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251 records · Page 14

Finding Evidence of a Prebiotic Pyruvate Reaction Network in Meteorites

The organic compounds of carbonaceous chondrites are structurally diverse products of the prolonged abiotic chemistry that occurred before and during the early stages of the solar system. They also represent the types of prebiotic compounds that may have participated in chemical evolutionary processes that gave rise to life. Pyruvic acid is a key component of primary metabolism that has recently been found in carbonaceous chondrites. Its prominence in biology has inspired us to explore its chemistry and possible role in the origin of metabolism. Our laboratory investigations have found that pyruvate (the ionized form of pyruvic acid) can serve as a single-source reactant to generate what we term a pyruvate reaction network (PRN). The core of the PRN is driven by pyruvate aldol polymerization and decay reactions. These decay reactions lead to the production of stable and unstable compounds which include mono, di, and tricarboxylic acids and a variety of keto acids. Importantly, many of these reactions apparently lead back to pyruvate, establishing a feedback process. Finding evidence of this reaction network in meteorites would establish that the chemistry of pyruvate did occur in a prebiotic environment and indeed many of the known compounds found in pyruvate reaction mixtures were detected in the Murchison and Murray meteorites. However, multiple synthetic origins, not tied to pyruvate chemistry, might also explain their meteoritic presence. Thus finding compounds unique to the PRN, such as reaction intermediates, would provide stronger evidence in support of its prebiotic relevance. The identity of these intermediates can only be con-firmed by acquiring chemical standards, however, as most are not available commercially, we engaged in extensive chemical synthesis. Obtaining these standards also provides the opportunity to study their subsequent chemistry and roles in the PRN.

Rios, A. C.↗

The Potential for a Ka-band (32 GHz) Worldwide VLBI Network

Ka-band (32 GHz, 9mm) Very Long Baseline Interferometric (VLBI) networking has now begun and has tremendous potential for expansion over the next few years. Ka-band VLBI astrometry from NASA's Deep Space Network has already developed a catalog of 470 observable sources with highly accurate positions. Now, several antennas worldwide are planning or are considering adding Ka-band VLBI capability. Thus, there is now an opportunity to create a worldwide Ka-band network with potential for high resolution imaging and astrometry. With baselines approaching a Giga-lambda, a Ka-band network would be able to probe source structure at the nano-radian (200 as) level ( 100X better than Hubble) and thus gain insight into the astrophysics of the most compact regions of emission in active galactic nuclei. We discuss the advantages of Ka-band, show the known sources and candidates, simulate projected baseline (uv) coverage, and discuss potential radio frequency feeds. The combination of these elements demonstrates the feasibility of a worldwide Ka network within the next few years!

quasar↗

INTEX-NA: Intercontinental Chemical Transport Experiment - North America

INTEX-NA is an integrated atmospheric chemistry field experiment to be performed over North America using the NASA DC-8 and P-3B aircraft as its primary platforms. It seeks to understand the exchange of chemicals and aerosols between continents and the global troposphere. The constituents of interest are ozone and its precursors (hydrocarbons, NOX and HOX), aerosols, and the major greenhouse gases (CO2, CH4, N2O). INTEX-NA will provide the observational database needed to quantify inflow, outflow, and transformations of chemicals over North America. INTEX-NA is to be performed in two phases. Phase A will take place during the period of May-August 2004 and Phase B during March-June 2006. Phase A is in summer when photochemistry is most intense and climatic issues involving aerosols and carbon cycle are most pressing, and Phase B is in spring when Asian transport to North America is at its peak. INTEX-NA will coordinate its activities with concurrent measurement programs including satellites (e. g. Terra, Aura, Envisat), field activities undertaken by the North American Carbon Program (NACP), and other U.S. and international partners. However, it is being designed as a 'stand alone' mission such that its successful execution is not contingent on other programs. Synthesis of the ensemble of observation from surface, airborne, and space platforms, with the help of global/regional models is an important It is anticipated that approximately 175 flight hours for each of the aircraft (DC-8 and P-3B) will be required for each Phase. Principal operational sites are tentatively selected to be Bangor, ME; Wallops Island, VA; Seattle, WA; Rhinelander, WI; Lancaster, CA; and New Orleans, LA. These coastal and continental sites can support large missions and are suitable for INTEX-NA objectives. The experiment will be supported by forecasts from meteorological and chemical models, satellite observations, surface networks, and enhanced O3,-sonde releases. In addition to characterizing Atlantic-outflow and Pacific-inflow, INTEX-NA will characterize air masses transported between the U.S., Canada, and Mexico. INTEX-NA will be the first continental scale inflow, outflow, and transformation experiment to be performed over North America. It will provide the most comprehensive observational data set to date to understand the O3/NOX/HOX/aerosol photochemical system and the carbon cycle. One of the critical needs of the carbon cycle research is to obtain large-scale vertical and horizontal concentration gradients of CO2, throughout the troposphere over continental source/sink regions. INTEX-NA is ideally suited to perform this role. Coastal and continental operational sites will allow us to develop a curtain profile of greenhouse gases (e. g. CO2,) and other key pollutants across North America. Such information is central to our quantitative understanding of chemical budgets on the continental scale. We expect to provide a number of satellite under-flights over land and water to test and validate observations from the appropriate satellite platform (e. g. Aura). We plan to develop strong collaborations with other national and international observational programs. Results from INTEX-NA should directly benefit the development of environmental policy for air quality and climate change.

Singh, Hanwant B.↗

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

NASA Discipline Musculoskeletal↗

Two Variable Radio Sources Near the Position of GRB 940301

We report on the results of a search for a radio counterpart to the strong gamma-ray burst GRB 940301. Observations with the Westerbork Synthesis Radio Telescope of the Compton Telescope error box region of GRB 940301 began on March 4, 1994, at 21 cm and April 2, 1994, at 92 cm. No flux density variations were detected at 92 cm above S= 10 mJy (5 (sigma)) within a period of 1 to 4 months after the burst. However, when we compared the field with Westerbork Northern Sky Survey data, taken two years prior to GRB 940301, we found two radio sources with significantly increased flux densities. These sources, only 17 min. apart, are located at the 2.3 and 2.6(sigma) Compton Telescope confidence contours. Their separation from the Inter Planetary Network annulus virtually excludes association with GRB 940301. Further observations in January 1996 reveal that the sources continued to change in flux density. The relatively large flux density variations at 92 cm, compared to those at higher frequencies, and the inverted spectra in the frequency range from 325-38O MHz make the sources somewhat unusual. Because the sources were already detected at 5 GHz in 1986 most, if not all, of the radio emission is probably associated with activity in Active Galactic Nuclei in distant galaxies.

Galama, T. J.↗

Europa's Northern Trailing Hemisphere: Lineament Stratigraphic Framework

Knowledge of the global distribution of Europan geologic units in time and space is a necessary step for the synthesis of the results of the Galileo mission and in preparation for future exploration (namely, by JIMO) of the satellite. We have initiated the production of the first Global Geological Map of Europa. As a base map, we use the recently published global photomosaic of Europa (U.S.G.S. Map I-2757) and additional Galileo SSI images at their original resolution. The map is being produced entirely on GIS format for analysis and combination with other datasets [1]. One of the main objectives of this project is to establish a global stratigraphic framework for Europa. In the absence of a well-developed cratering record, this goal will be achieved using the satellite s global network of lineaments (ridges, ridge complexes and bands; cf. [2]). Here we present the preliminary stratigraphic framework synthesized from the sequence of lineaments derived for the northern trailing hemisphere of Europa (Figure 1, below), and we discuss its significance and some emerging implications.

Figueredo, P. H.↗

Systems Architecture for Fully Autonomous Space Missions

The NASA Goddard Space Flight Center is working to develop a revolutionary new system architecture concept in support of fully autonomous missions. As part of GSFC's contribution to the New Millenium Program (NMP) Space Technology 7 Autonomy and on-Board Processing (ST7-A) Concept Definition Study, the system incorporates the latest commercial Internet and software development ideas and extends them into NASA ground and space segment architectures. The unique challenges facing the exploration of remote and inaccessible locales and the need to incorporate corresponding autonomy technologies within reasonable cost necessitate the re-thinking of traditional mission architectures. A measure of the resiliency of this architecture in its application to a broad range of future autonomy missions will depend on its effectiveness in leveraging from commercial tools developed for the personal computer and Internet markets. Specialized test stations and supporting software come to past as spacecraft take advantage of the extensive tools and research investments of billion-dollar commercial ventures. The projected improvements of the Internet and supporting infrastructure go hand-in-hand with market pressures that provide continuity in research. By taking advantage of consumer-oriented methods and processes, space-flight missions will continue to leverage on investments tailored to provide better services at reduced cost. The application of ground and space segment architectures each based on Local Area Networks (LAN), the use of personal computer-based operating systems, and the execution of activities and operations through a Wide Area Network (Internet) enable a revolution in spacecraft mission formulation, implementation, and flight operations. Hardware and software design, development, integration, test, and flight operations are all tied-in closely to a common thread that enables the smooth transitioning between program phases. The application of commercial software development techniques lays the foundation for delivery of product-oriented flight software modules and models. Software can then be readily applied to support the on-board autonomy required for mission self-management. An on-board intelligent system, based on advanced scripting languages, facilitates the mission autonomy required to offload ground system resources, and enables the spacecraft to manage itself safely through an efficient and effective process of reactive planning, science data acquisition, synthesis, and transmission to the ground. Autonomous ground systems in turn coordinate and support schedule contact times with the spacecraft. Specific autonomy software modules on-board include mission and science planners, instrument and subsystem control, and fault tolerance response software, all residing within a distributed computing environment supported through the flight LAN. Autonomy also requires the minimization of human intervention between users on the ground and the spacecraft, and hence calls for the elimination of the traditional operations control center as a funnel for data manipulation. Basic goal-oriented commands are sent directly from the user to the spacecraft through a distributed internet-based payload operations "center". The ensuing architecture calls for the use of spacecraft as point extensions on the Internet. This paper will detail the system architecture implementation chosen to enable cost-effective autonomous missions with applicability to a broad range of conditions. It will define the structure needed for implementation of such missions, including software and hardware infrastructures. The overall architecture is then laid out as a common thread in the mission life cycle from formulation through implementation and flight operations.

Esper, Jamie↗

On principles, methods and recent advances in studies towards a GPS-based control system for geodesy and geodynamics

Although Very Long Baseline Interferometry (VLBI) and Satellite Laser Ranging (SLR) are becoming increasingly important tools for geodynamic studies, their future role may well be fulfilled by using alternative techniques such as those utilizing the signals from the Global Positioning System (GPS). GPS, without the full implementation of the system, already offers a favorable combination of cost and accuracy and has consistently demonstrated the capability to provide high precision densification control in the regional and local areas of the VLBI and SLR networks. This report reviews VLBI and SLR vis-a-vis GPS and outlines the capabilities and limitations of each technique and how their complementary application can be of benefit to geodetic and geodynamic operations. It demonstrates, albeit with a limited data set, that dual-frequency GPS observations and interferometric type analysis techniques make possible the modelling of the GPS orbits for several days with an accuracy of a few meters. The use of VLBI or SLR sites as fiducial stations together with refinements in the orbit determination procedures can greatly reduce the systematic errors in the GPS satellite orbits used to compute the positions of non-fiducial locations. In general, repeatability and comparison with VLBI of the GPS determined locations are of the order of between 2 parts in 10 to the 7th power and 5 parts in 10 to the 8th power for baseline lengths less than 2000 km. This report is mainly a synthesis of problems, assumptions, methods and recent advances in the studies towards the establishment of a GPS-based system for geodesy and geodynamics and is one phase in the continuing effort for the development of such a system. To some, including the author, it seems reasonable to expect within the next few years that more evidence will show GPS to be as a powerful and reliable a tool as mobile VLBI and SLR are today, but largely more economical.

Delikaraoglou, Demitris↗

Evidence that the protocell was also a protoneuron

A blueprint for a protocell was presented in 1960 (Fox) as a consequence of the newly discovered self-ordering of amino acids and the self-organization of the resultant thermal proteins into cellular structures. The biofunctions of the laboratory protocells (proteinoid microspheres) have since been cataloged; they display roots of many phenomena of modern cells, e.g. synthesis of internucleotide and peptide bonds in aqueous media. These results are inconsistent with a popular assumption that DNA/RNA preceded protein in earliest molecular evolution. The necessity for synthetic research on molecules and cells to 'begin at the beginning' is being realized. Ivanov and Fortsch (1986) have described by analysis how the self-ordering mechanism of amino acids into informed thermal proteins was conserved in evolution from the earliest stage as modern (reverse) mechanisms assumed control. Tyagi and Ponnamperuma (1990) have negated assumptions corollary to DNA/RNA. Ponnamperuma has demonstrated the powerful effect of self-ordering of amino acids in polymerization of aminoacyl nucleotides and the irrelevance of mononucleotide residues. Excitable thermal proteins (Vaughan et al, 1987) are neurotrophic and antiaging when added to cultures of real neurons (Hefti et al, 1991) and are memory enhancers in mice (Fox and Flood, 1992). Proteinoid microspheres of dominant hydrophobic constitution form 'gap junctions', sprout axon-like outgrowths, and form dendritic networks spontaneously. In the latest studies, phenylalanine-rich equimolar proteinoid or the leucine analog (Ishima et al 1981), is found to produce electrical signals for several days when lecithin is included in the assembly with the thermal polymer.

Bi, YU↗

Vision-Based Distributed Sensing at Vertiports for Advanced Air Mobility and Urban Air Mobility Approach and Landing

Advanced Air Mobility (AAM) encompasses a broad vision for air transportation, including Urban Air Mobility (UAM) as a subset. AAM aims to create a more connected and efficient transportation network across various geographical settings. However, navigating AAM aircraft in GPS-denied or degraded environments during approach and landing is challenging. Traditional vision aids like glideslopes and localizers are limited in vertiport environments due to narrow beam constraints and reduced landing angle options. This paper addresses the need for accurate navigation solutions at vertiports by proposing a vision-based distributed sensing (VIDIS) system utilizing cameras with bundle adjustment to assist incoming AAM aircraft during approach and landing while monitoring surface movements to enhance safety and efficiency. Key focus areas for current and future vertiport developers include identifying suitable sensor types and infrastructure standards to support AAM operations and including vertiport markings as vision-based navigation aids. The proposed system offers a novel approach to overcoming navigation challenges in AAM operations, particularly in urban settings where traditional aids may be insufficient. Preliminary simulation results with distributed cameras demonstrate promising outcomes for implementing bundle adjustment techniques to enhance vision-based navigation solutions at vertiports. Generating waypoint-based trajectories via waypoint integration using explicit guidance synthesis (WINGS) creates smooth AAM trajectories for landing at vertiports by using the current waypoint's terminal conditions as the initial conditions for the next waypoint. Combining bundle adjustment's ground-based solution of vertiport features with WINGS, Coplanar Pose from Orthography and Scaling with Iterations (COPOSIT), and an extended Kalman filter (EKF) estimates the state of an incoming aircraft during approach and landing at vertiports. Future work includes testing VIDIS in a high-fidelity simulation and with real-world data.

Distributed sensing↗

Vision-Based Distributed Sensing at Vertiports for Advanced Air Mobility and Urban Air Mobility Approach and Landing

Advanced Air Mobility (AAM) encompasses a broad vision for air transportation, including Urban Air Mobility (UAM) as a subset. AAM aims to create a more connected and efficient transportation network across various geographical settings. However, navigating AAM aircraft in GPS-denied or degraded environments during approach and landing is challenging. Traditional vision aids like glideslopes and localizers are limited in vertiport environments due to narrow beam constraints and reduced landing angle options. This paper addresses the need for accurate navigation solutions at vertiports by proposing a vision-based distributed sensing (VIDIS) system utilizing cameras with bundle adjustment to assist incoming AAM aircraft during approach and landing while monitoring surface movements to enhance safety and efficiency. Key focus areas for current and future vertiport developers include identifying suitable sensor types and infrastructure standards to support AAM operations and including vertiport markings as vision-based navigation aids. The proposed system offers a novel approach to overcoming navigation challenges in AAM operations, particularly in urban settings where traditional aids may be insufficient. Preliminary simulation results with distributed cameras demonstrate promising outcomes for implementing bundle adjustment techniques to enhance vision-based navigation solutions at vertiports. Generating waypoint-based trajectories via waypoint integration using explicit guidance synthesis (WINGS) creates smooth AAM trajectories for landing at vertiports by using the current waypoint's terminal conditions as the initial conditions for the next waypoint. Combining bundle adjustment's ground-based solution of vertiport features with WINGS, Coplanar Pose from Orthography and Scaling with Iterations (COPOSIT), and an extended Kalman filter (EKF) estimates the state of an incoming aircraft during approach and landing at vertiports. Future work includes testing VIDIS in a high-fidelity simulation and with real-world data.

Distributed sensing↗

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

Non-NASA Center↗

Non-Genomic Origins of Proteins and Metabolism

It is proposed that evolution of inanimate matter to cells endowed with a nucleic acid- based coding of genetic information was preceded by an evolutionary phase, in which peptides not coded by nucleic acids were able to self-organize into networks capable of evolution towards increasing metabolic complexity. Recent findings that truly different, simple peptides (Keefe and Szostak, 2001) can perform the same function (such as ATP binding) provide experimental support for this mechanism of early protobiological evolution. The central concept underlying this mechanism is that the reproduction of cellular functions alone was sufficient for self-maintenance of protocells, and that self- replication of macromolecules was not required at this stage of evolution. The precise transfer of information between successive generations of the earliest protocells was unnecessary and, possibly, undesirable. The key requirement in the initial stage of protocellular evolution was an ability to rapidly explore a large number of protein sequences in order to discover a set of molecules capable of supporting self- maintenance and growth of protocells. Undoubtedly, the essential protocellular functions were carried out by molecules not nearly as efficient or as specific as contemporary proteins. Many, potentially unrelated sequences could have performed each of these functions at an evolutionarily acceptable level. As evolution progressed, however proteins must have performed their functions with increasing efficiency and specificity. This, in turn, put additional constraints on protein sequences and the fraction of proteins capable of performing their functions at the required level decreased. At some point, the likelihood of generating a sufficiently efficient set of proteins through a non-coded synthesis was so small that further evolution was not possible without storing information about the sequences of these proteins. Beyond this point, further evolution required coupling between proteins and informational polymers that is characteristic to all known forms of life. The emergence of such coupling must be postulated in any scenario of the origin of life, no matter whether it starts with RNA or proteins. To examine the evolutionary potential of non-genomic systems, a simple, computationally tractable model, which is still capable of capturing the essential features of the real system, has been studied computationally. Both constructive and destructive processes have been introduced into the model in a stochastic manner. Instead of assuming random reaction sets, only a suite of protobiologically plausible reactions has been considered. Peptides have been explicitly considered as protoenzymes and their catalytic efficiencies have been assigned on the basis of biochemical principles and experimental estimates. Simulations have been carried out using a novel approach (The Next Reaction Method) that is appropriate even for very low concentrations of reactants. Studies have focused on global autocatalytic processes and their diversity.

Pohorille, Andrew↗

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

manned↗

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

mechanical loading↗

Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture

Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesionattachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by immunohistological observation. Increased CX43 expression is also observed with the three-dimensional co-cultures with MC3T3-E1 MCS stimulation but the increased gap-junction protein presence was limited to the osteoblast-osteocyte interface region. Previously reported PCR evaluation of osteogenic markers further corroborate that the co-cultured populations communicative networks play a role in translating mechanical signals to molecular messaging. These findings suggests an osteocyte-osteoblast gap-junction signaling feedback mechanism may regulate mechanotransduction of apoptosis initiation and transcription of cytokine signaling proteins responsible for stem cell niche recruitment much more directly than previously believed.

Juran, C. M.↗

New Material for Surface-Enhanced Raman Spectroscopy

A chemical method of synthesis and application of coating materials that are especially suitable for surface-enhanced Raman spectroscopy (SERS) has been developed. The purpose of this development is to facilitate the utilization of the inherently high sensitivity of SERS to detect chemicals of interest (analytes) in trace amounts, without need for lengthy sample preparation. Up to now, the use of SERS has not become routine because the methods available have not been able to reproduce sampling conditions and provide quantitative measurements. In contrast, the coating materials of the present method enable analysis with minimum preparation of samples, and SERS measurements made using these materials are reproducible and reversible. Moreover, unlike in methods investigated in prior efforts to implement SERS, sampling is not restricted to such specific environments as electrolytes or specific solvents. The coating materials of this method are porous glasses, formed in sol-gel processes, that contain small particles of gold or silver metal. Materials of this type can be applied to the sample-contact surfaces of a variety of sampling and sensing devices, including glass slides, glass vials, fiber-optic probes, and glass tubes. Glass vials with their insides coated according to this method are particularly convenient for SERS to detect trace chemicals in solutions: One simply puts a sample solution containing the analyte(s) into a vial, then puts the vial into a Raman spectrometer for analysis. The chemical ingredients and the physical conditions of the sol-gel process have been selected so that the porous glass formed incorporates particles of the desired metal with size(s) to match the wavelength(s) of the SERS excitation laser in order to optimize the generation of surface plasmons. The ingredients and processing conditions have further been chosen to tailor the porosity and polarity of the glass to optimize the sample flow and the interaction between the analyte(s) and the plasmon field that generates Raman photons. The porous silica network of a sol-gel glass creates a unique environment for stabilizing SERS-active metal particles. Relative to other material structures that could be considered for SERS, the porous silica network offers higher specific surface area and thus greater interaction between analyte molecules and metal particles. Efforts to perform SERS measurements with the help of sampling devices coated by this method have been successful. In tests, numerous organic and inorganic chemicals were analyzed in several solvents, including water. The results of the tests indicate that the SERS measurements were reproducible within 10 percent and linear over five orders of magnitude. One measure of the limits of detectability of chemicals in these tests was found to be a concentration of 300 parts per billion. Further development may eventually make it possible to realize the full potential sensitivity of SERS for detecting some analytes in quantities as small as a single molecule.

Farquharson, Stuart↗