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All sourcesNASA NTRSDOE OSTI
NASA NTRS2017

Modulation of Bone Marrow Primary Cell Osteoblastogenesis and Cell Senescence by Mechanical Stimulation

Cell and animal studies conducted onboard the International Space Station and formerly the Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. Specifically CDKN1A/p-21 a cell senescence protein, was found to be upregulated in osteoprecursor cells of the femur during 15-day spaceflight, leading to the working hypothesis that CDKN1A/p-21 plays a role in inhibition of bone formation via mechanical regulation. To evaluate this hypothesis, utilizing a p-21 knockout mouse-line and relevant wildtype control, we cultured femoral bone marrow primary cells under unloaded (static) and cyclically stretched loading through a 30 day osteoblastogenesis protocol. Morphologic evaluation of the cultures demonstrated that mechanical stretching aligned the cells and increased the presence of defined focal adhesion expressing talin, integrin αvβ3, and PTK2 protein tyrosine kinase 2, also known as focal adhesion kinase (FAK) in both mouse strains. In corroboration with previous investigations of cell survival signals relation to FAK, our study found that with greater concentration of focal adhesions via stretch stimulation the live cell percentage was significantly higher than the unloaded controls (p-21 knockout line: +49.70%, p*=0.009, wildtype control: +18.14%, p*=.01). Also evaluated was the mineralization and ECM secretion capability of the differentiating cells. Von Kossa staining has shown that in the p-21 knockout cells unloaded cells produce more matrix that the stretch stimulated, however the matrix is unorganized presenting in sporadic nodules covering approximately 30% of the culture area at day 14 (n=6 wells) while the stretch stimulated cultures have less mineralization content the surface area containing mineralized matrix is greater (~68% at day 14). Q-PCR evaluation of the p-21 knockout cells revealed that canonical (β-catenin cascade) and non-canonical wnt11 and downstream planar cell polarity (wnt/PCP) pathway molecule RAC1 are prevalently upregulated with mechanical stimulation. Immunofluorescence for β-catenin and RAC1 showed co-localization at the nuclear membrane of the p-21 knockout cells but not the wildtype (n=1) suggesting that molecular communication via the canonical and wnt/PCP pathway are initiated by mechanical loading and experience regulation along the signaling cascade by CDKN1A/p-21. Future investigations will further elucidate this relationship and provide causal data demonstrating mechanical loading’s modulatory effect on p-21 expression change.

Juran, Cassandra M.↗
NASA NTRS2004

Software Assurance of PLCs Training Course

Being heavily visually-oriented, I am a firm believer in communication and conveying emotions through the art of color, motion, and transformation. A four-part online training course was created in PowerPoint and needed to be translated over into a Flash format. Issues with the Powerpoint were that the size of the files caused noticeable delays when placed online, there were compatibility issues, and from a composition and design perspective, color schemes and layout left much to be desired. High contrast, pixilated yellow text spiraling and flying on to a background of overly rich hues of blue with cheesy gradient patterns was just not appeasing to my eye, along with the menu directory buttons located at the top resembling blue pills of NyQuil on top of a stale gray border that had nothing to do with the background. The course itself is extremely broad and verbose, and will get monotonous very soon after starting. Moving about the course was very cumbersome as well. efficient by drastically reducing the size (The file size of all four parts of the actual course combined will ultimately not even be a fifth as big as one part of the original PowerPoint alone!); along with that, the course was made to be more interactive and user-friendly, as well as pleasing to the eye. Upon being viewed by fellow co-workers, nothing but positive feedback has been received. When beginning the presentation, onscreen comes a 3' 2" chubby, balding professor, who is a master in his knowledge of Programmable Logic Controllers (PLCs). He introduces himself and presents all of his vast knowledge over PLCs in a fun and innovative manner, making it much easier to acquire the information presented. A Scene Selection feature has been added making it a lot easier to jump from part to part and the back and forth arrows are much easier to utilize, and they are both less obtrusive than its PowerPoint predecessor. The user can also go at their pace, as the presentation pauses after at the end of each statement. computer animation, it was.. .still. ..animation done on a computer. I was able to incorporate my artistic talent and intuitive creativity into it, one thing I am very proficient at doing when it comes to what I do and what I will do in my profession as an artist/computer animator. At first, I felt that there was no place for an artist within a faculty of scientists, engineers, chemists, mathematicians, and programmers, but I managed to fit in quite successfully. My task was to convert the course into a Flash format, which would make it much more My project surprisingly somewhat dealt with my field of interest-though it was not

Threatt, Jamarr↗
NASA NTRS2000

Changes in ribbon synapses and rough endoplasmic reticulum of rat utricular macular hair cells in weightlessness

This study combined ultrastructural and statistical methods to learn the effects of weightlessness on rat utricular maculae. A principle aim was to determine whether weightlessness chiefly affects ribbon synapses of type II cells, since the cells communicate predominantly with branches of primary vestibular afferent endings. Maculae were microdissected from flight and ground control rat inner ears collected on day 13 of a 14-day spaceflight (F13), landing day (R0) and day 14 postflight (R14) and were prepared for ultrastructural study. Ribbon synapses were counted in hair cells examined in a Zeiss 902 transmission electron microscope. Significance of synaptic mean differences was determined for all hair cells contained within 100 section series, and for a subset of complete hair cells, using SuperANOVA software. The synaptic mean for all type II hair cells of F13 flight rats increased by 100%, and that for complete cells by 200%. Type I cells were less affected, with synaptic mean differences statistically insignificant in complete cells. Synapse deletion began within 8 h upon return to Earth. Additionally, hair cell laminated rough endoplasmic reticulum of flight rats was reversibly disorganized on R0. Results support the thesis that synapses in type II hair cells are uniquely affected by altered gravity. Type II hair cells may be chiefly sensors of gravitational and type I cells of translational linear accelerations.

short duration↗
NASA NTRS2001

Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

NASA Discipline Neuroscience↗
NASA NTRS2017

Operating Small Sat Swarms as a Single Entity: Introducing SODA

Swarm concepts are a growing topic of interest in the small satellite community. Compared to a small satellite constellation, a swarm has the distinction of being multiple spacecraft in close proximity, in approximately the same orbit. Furthermore, we envision swarms to have capabilities for cross-link communication and station-keeping. Of particular interest is a means to maintain operator-specified geometry, alignment, and/or separation.From NASA's decadal survey, it is clear that simultaneous measurements from a 3D volume of space are desired for a variety of Earth scientific studies. As this mission concept is ultimately extended to deep space, some degree of local control for the swarm to self-correct its configuration is required. We claim that the practicality of ground commanding each individual satellite in the swarm is simply not a feasible concept of operations. In other words, the current state-of-practice does not scale to very large swarms (e.g. 100 spacecraft or more) without becoming cost prohibitive. To contain the operations costs and complexity, a new approach is required: the swarm must be operated as a unit, responding to high-level specifications for relative position and velocity.The Mission Design Division at NASA Ames Research Center is looking to the near future for opportunities to develop satellite swarm technology. As part of this effort, we are developing SODA (Swarm Orbital Dynamics Advisor), a tool that provides the orbital maneuvers required to achieve a desired type of relative swarm motion. The purpose of SODA is two-fold. First, it encompasses the algorithms and orbital dynamics model to enable the desired relative motion of the swarm satellites. The process starts with the user specifying the properties of a swarm configuration. This could be as simple as varying in-track spacing of the swarm in one orbit, or as complex as maintaining a specified 3D geometrical orientation. We presume that science objectives will drive this choice. Given these inputs, the tool provides the most efficient maneuver(s) to achieve the objective.Second, SODA provides a variety of visualization tools. We acknowledge that the relationship between a desired relative motion amongst the swarm, and the corresponding orbital parameters for each individual satellite may not be immediately apparent for ground controllers and mission planners. The purpose of SODA's visualization tools is to illustrate this concept clearly with a variety of graphics and animations. After computing the optimal orbital maneuvers to modify the swarm, these results are simulated to demonstrate successful swarm control.Our emphasis in this paper is on the importance of relating the desired motion of the swarm satellites relative to one another with the required orbital element changes. One cannot joystick a drifting swarm satellite back into position; the underlying orbital mechanics dictate the most efficient recovery maneuvers. To illustrate this point, results from several case study simulations are presented. We conclude with our forward work for ongoing SODA development and potential science applications.

small satellites↗
NASA NTRS2002

Neural substrates linking balance control and anxiety

This communication provides an update of our understanding of the neurological bases for the close association between balance control and anxiety. New data suggest that a vestibulo-recipient region of the parabrachial nucleus (PBN) contains cells that respond to body rotation and position relative to gravity. The PBN, with its reciprocal relationships with the extended central amygdaloid nucleus, infralimbic cortex, and hypothalamus, appears to be an important node in a primary network that processes convergent vestibular, somatic, and visceral information processing to mediate avoidance conditioning, anxiety, and conditioned fear responses. Noradrenergic and serotonergic projections to the vestibular nuclei also have parallel connections with anxiety pathways. The coeruleo-vestibular pathway originates in caudal locus coeruleus (LC) and provides regionally specialized noradrenergic input to the vestibular nuclei, which likely mediate effects of alerting and vigilance on the sensitivity of vestibulo-motor circuits. Both serotonergic and nonserotonergic pathways from the dorsal raphe nucleus and the nucleus raphe obscurus also project differentially to the vestibular nuclei, and 5-HT(2A) receptors are expressed in amygdaloid and cortical targets of the PBN. It is proposed that the dorsal raphe nucleus pathway contributes to both (a) a tradeoff between motor and sensory (information gathering) aspects of responses to self-motion and (b) a calibration of the sensitivity of affective responses to aversive aspects of motion. This updated neurologic model continues to be a synthetic schema for investigating the neurological and neurochemical bases for comorbidity of balance disorders and anxiety disorders.

Review↗
NASA NTRS1997

Activation and proliferation of lymphocytes and other mammalian cells in microgravity

The experimental findings reviewed in this chapter support the following conclusions: Proliferation. Human T-lymphocytes, associated with monocytes as accessory cells, show dramatic changes in the centrifuge, in the clinostat and in space. In free-floating cells the mitogenic response is depressed by 90% in microgravity, whereas in cells attached to a substratum activation is enhanced by 100% compared to 1-G ground and inflight controls. The duration of phase G1 of the mitotic cycle of HeLa cells is reduced in hypergravity, resulting in an increased proliferation rate. Other systems like Friend cells and WI38 human embryonic lung cells do not show significant changes. Genetic expression and signal transduction. T-lymphocytes and monocytes show important changes in the expression of cytokines like interleukin-1, interleukin-2, interferon-gamma and tumor necrosis factor. The data from space experiments in Spacelab, Space Shuttle mid-deck, and Biokosmos have helped to clarify certain aspects of the mechanism of T-cell activation. Epidermoid A431 cells show changes in the genetic expression of the proto-oncogenes c-fos and c-jun in the clinostat and in sounding rockets. Membrane function, in particular the binding of ligates as first messengers of a signal, is not changed in most of the cell systems in microgravity. Morphology and Mortility. Free cells, lymphocytes in particular, are able to move and form aggregates in microgravity, indicating that cell-cell contacts and cell communications do take place in microgravity. Dramatic morphological and ultrastructural changes are not detected in cells cultured in microgravity. Important experiments with single mammalian cells, including immune cells, were carried out recently in three Spacelab flights, (SL-J, D-2, and IML-2 in 1992, 1993, and 1994, respectively). The results of the D-2 mission have been published in ref. 75; those of the IML-2 mission in ref. 76. Finally, many cell biology experiments in space have suffered in the past from a lack of adequate controls (like 1-G centrifuges) and of proper experimental conditions (like well-controlled temperature). In this respect the availability of Biorack, outfitted with proper incubators with 1-G control centrifuge as well as a glovebox with a microscope, is a great advantage. It is also desirable that cell biology experiments in space are accompanied or even preceded by a program of ground-based investigations in the fast rotating clinostat and in the centrifuge, and that preparatory experiments be done in parabolic flights and sounding rockets, whenever possible. Proper publication of the results of space experiments is another important need. A great number of data have been published in proceedings and reports that are not available to the broad scientific community. To guarantee the credibility and the international recognition of space biology it is important that the results be published in international, peer reviewed journals.

manned↗
NASA NTRS2001

Extending unbiased stereology of brain ultrastructure to three-dimensional volumes

OBJECTIVE: Analysis of brain ultrastructure is needed to reveal how neurons communicate with one another via synapses and how disease processes alter this communication. In the past, such analyses have usually been based on single or paired sections obtained by electron microscopy. Reconstruction from multiple serial sections provides a much needed, richer representation of the three-dimensional organization of the brain. This paper introduces a new reconstruction system and new methods for analyzing in three dimensions the location and ultrastructure of neuronal components, such as synapses, which are distributed non-randomly throughout the brain. DESIGN AND MEASUREMENTS: Volumes are reconstructed by defining transformations that align the entire area of adjacent sections. Whole-field alignment requires rotation, translation, skew, scaling, and second-order nonlinear deformations. Such transformations are implemented by a linear combination of bivariate polynomials. Computer software for generating transformations based on user input is described. Stereological techniques for assessing structural distributions in reconstructed volumes are the unbiased bricking, disector, unbiased ratio, and per-length counting techniques. A new general method, the fractional counter, is also described. This unbiased technique relies on the counting of fractions of objects contained in a test volume. A volume of brain tissue from stratum radiatum of hippocampal area CA1 is reconstructed and analyzed for synaptic density to demonstrate and compare the techniques. RESULTS AND CONCLUSIONS: Reconstruction makes practicable volume-oriented analysis of ultrastructure using such techniques as the unbiased bricking and fractional counter methods. These analysis methods are less sensitive to the section-to-section variations in counts and section thickness, factors that contribute to the inaccuracy of other stereological methods. In addition, volume reconstruction facilitates visualization and modeling of structures and analysis of three-dimensional relationships such as synaptic connectivity.

NASA Discipline Neuroscience↗
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.↗