Myocyte Contractility Can Be Maintained by Storing Cells with the Myosin ATPase Inhibitor 2,3 Butanedione Monoxime

Isolated intact myocytes can be used to investigate contractile mechanisms and to screen new therapeutic compounds. These experiments typically require euthanizing an animal and isolating fresh cells each day or analyzing cultured myocytes, which quickly lose their rod-shaped morphology. Recent data suggest that the viability of canine myocytes can be prolonged using low temperature and N-benzyl-p-toluene sulfonamide (an inhibitor of skeletal myosin ATPase). We performed similar studies in rat myocytes in order to test whether the cardiac myosin ATPase inhibitors 2,3-Butanedione monoxime (BDM) and blebbistatin help to maintain cell-level function over multiple days. Myocytes were isolated from rats and separated into batches that were stored at 4°C in a HEPES-buffered solution that contained 0.5 mmol L-1 Ca2+ and (1) no myosin ATPase inhibitors; (2) 10 mmol L-1 BDM; or (3) 3 μmol L-1 blebbistatin. Functional viability of myocytes was assessed up to 3 days after the isolation by measuring calcium transients and unloaded shortening profiles induced by electrical stimuli in inhibitor-free Tyrode\u27s solution. Cells stored without myosin ATPase inhibitors had altered morphology (fewer rod-shaped cells, shorter diastolic sarcomere lengths, and membrane blebbing) and were not viable for contractile assays after 24 h. Cells stored in BDM maintained morphology and contractile function for 48 h. Storage in blebbistatin maintained cell morphology for 72 h but inhibited contractility. These data show that storing cells with myosin ATPase inhibitors can extend the viability of myocytes that will be used for functional assays. This may help to refine and reduce the use of animals in experiments

Myocardial Relaxation Is Accelerated by Fast Stretch, Not Reduced Afterload

Fast relaxation of cross-bridge generated force in the myocardium facilitates efficient diastolic function. Recently published research studying mechanisms that modulate the relaxation rate has focused on molecular factors. Mechanical factors have received less attention since the 1980s when seminal work established the theory that reducing afterload accelerates the relaxation rate. Clinical trials using afterload reducing drugs, partially based on this theory, have thus far failed to improve outcomes for patients with diastolic dysfunction. Therefore, we reevaluated the protocols that suggest reducing afterload accelerates the relaxation rate and identified that myocardial relengthening was a potential confounding factor. We hypothesized that the speed of myocardial relengthening at end systole (end systolic strain rate), and not afterload, modulates relaxation rate and tested this hypothesis using electrically-stimulated trabeculae from mice, rats, and humans. We used load-clamp techniques to vary afterload and end systolic strain rate independently. Our data show that the rate of relaxation increases monotonically with end systolic strain rate but is not altered by afterload. Computer simulations mimic this behavior and suggest that fast relengthening quickens relaxation by accelerating the detachment of cross-bridges. The relationship between relaxation rate and strain rate is novel and upends the prevailing theory that afterload modifies relaxation. In conclusion, myocardial relaxation is mechanically modified by the rate of stretch at end systole. The rate of myocardial relengthening at end systole may be a new diagnostic indicator or target for treatment of diastolic dysfunction

The Stress Tensor for Simple Shear Flows of a Granular Material,"

The complete stress tensor has been measured using a computer simulation of an assemblage of rough, inelastic spheres in an imposed simple shear flow. Only five components of the stress tensor were found to be significantly different from zero. These represent the disperssive normal stresses 7,,, 7yy and 7,, and the in-the-shearplane shear stresses 7,y and 7yz; furthermore, the two off-diagonal stresses, r X y and rYz, were found to be equal so that the resultant stress tensor is symmetric. Two modes of microscopic momentum transport produce the final macroscopic stress tensor: the streaming or kinetic mode by which particles carry the momentum of their motion as they move through the bulk material, and the collisional mode by which momentum is transported by interparticle collisions. The contribution of each to the final result is examined separately. The friction coefficient, the ratio of shear to normal force, is shown to decrease at dense packings for both the collisional and streaming modes. Also observed were normal stress differences, both in and out of the shear plane, reflecting anisotropies in the granular temperature

Existence of multi-site intrinsic localized modes in one-dimensional Debye crystals

The existence of highly localized multi-site oscillatory structures (discrete multibreathers) in a nonlinear Klein-Gordon chain which is characterized by an inverse dispersion law is proven and their linear stability is investigated. The results are applied in the description of vertical (transverse, off-plane) dust grain motion in dusty plasma crystals, by taking into account the lattice discreteness and the sheath electric and/or magnetic field nonlinearity. Explicit values from experimental plasma discharge experiments are considered. The possibility for the occurrence of multibreathers associated with vertical charged dust grain motion in strongly-coupled dusty plasmas (dust crystals) is thus established. From a fundamental point of view, this study aims at providing a first rigorous investigation of the existence of intrinsic localized modes in Debye crystals and/or dusty plasma crystals and, in fact, suggesting those lattices as model systems for the study of fundamental crystal properties.Comment: 12 pages, 8 figures, revtex forma

Diffuse flow environments within basalt- and sediment-based hydrothermal vent ecosystems harbor specialized microbial communities

Hydrothermal vents differ both in surface input and subsurface geochemistry. The effects of these differences on their microbial communities are not clear. Here, we investigated both alpha and beta diversity of diffuse flow-associated microbial communities emanating from vents at a basalt-based hydrothermal system along the East Pacific Rise (EPR) and a sediment-based hydrothermal system, Guaymas Basin. Both Bacteria and Archaea were targeted using high throughput 16S rRNA gene pyrosequencing analyses. A unique aspect of this study was the use of a universal set of 16S rRNA gene primers to characterize total and diffuse flow-specific microbial communities from varied deep-sea hydrothermal environments. Both surrounding seawater and diffuse flow water samples contained large numbers of Marine Group I (MGI) Thaumarchaea and Gammaproteobacteria taxa previously observed in deep-sea systems. However, these taxa were geographically distinct and segregated according to type of spreading center. Diffuse flow microbial community profiles were highly differentiated. In particular, EPR dominant diffuse flow taxa were most closely associated with chemolithoautotrophs, and off axis water was dominated by heterotrophic-related taxa, whereas the opposite was true for Guaymas Basin. The diversity and richness of diffuse flow-specific microbial communities were strongly correlated to the relative abundance of Epsilonproteobacteria, proximity to macrofauna, and hydrothermal system type. Archaeal diversity was higher than or equivalent to bacterial diversity in about one third of the samples. Most diffuse flow-specific communities were dominated by OTUs associated with Epsilonproteobacteria, but many of the Guaymas Basin diffuse flow samples were dominated by either OTUs within the Planctomycetes or hyperthermophilic Archaea. This study emphasizes the unique microbial communities associated with geochemically and geographically distinct hydrothermal diffuse flow environments

Experimental Space Shuttle Orbiter Studies to Acquire Data for Code and Flight Heating Model Validation

In an experimental study to obtain detailed heating data over the Space Shuttle Orbiter, CUBRC has completed an extensive matrix of experiments using three distinct models and two unique hypervelocity wind tunnel facilities. This detailed data will be employed to assess heating augmentation due to boundary layer transition on the Orbiter wing leading edge and wind side acreage with comparisons to computational methods and flight data obtained during the Orbiter Entry Boundary Layer Flight Experiment and HYTHIRM during STS-119 reentry. These comparisons will facilitate critical updates to be made to the engineering tools employed to make assessments about natural and tripped boundary layer transition during Orbiter reentry. To achieve the goals of this study data was obtained over a range of Mach numbers from 10 to 18, with flight scaled Reynolds numbers and model attitudes representing key points on the Orbiter reentry trajectory. The first of these studies were performed as an integral part of Return to Flight activities following the accident that occurred during the reentry of the Space Shuttle Columbia (STS-107) in February of 2003. This accident was caused by debris, which originated from the foam covering the external tank bipod fitting ramps, striking and damaging critical wing leading edge heating tiles that reside in the Orbiter bow shock/wing interaction region. During investigation of the accident aeroheating team members discovered that only a limited amount of experimental wing leading edge data existed in this critical peak heating area and a need arose to acquire a detailed dataset of heating in this region. This new dataset was acquired in three phases consisting of a risk mitigation phase employing a 1.8% scale Orbiter model with special temperature sensitive paint covering the wing leading edge, a 0.9% scale Orbiter model with high resolution thin-film instrumentation in the span direction, and the primary 1.8% scale Orbiter model with detailed thin-film resolution in both the span and chord direction in the area of peak heating. Additional objectives of this first study included: obtaining natural or tripped turbulent wing leading edge heating levels, assessing the effectiveness of protuberances and cavities placed at specified locations on the orbiter over a range of Mach numbers and Reynolds numbers to evaluate and compare to existing engineering and computational tools, obtaining cavity floor heating to aid in the verification of cavity heating correlations, acquiring control surface deflection heating data on both the main body flap and elevons, and obtain high speed schlieren videos of the interaction of the orbiter nose bow shock with the wing leading edge. To support these objectives, the stainless steel 1.8% scale orbiter model in addition to the sensors on the wing leading edge was instrumented down the windward centerline, over the wing acreage on the port side, and painted with temperature sensitive paint on the starboard side wing acreage. In all, the stainless steel 1.8% scale Orbiter model was instrumented with over three-hundred highly sensitive thin-film heating sensors, two-hundred of which were located in the wing leading edge shock interaction region. Further experimental studies will also be performed following the successful acquisition of flight data during the Orbiter Entry Boundary Layer Flight Experiment and HYTHIRM on STS-119 at specific data points simulating flight conditions and geometries. Additional instrumentation and a protuberance matching the layout present during the STS-119 boundary layer transition flight experiment were added with testing performed at Mach number and Reynolds number conditions simulating conditions experienced in flight. In addition to the experimental studies, CUBRC also performed a large amount of CFD analysis to confirm and validate not only the tunnel freestream conditions, but also 3D flows over the orbiter acreage, wing leading edge, and controlurfaces to assess data quality, shock interaction locations, and control surface separation regions. This analysis is a standard part of any experimental program at CUBRC, and this information was of key importance for post-test data quality analysis and understanding particular phenomena seen in the data. All work during this effort was sponsored and paid for by the NASA Space Shuttle Program Office at the Johnson Space Center in Houston, Texas

BLIMPK/Streamline Surface Catalytic Heating Predictions on the Space Shuttle Orbiter

This paper describes the results of an analysis of localized catalytic heating effects to the U.S. Space Shuttle Orbiter Thermal Protection System (TPS). The analysis applies to the High-temperature Reusable Surface Insulation (HRSI) on the lower fuselage and wing acreage, as well as the critical Reinforced Carbon-Carbon on the nose cap, chin panel and the wing leading edge. The object of the analysis was to use a modified two-layer approach to predict the catalytic heating effects on the Orbiter windward HRSI tile acreage, nose cap, and wing leading edge assuming localized highly catalytic or fully catalytic surfaces. The method incorporated the Boundary Layer Integral Matrix Procedure Kinetic (BLIMPK) code with streamline inputs from viscous Navier-Stokes solutions to produce heating rates for localized fully catalytic and highly catalytic surfaces as well as for nominal partially catalytic surfaces (either Reinforced Carbon-Carbon or Reaction Cured Glass) with temperature-dependent recombination coefficients. The highly catalytic heating results showed very good correlation with Orbiter Experiments STS-2, -3, and -5 centerline and STS-5 wing flight data for the HRSI tiles. Recommended catalytic heating factors were generated for use in future Shuttle missions in the event of quick-time analysis of damaged or repaired TPS areas during atmospheric reentry. The catalytic factors are presented along the streamlines as well as a function of stagnation enthalpy so they can be used for arbitrary trajectories

Computer Simulation of Granular Shear Flows

Detailed understanding of flowing granular materials is severly hampered by the deficiencies of present experimental methods. To help increase the information base, a computer simulation has been developed to describe two-dimensional unidirectional flows of inelastic fully rough particles. This paper presents the results of a Couette shear-flow simulation. The results include distributions of velocity, density and granular temperature (a measure of the kinetic energy contained in the random particle motions). The effects of density and shear rate on the granular temperature are explored. Shear and normal forces on the solid walls are compared with experimental and theoretical results. The behaviour of the particles in the simulated flow is examined and assessments are made of the collision angle and velocity distributions. The development of a distinct, 'layered' microstructure is observed in high-density granular flows

Model-independent extraction of ∣Vtq∣|V_{tq}| matrix elements from top-quark measurements at hadron colliders

Current methods to extract the quark-mixing matrix element $|V_{tb}|$ from single-top production measurements assume that $|V_{tb}|\gg |V_{td}|, |V_{ts}|$: top quarks decay into $b$ quarks with 100% branching fraction, s-channel single-top production is always accompanied by a $b$ quark and initial-state contributions from $d$ and $s$ quarks in the $t$-channel production of single top quarks are neglected. Triggered by a recent measurement of the ratio $R=\frac{|V_{tb}|^{2}}{|V_{td}|^{2}+|V_{ts}|^{2}+|V_{tb}|^{2}}=0.90 \pm 0.04$ performed by the D0 collaboration, we consider a $|V_{tb}|$ extraction method that takes into account non zero d- and s-quark contributions both in production and decay. We propose a strategy that allows to extract consistently and in a model-independent way the quark mixing matrix elements $|V_{td}|$, $|V_{ts}|$, and $|V_{tb}|$ from the measurement of $R$ and from single-top measured event yields. As an illustration, we apply our method to the Tevatron data using a CDF analysis of the measured single-top event yield with two jets in the final state one of which is identified as a $b$-quark jet. We constrain the $|V_{tq}|$ matrix elements within a four-generation scenario by combining the results with those obtained from direct measurements in flavor physics and determine the preferred range for the top-quark decay width within different scenarios.Comment: 36 pages, 17 figure