<0.05). The amount of 2-DG injected was associated with an increase in TNF-alpha, IL-1beta, and IL-6 concentrations in the blood of mice after one or three injections of 2-DG (p<0.05). A significant decrease in in vitro proliferation of partially purified splenocytes in the presence of ConA was associated with a decrease in IFN-gamma production in the culture supernatants and an increase in IL-1 receptor expression on the cell surface (p<0.05).

NASA Discipline Regulatory Physiology↗
NASA NTRS1997

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica

To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

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