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At least 523 records · Page 29

A vision system planner for increasing the autonomy of the Extravehicular Activity Helper/Retriever

The Extravehicular Activity Retriever (EVAR) is a robotic device currently being developed by the Automation and Robotics Division at the NASA Johnson Space Center to support activities in the neighborhood of the Space Shuttle or Space Station Freedom. As the name implies, the Retriever's primary function will be to provide the capability to retrieve tools and equipment or other objects which have become detached from the spacecraft, but it will also be able to rescue a crew member who may have become inadvertently de-tethered. Later goals will include cooperative operations between a crew member and the Retriever such as fetching a tool that is required for servicing or maintenance operations. This paper documents a preliminary design for a Vision System Planner (VSP) for the EVAR that is capable of achieving visual objectives provided to it by a high level task planner. Typical commands which the task planner might issue to the VSP relate to object recognition, object location determination, and obstacle detection. Upon receiving a command from the task planner, the VSP then plans a sequence of actions to achieve the specified objective using a model-based reasoning approach. This sequence may involve choosing an appropriate sensor, selecting an algorithm to process the data, reorienting the sensor, adjusting the effective resolution of the image using lens zooming capability, and/or requesting the task planner to reposition the EVAR to obtain a different view of the object. An initial version of the Vision System Planner which realizes the above capabilities using simulated images has been implemented and tested. The remaining sections describe the architecture and capabilities of the VSP and its relationship to the high level task planner. In addition, typical plans that are generated to achieve visual goals for various scenarios are discussed. Specific topics to be addressed will include object search strategies, repositioning of the EVAR to improve the quality of information obtained from the sensors, and complementary usage of the sensors and redundant capabilities.

Magee, Michael↗

Theoretical spectra of circumstellar dust shells around carbon-rich asymptotic giant branch stars

Realistic modeling of circumstellar dust shells around evolved stars has to include a physical treatment of the interaction among hydrodynamics, thermodynamics, radiative transfer, chemistry and dust formation and -growth. A self-consistent solution of this problem is presented in the case of stationary, spherical symmetric dust-driven winds. The resulting shell structure and the mass-loss rate are completely determined by the three fundamental stellar parameters stellar mass M(stellar), stellar luminosity L(stellar) and effective temperature T(sub eff) and by the element abundances epsilon(sub i). A detailed calculation of the transport coefficients of the dust component by means of the particle size distribution function and the solution of the non-grey radiative transfer problem provide realistic synthetic spectra of the dust shell models. We discuss the dependence of the resulting spectra on the stellar parameters in terms of infrared two color diagrams. Application of these model calculations to the prominent infrared object IRC +10216 yields best agreement with the observed spectrum and the visibility data at maximum light for the stellar parameters M(stellar) = 0.7 solar mass, L(stellar) = 2.4 x 10(exp 4) solar luminosity, T(stellar) = 2010K and a carbon to oxygen ratio of epsilon(sub c)/epsilon(sub o) = 1.40, which corresponds to a mass-loss rate of M-dot = 8 x 10(exp -5) solar mass/yr. In this model only amorphous carbon grains are considered as the main opacity source. From this model a distance to IRC +10216 of d = 170pc is deduced. The total mass contained in the circumstellar dust shell implies and initial main sequence mass of M(sub ZAMS) greater than or = 1.3 solar mass for IRC +10216.

Winters, J. M.↗

General Mission Analysis Tool (GMAT) Architectural Specification. Draft

Early in 2002, Goddard Space Flight Center (GSFC) began to identify requirements for the flight dynamics software needed to fly upcoming missions that use formations of spacecraft to collect data. These requirements ranged from low level modeling features to large scale interoperability requirements. In 2003 we began work on a system designed to meet these requirement; this system is GMAT. The General Mission Analysis Tool (GMAT) is a general purpose flight dynamics modeling tool built on open source principles. The GMAT code is written in C++, and uses modern C++ constructs extensively. GMAT can be run through either a fully functional Graphical User Interface (GUI) or as a command line program with minimal user feedback. The system is built and runs on Microsoft Windows, Linux, and Macintosh OS X platforms. The GMAT GUI is written using wxWidgets, a cross platform library of components that streamlines the development and extension of the user interface Flight dynamics modeling is performed in GMAT by building components that represent the players in the analysis problem that is being modeled. These components interact through the sequential execution of instructions, embodied in the GMAT Mission Sequence. A typical Mission Sequence will model the trajectories of a set of spacecraft evolving over time, calculating relevant parameters during this propagation, and maneuvering individual spacecraft to maintain a set of mission constraints as established by the mission analyst. All of the elements used in GMAT for mission analysis can be viewed in the GMAT GUI or through a custom scripting language. Analysis problems modeled in GMAT are saved as script files, and these files can be read into GMAT. When a script is read into the GMAT GUI, the corresponding user interface elements are constructed in the GMAT GUI. The GMAT system was developed from the ground up to run in a platform agnostic environment. The source code compiles on numerous different platforms, and is regularly exercised running on Windows, Linux and Macintosh computers by the development and analysis teams working on the project. The system can be run using either a graphical user interface, written using the open source wxWidgets framework, or from a text console. The GMAT source code was written using open source tools. GSFC has released the code using the NASA open source license.

Hughes, Steven P.↗

One Step Closer to Mars with Aquaponics: Cultivating Citizen Science in K12 Schools

The Microbial Ecology and Biogeochemistry Research Laboratory at NASA Ames Research Center focuses primarily on the nutrient cycling and diversity of complex microbial communities. NASA is interested in the composition and functioning of microbial mat communities as these processes fundamentally shape the form and function of these analogs for the earliest forms of life on Earth (3.6 billion years ago), and likely will on other planets as well. Aquaponics systems are supported by microbial communities who perform many complex ecosystem services, including cycling nitrogen. Microbes are integral to the stability and productivity of aquaponics systems, which are analogous to microbial communities in food production systems that are essential for building efficient life support systems for long-distance space travel. Students at Meadow Park Middle School created 10 parallel aquaponics systems and took temporal microbial samples to characterize whether any macro-ecology variables impacted or changed the microbial diversity of these systems. Students additionally created a website so that other classrooms can pursue similar projects in their own schools (https://go.nasa.gov/2uJhxmF). Our lab at NASA Ames has sequenced water samples from each of the 10 tanks at 3 timepoints using a MinION sequencer. MPMS students will be involved in the analysis of the bioinformatics data generated through this collaboration. Our ongoing collaboration aims to collect and analyze data in the classroom setting that has utility for research scientists, while involving students as collaborators in the research process.

Kolattukudy, Maria↗

Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

Bacterial Proteins/chemistry/genetics/metabolism↗

Alkaloids are associated with increased microbial diversity and metabolic function in poison frogs

Shifts in host-associated microbiomes can impact both host and microbes. It is of interest to understand how perturbations, like the introduction of exogenous chemicals, impact microbiomes. In poison frogs (family Dendrobatidae), the skin microbiome is exposed to alkaloids that the frogs sequester for defense. These alkaloids are antimicrobial; however, their effect on the frogs’ skin microbiome is unknown. To test this, we characterized microbial communities from field-collected dendrobatid frogs. Then, we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of two frog species with contrasting alkaloid loads in nature. In both datasets, we found that alkaloid-exposed microbiomes were more phylogenetically diverse, with an increase in diversity among rare taxa. Further, to better understand the isolate-specific response to alkaloids, we cultured microbial isolates from poison frog skin and found that many isolates exhibited enhanced growth or were not impacted by the addition of DHQ. To further explore the microbial response to alkaloids, we sequenced the metagenomes from high- and low-alkaloid frogs and observed a greater diversity of genes associated with nitrogen and carbon metabolism in high-alkaloid frogs. From these data, we hypothesized that some strains may metabolize the alkaloids. We used stable isotope tracing coupled to nanoSIMS (nanoscale secondary ion mass spectrometry), which supported the idea that some of these isolates are able to metabolize DHQ. Together, these data suggest that poison frog alkaloids open new niches for skin-associated microbes with specific adaptations, such as alkaloid metabolism, that enable survival in this environment.

59 BASIC BIOLOGICAL SCIENCES↗

Gene expression changes in peripheral blood mononuclear cells of ISS crewmembers suggest impacts of spaceflight on cell death

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 24 hrs (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the data from all of the time points from the ground control subjects as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of several cell cycle related genes including CDKN1A and VEGFA at MF and LF. Pathway analysis of these differentially expressed genes indicated that, in space, pathways associated with autophagy and senescence were affected. Our analysis also indicated that the genes related to metabolisms were downregulated in the microgravity environment. Taken together, our data suggests that PBMC in the ISS crewmembers may be starved, resulting in autophagy and delayed senescence in space. Such findings are in agreement with delayed cell death in PBMC under simulated microgravity conditions on the ground and offer an explanation for telomere lengthening that has been reported among the ISS astronauts in flight.

Maria Moreno-Villanueva↗

Gene Expression Changes in Peripheral Blood Mononuclear Cells of ISS Crewmembers Suggest Impacts of Spaceflight on Cell Death

In space, living organisms are exposed to numerous stress factors including microgravity and space radiation. For humans, these harmful environmental factors have been known to cause negative health impacts such as immune dysfunction. Understanding the mechanisms by which spaceflight impacts human health at the molecular level is critical not only for accurately assessing the risks associated with spaceflight, but also for developing effective countermeasures. This study is part of the Functional Immune Project, intended to determine alterations in crewmembers` immunobiology before, during, and after spaceflight. For this project, blood samples were collected from International Space Station (ISS) crewmembers at the following time points: i) at two pre-flight time points of 180 days (L180) and 45 days (L45) before launch. ii) During flight, blood was drawn at approximately the midpoint (mid-flight, MF) of the mission, and shortly before egress from the ISS (late-flight, LF). iii) Post-flight blood samples were collected within 24 hrs (R0), 30 days (R30) and 90 days (R90) after landing. For each crewmember, blood was also drawn from a matching test subject on the ground at the corresponding time point. For both the ISS crewmembers and the ground control subjects, total RNA was isolated from peripheral blood mononuclear cells (PBMC) and mRNA was analysed using next generation RNA-sequencing (NGS). Differentially expressed genes were determined by performing contrast analysis. Using the data from all of the time points from the ground control subjects as a control, a number of dysregulated genes were identified in astronauts at MF, LF and R0, including downregulations of several cell cycle related genes including CDKN1A and VEGFA at MF and LF. Pathway analysis of these differentially expressed genes indicated that, in space, pathways associated with autophagy and senescence were affected. Our analysis also indicated that the genes related to metabolisms were downregulated in the microgravity environment. Taken together, we hypothesize that PBMC in the ISS crewmembers may be starved, resulting in autophagy and delayed senescence in space. Such findings are in agreement with delayed cell death in PBMC under simulated microgravity conditions on the ground and offer an explanation for telomere lengthening that has been reported among the ISS astronauts in flight

Maria Moreno-Villanueva↗

Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at approximately 40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle alpha-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

NASA Discipline Cardiopulmonary↗

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

Non-NASA Center↗

Damage Initiation and Ultimate Tensile Strength of Scaled [0 deg n/90 deg n/0 deg n]sub T Graphite-Epoxy Coupons

Previous research on scaling effects in composite materials has demonstrated that the stress levels at first ply failure and ultimate failure of composite laminates are dependent on the size of the laminate. In particular, the thickness dimension has been shown to be the most influential parameter in strength scaling of composite coupons loaded in tension. Geometrically and constitutively scaled laminates exhibit decreasing strength with increasing specimen size, and the magnitude of the strength-size effect is a function of both material properties and laminate stacking sequence. Some of the commonly used failure criteria for composite materials such as maximum stress, maximum strain, and tensor polynomial (e.g., Tsai-Wu) cannot account for the strength-size effect. In this paper, three concepts are developed and evaluated for incorporating size dependency into failure criteria for composite materials. An experimental program of limited scope was performed to determine the first ply failure stress in scaled cross-ply laminates loaded in tension. Test specimens were fabricated of AS-4/3502 graphite-epoxy composite material with laminate stacking sequences of [0 deg n/90 deg n/o deg n]subT where n=1-6. Two experimental techniques were used to determine first ply failure, defined as a transverse matrix crack in the 90 deg ply: (1) step loading with dye penetrant x-ray of the specimen at each load interval, and (2) acoustic emission. The best correlation between first ply failure analysis and experimental data was obtained using a modified Weibull approach which incorporated the residual thermal stress and the outer ply constraint, as well as the ply thickness effect. Finally, a second set of experiments was performed to determine the tensile response and ultimate failure of the scaled cross-ply laminates. The results of these experiments indicated no influence of specimen size on tensile response or ultimate strength.

Jackson, Karen E.↗

Hyperspectral Sun Photometer for Atmospheric Characterization and Vicarious Calibrations

A hyperspectral sun photometer and associated methods have been developed and demonstrated. Accurate sun photometer calibration is critical to properly measure the solar irradiance and characterize the atmosphere. Traditional sun photometer calibration requires solar observations over several hours. In contrast, the procedures for operating this photometer entail less data acquisition time and embody a more direct approach to calibration. The scientific value of the measurement data produced by this instrument is not adversely affected by atmospheric instability. In addition, this instrument yields hyperspectral data covering a large spectral range (350-2,500 nm) not available from most traditional sun photometers. The hyperspectral sun photometer components include (1) a commercially available spectroradiometer that has been laboratory-calibrated and (2) a commercially available reflectance standard panel that exhibits nearly Lambertian 99% reflectance. The spectroradiometer is positioned above, and aimed downward at, the panel. The procedure for operating this instrument calls for a series of measurements: one in which the panel is fully illuminated by the sun, one in which a shade is positioned between the panel and the sun, and two in which the shade is positioned to cast a shadow to either side of the panel. The total sequence of measurements can be performed in less than a minute. From these measurements, the total radiance, the diffuse radiance, and the direct solar radiance are calculated. The direct solar irradiance is calculated from the direct solar radiance and the known reflectance factor of the panel as a function of the solar zenith angle. Atmospheric characteristics are estimated from the optical depth at various wavelengths calculated from (1) the direct solar irradiance obtained as described above, (2) the air mass along a column from the measurement position to the Sun, and (3) the top-of-atmosphere solar irradiance. The instrumentation used to implement the sun photometer is the same as that used to characterize targets used in radiometric vicarious calibrations. Utilizing this type of sun photometer thus reduces the amount of instrumentation and labor required to perform these studies.

Pagnutti, Mary↗

Extracellular calcium (Ca2+o)-sensing receptor in a mouse monocyte-macrophage cell line (J774): potential mediator of the actions of Ca2+o on the function of J774 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca2+o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 microM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.

NASA Discipline Musculoskeletal↗

Effect of GOES-R Image Navigation and Registration Errors on Atmospheric Motion Vectors

High temporal frequency imagery from geostationary satellites allows for the continuous monitoring of rapidly changing atmospheric constituents such as smoke, dust, water vapor and clouds. The image sequences are often used to quantify the displacement of image features such as water vapor and clouds to produce atmospheric motion vectors (AMVs) which are used as diagnostic tools and also assimilated into numerical weather forecast models. The basic principle behind the determination of AMVs is the calculation of the physical displacement of features from one image (time) to the next. This process assumes that the features being tracked do not change as a function of time, usually requiring the use of short time interval imagery to minimize substantial change in size and shape of the features being tracked. High spatial resolution imagery also is required for reliable feature identification. While these image resolution and temporal sampling requirements often provide major drivers for space-based instrument design requirements, accurate image navigation and registration, INn (between a sequence of images), is also critical to the derivation of useful AMVs. In this paper and poster to be presented at the conference, the image navigation and registration (INR) accuracy expected for the Advanced Baseline Imager (ABI) on the GOES-R series of satellites will be discussed in light of its impact on AMV accuracy. Significant satellite platform and modeling enhancements are planned which should significantly improve INn performance of the GOES-R instruments. Some of these improvements have been demonstrated for the GOES-13 satellite which was launched in summer of 2006. An analysis of GOES-13 INR data, from the special satellite check out period, will be used in the assessment.

Jedlovec, Gary↗

Pilot Field Test: Performance of a Sit-to-Stand Test After Long-Duration Space Flight

BACKGROUND: Astronauts returning from the International Space Station are met by a team of recovery personnel typically providing physical assistance and medical support immediately upon landing. That is because long-duration spaceflight impacts astronauts' functional abilities. Future expeditions to planets or asteroids beyond the low Earth orbit, however, may require crewmembers to egress the vehicle and perform other types of physical tasks unassisted. It is therefore important to characterize the extent and longevity of functional deficits experienced by astronauts in order to design safe exploration class missions. Pilot Field Test (PFT) experiment conducted with participation of ISS crewmembers traveling on Soyuz expeditions 34S - 41S comprised several tasks designed to study the recovery of sensorimotor abilities of astronauts during the first 24 hours after landing and beyond. METHODS: The first test in the PFT battery sequence, and also the least demanding one from the sensorimotor perspective, was a Sit-to-Stand test. Test subjects were seated in the chair and had to stand up on command and remain standing for ten seconds. The subjects were instructed to stand up unassisted as quickly as they were able to, while maintaining postural control. Synchronized wireless inertial sensors mounted on the head, chest, lower back, wrists, and ankles were used to continuously log body kinematics. Crewmembers' blood pressure and heart rate were monitored and recorded with the Portapres and Polar systems. Each session was recorded with a digital video camera. During data collections occurring within the 24-hour postflight period, crewmembers were also asked to (1) evaluate their perceived motion sickness symptoms on a 20-point scale before and after completion of the test and (2) estimate how heavy they felt compared to their normal (preflight) body weight. Consent to participate in PFT was obtained from 18 crewmembers (11 US Orbital Segment [USOS] astronauts and 7 Russian cosmonauts). For 10 subjects, the first set of data was collected in the medical tent in Soyuz landing zone (1-2 hours after landing); the other 8 subjects were tested at the Kazakhstan deployment airport (4-5 hours after landing). 8 of the 11 astronauts were tested twice more within the first 24 hours postflight, at a refueling stop on the way to Houston (approximately 13 hours after landing) and at the Johnson Space Center (approximately 24 hours after landing). Later postflight data were collected in the first two weeks on some crewmembers. Finally, 6 astronauts were tested 60+ days after landing to establish a delayed baseline. RESULTS/DISCUSSION: Two of the 18 PFT participants felt too ill to attempt any tests in Kazakhstan (at either the landing zone or deployment airport). The remaining test subjects completed the Sit-to-Stand test and their reported motion sickness scores were unaffected by this task. The task completion times and body kinematics data analysis are currently underway. Preliminary analysis of astronaut data shows a steep improvement in the time to complete the task on the second data take, and in some cases, the trend continues through day six postflight. Head and trunk pitch angles and pitch rates were also examined and increases in all measures are evident throughout the observed recovery period (60+ days postflight). Interesting patterns of head and trunk pitch coordination have also emerged. One of the data analysis objectives is comparison of initial postflight performance and recovery of experienced crewmembers and first-time fliers. Another one - possible differences in performance between USOS and Russian crewmembers.

Kofman, I. S.↗

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

NASA Discipline Musculoskeletal↗

Identification of a new EF-hand superfamily member from Trypanosoma brucei

We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.

Non-NASA Center↗

Space Suit Portable Life Support System (PLSS) 2.0 Unmanned Vacuum Environment Testing

For the first time in more than 30 years, an advanced space suit Portable Life Support System (PLSS) design was operated inside a vacuum chamber representative of the flight operating environment. The test article, PLSS 2.0, was the second system-level integrated prototype of the advanced PLSS design, following the PLSS 1.0 Breadboard that was developed and tested throughout 2011. Whereas PLSS 1.0 included five technology development components with the balance the system simulated using commercial-off-the-shelf items, PLSS 2.0 featured first generation or later prototypes for all components less instrumentation, tubing and fittings. Developed throughout 2012, PLSS 2.0 was the first attempt to package the system into a flight-like representative volume. PLSS 2.0 testing included an extensive functional evaluation known as Pre-Installation Acceptance (PIA) testing, Human-in-the-Loop testing in which the PLSS 2.0 prototype was integrated via umbilicals to a manned prototype space suit for 19 two-hour simulated EVAs, and unmanned vacuum environment testing. Unmanned vacuum environment testing took place from 1/9/15-7/9/15 with PLSS 2.0 located inside a vacuum chamber. Test sequences included performance mapping of several components, carbon dioxide removal evaluations at simulated intravehicular activity (IVA) conditions, a regulator pressure schedule assessment, and culminated with 25 simulated extravehicular activities (EVAs). During the unmanned vacuum environment test series, PLSS 2.0 accumulated 378 hours of integrated testing including 291 hours of operation in a vacuum environment and 199 hours of simulated EVA time. The PLSS prototype performed nominally throughout the test series, with two notable exceptions including a pump failure and a Spacesuit Water Membrane Evaporator (SWME) leak, for which post-test failure investigations were performed. In addition to generating an extensive database of PLSS 2.0 performance data, achievements included requirements and operational concepts verification, as well as demonstration of vehicular interfaces, consumables sizing and recharge, and water quality control.

Watts, Carly↗