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Study of Fluid Flow Control in Protein Crystallization using Strong Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in "microgravity", researchers have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. Whether this limited convection in a magnetic field will provide the environment for the growth of high quality crystals is still a matter of conjecture that our research will address. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. Once the basic understanding is obtained, the study will focus on testing the hypothesis on proteins of pyruvate dehydrogenase complex (PDC), proteins E1 and E3. Obtaining high crystal quality of these proteins is of great importance to structural biologists since their structures need to be determined. Specific goals for the investigation are: 1. To develop an understanding of convection control in diamagnetic fluids with concentration gradients through experimentation and numerical modeling. Specifically solutal buoyancy driven convection due to crystal growth will be considered. 2. To develop predictive measures for successful crystallization in a magnetic field using analyses and numerical modeling for use in future protein crystal growth experiments. This will establish criteria that can be used to estimate the efficacy of magnetic field flow damping on crystallization of candidate proteins. 3. To demonstrate the understanding of convection damping by high magnetic fields to a class of proteins that is of interest and whose structure is as yet not determined. 4. To compare quantitatively, the quality of the grown crystals with and without a magnetic field. X-ray diffraction techniques will be used for the comparative studies. In a preliminary set of experiments, we studied crystal dissolution effects in a 5 Tesla magnet available at NASA Marshall Space Flight Center (MSFC). Using a Schlieren setup, a 1mm crystal of Alum (Aluminum-Potassium Sulfate) was introduced in a 75% saturated solution and the resulting dissolution plume was observed. The experiment was conducted both in the presence and absence of a magnetic field gradient. The magnet produces a gradient field of approx. 1 Tesla2/cm. Image analysis of the recorded images indicated an enhanced plume velocity that was of the order of the measurement limit. For this experiment, both the gradient and gravity fields are in the same direction resulting in an enhanced effective gravity that tends to accelerate the observed plume velocity. While the results are not conclusive, pending further tests, it clearly points out the inadequacy of the MSFC magnet for conducting protein crystallization experiments and the need for a stronger magnet. In spacebased experiments, however, where the gravitational effects are small, only a weak magnetic field will be required to control or mitigate the effects of convective contamination.

Ramachandran, Narayanan↗

Countering Solutal Buoyant Convection with High Magnetic Fields

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemist, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitant, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity, we have been able to dramatically effect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the current status of the investigation and discuss results from the experimental and modeling efforts.

Ramachandran, N.↗

Using Strong Magnetic Fields to Control Solutal Convection

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular crystals for diffraction analyses has been the central focus for biochemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of the container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and sedimentation, as is achieved in microgravity , we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, formation of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with concentration for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteracts terrestrial gravity. The general objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagnetic salts and solutions in magnetic fields and compare them to analytical predictions.

Ramachandran, N.↗

Deviations from uniform power law scaling in nonstationary time series

A classic problem in physics is the analysis of highly nonstationary time series that typically exhibit long-range correlations. Here we test the hypothesis that the scaling properties of the dynamics of healthy physiological systems are more stable than those of pathological systems by studying beat-to-beat fluctuations in the human heart rate. We develop techniques based on the Fano factor and Allan factor functions, as well as on detrended fluctuation analysis, for quantifying deviations from uniform power-law scaling in nonstationary time series. By analyzing extremely long data sets of up to N = 10(5) beats for 11 healthy subjects, we find that the fluctuations in the heart rate scale approximately uniformly over several temporal orders of magnitude. By contrast, we find that in data sets of comparable length for 14 subjects with heart disease, the fluctuations grow erratically, indicating a loss of scaling stability.

Non-NASA Center↗

Magnetic Control of Convection during Protein Crystallization

An important component in biotechnology, particularly in the area of protein engineering and rational drug design is the knowledge of the precise three-dimensional molecular structure of proteins. The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the protein crystals. As a consequence, the growth of high quality macromolecular Crystals for diffraction analyses has been the central focus for bio-chemists, biologists, and bioengineers. Macromolecular crystals are obtained from solutions that contain the crystallizing species in equilibrium with higher aggregates, ions, precipitants, other possible phases of the protein, foreign particles, the walls of container, and a likely host of other impurities. By changing transport modes in general, i.e., reduction of convection and Sedimentation as is achieved in "microgravity", we have been able to dramatically affect the movement and distribution of macromolecules in the fluid, and thus their transport, f o d o n of crystal nuclei, and adsorption to the crystal surface. While a limited number of high quality crystals from space flights have been obtained, as the recent National Research Council (NRC) review of the NASA microgravity crystallization program pointed out, the scientific approach and research in crystallization of proteins has been mainly empirical yielding inconclusive results. We postulate that we can reduce convection in ground-based experiments and we can understand the different aspects of convection control through the use of strong magnetic fields and field gradients. We postulate that limited convection in a magnetic field will provide the environment for the growth of high quality crystals. The approach exploits the variation of fluid magnetic susceptibility with counteracts on for this purpose and the convective damping is realized by appropriately positioning the crystal growth cell so that the magnetic susceptibility force counteract terrestrial gravity. The genera1 objective is to test the hypothesis of convective control using a strong magnetic field and magnetic field gradient and to understand the nature of the various forces that come into play. Specifically we aim to delineate causative factors and to quantify them through experiments, analysis and numerical modeling. The paper will report on the experimental results using paramagentic salts and solutions in magnetic fields and compare them to analyticalpr~ctions.

Ramachandran, N.↗

Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

Non-NASA Center↗

Lessons Learned from Seven Space Shuttle Missions

Much can be learned from well-written descriptions of the technical and organizational factors that lead to an accident. Subsequent analysis by third parties of investigation reports and associated evidence collected during the investigations can lead to additional insight. Much can also be learned from documented close calls that do not result in loss of life or a spacecraft, such as the Mars Exploration Rover Spirit software anomaly, the SOHO mission interruption, and the NEAR burn anomaly. Seven space shuttle incidents fall into the latter category: Rendezvous Target Failure On STS-41B; Rendezvous Radar Anomaly and Trajectory Dispersion-STS-32 ;Rendezvous Lambert Targeting Anomaly-STS-49; Rendezvous Lambert Targeting Anomaly-STS-51; Zero Doppler Steering Maneuver Anomaly-STS-59; Excessive Propellant Consumption During Rendezvous-STS-69; Global Positioning System Receiver and Associated Shuttle Flight Software Anomalies-STS-91 Procedural work-arounds or software changes prevented them from threatening mission success. Extensive investigations, which included the independent recreation of the anomalies by multiple Shuttle Program organizations, were the key to determining the cause, accurately assessing risk, and identifying software and software process improvements. Lessons learned from these incidents not only validated long-standing operational best practices, but serve to promote discussion and mentoring among Program personnel and are applicable to future space flight programs.

Goodman, John↗

Low-dimensional Representation of Error Covariance

Ensemble and reduced-rank approaches to prediction and assimilation rely on low-dimensional approximations of the estimation error covariances. Here stability properties of the forecast/analysis cycle for linear, time-independent systems are used to identify factors that cause the steady-state analysis error covariance to admit a low-dimensional representation. A useful measure of forecast/analysis cycle stability is the bound matrix, a function of the dynamics, observation operator and assimilation method. Upper and lower estimates for the steady-state analysis error covariance matrix eigenvalues are derived from the bound matrix. The estimates generalize to time-dependent systems. If much of the steady-state analysis error variance is due to a few dominant modes, the leading eigenvectors of the bound matrix approximate those of the steady-state analysis error covariance matrix. The analytical results are illustrated in two numerical examples where the Kalman filter is carried to steady state. The first example uses the dynamics of a generalized advection equation exhibiting nonmodal transient growth. Failure to observe growing modes leads to increased steady-state analysis error variances. Leading eigenvectors of the steady-state analysis error covariance matrix are well approximated by leading eigenvectors of the bound matrix. The second example uses the dynamics of a damped baroclinic wave model. The leading eigenvectors of a lowest-order approximation of the bound matrix are shown to approximate well the leading eigenvectors of the steady-state analysis error covariance matrix.

Tippett, Michael K.↗

High-Fidelity Buckling Analysis of Composite Cylinders Using the STAGS Finite Element Code

Results from previous shell buckling studies are presented that illustrate some of the unique and powerful capabilities in the STAGS finite element analysis code that have made it an indispensable tool in structures research at NASA over the past few decades. In particular, prototypical results from the development and validation of high-fidelity buckling simulations are presented for several unstiffened thin-walled compression-loaded graphite-epoxy cylindrical shells along with a discussion on the specific methods and user-defined subroutines in STAGS that are used to carry out the high-fidelity simulations. These simulations accurately account for the effects of geometric shell-wall imperfections, shell-wall thickness variations, local shell-wall ply-gaps associated with the fabrication process, shell-end geometric imperfections, nonuniform applied end loads, and elastic boundary conditions. The analysis procedure uses a combination of nonlinear quasi-static and transient dynamic solution algorithms to predict the prebuckling and unstable collapse response characteristics of the cylinders. Finally, the use of high-fidelity models in the development of analysis-based shell-buckling knockdown (design) factors is demonstrated.

Hilburger, Mark W.↗

Gene Expression Profiling of Lung Tissue of Rats Exposed to Lunar Dust Particles

The purpose of the study is to analyze the dynamics of global gene expression changes in the lung tissue of rats exposed to lunar dust particles. Multiple pathways and transcription factors were identified using the Ingenuity Pathway Analysis tool, showing the potential networks of these signaling regulations involved in lunar dust‐induced prolonged proflammatory response and toxicity. The data presented in this study, for the first time, explores the molecular mechanisms of lunar dust induced toxicity. This work contributes not only to the risk assessment for future space exploration, but also to the understanding of the dust‐induced toxicity to humans on earth.

Zhang, Ye↗

Advantage of Animal Models with Metabolic Flexibility for Space Research Beyond Low Earth Orbit

As the world's space agencies and commercial entities continue to expand beyond Low Earth Orbit (LEO), novel approaches to carry out biomedical experiments with animals are required to address the challenge of adaptation to space flight and new planetary environments. The extended time and distance of space travel along with reduced involvement of Earth-based mission support increases the cumulative impact of the risks encountered in space. To respond to these challenges, it becomes increasingly important to develop the capability to manage an organism's self-regulatory control system, which would enable survival in extraterrestrial environments. To significantly reduce the risk to animals on future long duration space missions, we propose the use of metabolically flexible animal models as "pathfinders," which are capable of tolerating the environmental extremes exhibited in spaceflight, including altered gravity, exposure to space radiation, chemically reactive planetary environments and temperature extremes. In this report we survey several of the pivotal metabolic flexibility studies and discuss the importance of utilizing animal models with metabolic flexibility with particular attention given to the ability to suppress the organism's metabolism in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of these factors to minimize damage caused by exposure to spaceflight and extreme planetary environments. Examples of microorganisms and animal models with dormancy capabilities suitable for space research are considered in the context of their survivability under hostile or deadly environments outside of Earth. Potential steps toward implementation of metabolic control technology in spaceflight architecture and its benefits for animal experiments and manned space exploration missions are discussed.

metabilic flexibility↗

Advantage of Animal Models with Metabolic Flexibility for Space Research Beyond Low Earth Orbit

As the worlds space agencies and commercial entities continue to expand beyond Low Earth Orbit (LEO), novel approaches to carry out biomedical experiments with animals are required to address the challenge of adaptation to space flight and new planetary environments. The extended time and distance of space travel along with reduced involvement of Earth-based mission support increases the cumulative impact of the risks encountered in space. To respond to these challenges, it becomes increasingly important to develop the capability to manage an organisms self-regulatory control system, which would enable survival in extraterrestrial environments. To significantly reduce the risk to animals on future long duration space missions, we propose the use of metabolically flexible animal models as pathfinders, which are capable of tolerating the environmental extremes exhibited in spaceflight, including altered gravity, exposure to space radiation, chemically reactive planetary environments and temperature extremes.In this report we survey several of the pivotal metabolic flexibility studies and discuss the importance of utilizing animal models with metabolic flexibility with particular attention given to the ability to suppress the organism's metabolism in spaceflight experiments beyond LEO. The presented analysis demonstrates the adjuvant benefits of these factors to minimize damage caused by exposure to spaceflight and extreme planetary environments. Examples of microorganisms and animal models with dormancy capabilities suitable for space research are considered in the context of their survivability under hostile or deadly environments outside of Earth. Potential steps toward implementation of metabolic control technology in spaceflight architecture and its benefits for animal experiments and manned space exploration missions are discussed.

metabolic flexibility↗

The NANOGrav Nine-Year Data Set: Limits on the Isotropic Stochastic Gravitational Wave Background

We compute upper limits on the nanohertz-frequency isotropic stochastic gravitational wave background (GWB) using the 9 year data set from the North American Nanohertz Observatory for Gravitational Waves (NANOGrav) collaboration. Well-tested Bayesian techniques are used to set upper limits on the dimensionless strain amplitude (at a frequency of 1 yr(exp -1) for a GWB from supermassive black hole binaries of A(sub gw) less than 1.5 x 10(exp -15). We also parameterize the GWB spectrum with a broken power-law model by placing priors on the strain amplitude derived from simulations of Sesana and McWilliams et al. Using Bayesian model selection we find that the data favor a broken power law to a pure power law with odds ratios of 2.2 and 22 to one for the Sesana and McWilliams prior models, respectively. Using the broken power-law analysis we construct posterior distributions on environmental factors that drive the binary to the GW-driven regime including the stellar mass density for stellar-scattering, mass accretion rate for circumbinary disk interaction, and orbital eccentricity for eccentric binaries, marking the first time that the shape of the GWB spectrum has been used to make astrophysical inferences. Returning to a power-law model, we place stringent limits on the energy density of relic GWs, OMEGA(sub gw) (f) h squared less than 4.2 x 10(exp -10). Our limit on the cosmic string GWB, OMEGA(sub gw) (f) h squared less than 2.2 x 10(exp -10), translates to a conservative limit on the cosmic string tension with G mu less than 3.3 x 10(exp -8), a factor of four better than the joint Planck and high-l‚ cosmic microwave background data from other experiments.

Arzoumanian, Z.↗

Controlled Rest: Profile of Use, Challenges, and Best Practices

Despite the introduction of flight, duty, and rest time regulations to reduce the risk of sleepiness, airline pilots often encounter elevated sleepiness during flight. To combat this sleepiness, in some instances, pilots can take a short nap on the flight deck (controlled rest) to improve their alertness. Little is known, however, as to when and how often this countermeasure is used operationally. Methods: Forty-four pilots from a European carrier wore actiwatches and filled in an electronic sleep and work diary for approximately 2 weeks resulting in data from 239 flights. Self-reported in-flight rest periods were used to set rest intervals and sleep was estimated within these intervals using Philips Actiware 6.0.9. Wake threshold selection was set to medium; sleep threshold detection algorithm was set to 10 immobile minutes at sleep onset and sleep end. Timing of sleep periods was analyzed relative to home base time. Results: Preliminary analyses showed that controlled rest was taken on 46% (n=110) of flights. On 23 flights (10%) pilots reported taking two controlled rest periods. Sleep, as estimated by actigraphy, was achieved during 80% (n=106) of controlled rest periods. The mean sleep duration was 32 (± 12) minutes estimated within successful controlled rest periods. Approximately two-thirds (67.5%, n=81) of all rest periods were initiated during home base time night (0000h-0800h). On 11% (n=26) of flights, pilots also reported taking bunk rest (longer rest period in a designated sleeping facility).Conclusion:This study shows that controlled rest is commonly used as a countermeasure to sleepiness on the flight deck. Further analysis is required to determine what other factors contribute to the decision to take controlled rest, and how effective it is in reducing sleepiness on the flight deck.

Hilditch, Cassie J.↗

Mars Science Laboratory interplanetary navigaton analysis

The Mars Science Laboratory (MSL) is a NASA rover mission that will be launched in late 2011 and will land on Mars in August of 2012. This paper describes the analyses performed to validate the navigation system for launch, interplanetary cruise, and approach. MSL will use guidance during its descent into Mars in order to minimize landing dispersions, and therefore will be able to use smaller landing zones that are closer to terrain of high scientific interest. This will require a more accurate delivery of the spacecraft to the atmospheric entry interface, and a late update of the state of the spacecraft at entry. During cruise and approach the spacecraft may perform up to six trajectory correction maneuvers (TCMs), to target to the desired landing site with the required flight path angle at entry. Approach orbit determination covariance analyses have been performed to evaluate the accuracy that can be achieved in delivering the spacecraft to the entry interface point, and to determine how accurately the state of the spacecraft can be predicted to initialize the guidance algorithm. In addition, a sensitivity analysis has been performed to evaluate which factors most contribute to the improvement or degradation of the navigation performance, for both entry flight path angle delivery and entry state knowledge.

Wong, Mau C.↗

Boundary Representation Tolerance Impacts on Mesh Generation and Adaptation

The control of discretization error is critical to obtaining reliable simulation results. Re-cent progress has matured anisotropic mesh adaptation, which automates discretization error control for complex geometries. However, the meshing process can fail when geometric model Boundary REPresentation (BREP) tolerances are larger than boundary layer surface nor-mal and tangential spacing requirements. Geometry sources are often created in Mechanical Computer-Aided Design (MCAD) systems, which are designed to produce models for manufacturing and not the stricter requirements of viscous flow analysis. BREP tolerances are strained by three factors: the inherent complexity of the model (e.g., large range of scales and complex topology), the numerical difficulties arising from surface/surface intersections, and the omission of crucial data during export and translation. Manual preparation of geometry is commonly employed to enable expert-guided mesh generation, which severely inhibits work-flow automation. Accommodation of loose BREP tolerances is required for automated mesh processes, because barrier issues may not be detected until well into the solution process. To raise awareness of this class of geometry issues and their impact on simulation, a survey of these barrier issues is presented with example mitigation techniques. This awareness may also impact geometry creation workflows to prevent the introduction of these artifacts.

CAD↗

Form Factors, Grey Bodies and Radiation Conductances (Radks)

With today's analysis tools, large, complex thermal radiation problems are easily solved; But, as with any analytical tool, lack of an understanding of the fundamental equations and technique limitations may leave you with the wrong answer; Whether you are a new engineer or a seasoned veteran, an understanding of the techniques employed by these powerful analysis tools is crucial.

Thermal↗

Refining the Definition of a Geothermal Exploration Success Rate

The geothermal exploration process impacts project development timelines, costs, and overall perceived risk. However, the success in the exploration phase of a geothermal project - and the calculation of a success rate - is universally ill-defined. In previous studies, the success of a geothermal process is loosely defined as the number of wells that result in commercially-viable production. This work focuses on refining the definition of success for the exploration process to represent an upper bound to the likelihood a project will proceed into commercial production. This research examines the topic of success from both a top-down and bottom-up approach: a review of previous assessments of success rate that identifies common conditions leading to successful exploration, and a project-level review of geothermal sites in the western United States focused on decision points in the geothermal exploration process. The analysis not only considers the impact of factors outside of the geothermal reservoir (i.e. delays due to financing or permitting), but also touches on the length of the exploration process and the symbiosis between exploration methods. Using a combination of these analyses, future work will investigate which levers within the geothermal exploration process have the strongest influence on overall project success.

15 GEOTHERMAL ENERGY↗

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