Numerical simulation of transom-stern waves

The flow field generated by a transom-stern hullform is a complex, broad-banded, three-dimensional phenomenon marked by a large breaking wave. This unsteady multiphase turbulent flow feature is difficult to study experimentally and simulate numerically. The results of a set of numerical simulations, which use the Numerical Flow Analysis (NFA) code, of the flow around the Model 5673 transom stern at speeds covering both wet- and dry-transom operating conditions are shown in the accompanying fluid dynamics video. The numerical predictions for wet-transom and dry-transom conditions are presented to demonstrate the current state of the art in the simulation of ship generated breaking waves. The interested reader is referred to Drazen et al. (2010) for a detailed and comprehensive comparison with experiments conducted at the Naval Surface Warfare Center Carderock Division (NSWCCD).Comment: Fluid Dynamics Video for 2010 APS Division of Fluid Dynamics Gallery of Fluid Motion include

The Effect of Inoculants on Nutrient Losses of Corn Silage and High-moisture Corn Stored in Mini Silos

Two experiments were conducted to determine the effects of inoculants (BONSILAGE CORN 200G and BONSILAGE HMC 200G) containing Lactobacillus buchneri, Lactobacillus plantarum and Pediococcus acidilactici on nutrient losses and aerobic stability of corn silage and high moisture corn. Corn silage and high moisture corn were inoculated and stored in mini silos with nutrient loss and spoilage characterizations at 30, 90, and 120 days with multiple inoculation levels. Longer ensiling times led to greater total acid production. The higher levels of inoculation led to lower total acid production and higher pH. Inoculating corn silage and high moisture corn also affected the fermentation process by decreasing lactic acid production and increasing acetic acid production. The increase in acetic acid production may be partially responsible for the increased aerobic stability observed for the inoculated feeds. Previous research would support our finding of greater stability and lower DM losses with L. buchneri inoculants

Evaluation of Latex Immunoturbidimetric Assay Thresholds and HIT in Cardiothoracic Surgery

Background Heparin-induced thrombocytopenia (HIT) is a common differential diagnosis in cardiothoracic surgery. The latex immunoturbidimetric assay (LIA) is an enhanced immunoassay that has recently been introduced for the detection of total HIT immunoglobulin and retains a higher specificity of 95% compared to the enzyme-linked immunosorbent assay. Objectives To investigate if a semiquantitative relationship exists between increasing LIA levels beyond the current positivity threshold and its correlation to positive serotonin release assay results in cardiothoracic surgery. Methods This was a multicenter, observational cohort of cardiothoracic surgery patients initiated on anticoagulation with heparin-based products. To conduct sensitivity and specificity analysis of LIA values, HIT positive was defined as a LIA value ≥1 unit/mL and HIT negative was defined as a LIA level <1 unit/mL. A receiver operating characteristic (ROC) analysis was utilized to evaluate the predictive performance of the LIA. Results At manufactures’ cutoffs of ≥1.0 unit/mL, LIA sensitivity and specificity was 93.8% and 22%, respectively, yielding a false positive rate of 78%. At a higher cutoff of 4.5 units/mL, LIA sensitivity and specificity was 75% and 71%, respectively, yielding a false positive rate of 29% and an area under the ROC curve of 0.75 (P = .01; 95% confidence interval: 0.621-0.889). Bivalirudin was initiated in 84.6% of false positive LIA results. Conclusion This study suggests that the diagnostic accuracy of the LIA can be optimized by increasing the LIA positivity threshold. Proposing a higher LIA cutoff, may mitigate unwarranted anticoagulation and bleeding outcomes

Computational Model Explains High Activity and Rapid Cycling of Rho GTPases within Protein Complexes

Formation of multiprotein complexes on cellular membranes is critically dependent on the cyclic activation of small GTPases. FRAP-based analyses demonstrate that within protein complexes, some small GTPases cycle nearly three orders of magnitude faster than they would spontaneously cycle in vitro. At the same time, experiments report concomitant excess of the activated, GTP-bound form of GTPases over their inactive form. Intuitively, high activity and rapid turnover are contradictory requirements. How the cells manage to maximize both remains poorly understood. Here, using GTPases of the Rab and Rho families as a prototype, we introduce a computational model of the GTPase cycle. We quantitatively investigate several plausible layouts of the cycling control module that consist of GEFs, GAPs, and GTPase effectors. We explain the existing experimental data and predict how the cycling of GTPases is controlled by the regulatory proteins in vivo. Our model explains distinct and separable roles that the activating GEFs and deactivating GAPs play in the GTPase cycling control. While the activity of GTPase is mainly defined by GEF, the turnover rate is a sole function of GAP. Maximization of the GTPase activity and turnover rate places conflicting requirements on the concentration of GAP. Therefore, to achieve a high activity and turnover rate at once, cells must carefully maintain concentrations of GEFs and GAPs within the optimal range. The values of these optimal concentrations indicate that efficient cycling can be achieved only within dense protein complexes typically assembled on the membrane surfaces. We show that the concentration requirement for GEF can be dramatically reduced by a GEF-activating GTPase effector that can also significantly boost the cycling efficiency. Interestingly, we find that the cycling regimes are only weakly dependent on the concentration of GTPase itself

Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons1

Phosphatidylinositol 4,5-bisphosphate (PIP2) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP2 levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons

Fluorescence changes reveal kinetic steps of muscarinic receptor–mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

G protein–coupled receptors initiate signaling cascades. M1 muscarinic receptor (M1R) activation couples through Gαq to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M1R activation, M1R/Gβ interaction, Gαq/Gβ separation, Gαq/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gαq/Gβ separation and Gαq/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gαq/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC

Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation