Nitric oxide mediates inactivation of glutathione S-transferase in suspension culture of Taxus cuspidata during shear stress

The importance of nitric oxide (NO) in regulating plant cell responses to environmental stresses is becoming evident. Here the possible role of NO in suspension cultures of Taxus cuspidata under shear stress was investigated in a Couette-type shear reactor. It was found that shear stress with 190 s(-1) caused NO generation in 8 h. NO formation can be inhibited by N-nitro-L-arginine, a nitric oxide synthase inhibitor. Moreover, the activity of glutathione S-transferase (GST), a principal enzyme responsible for detoxification, decreased during shear stress. This inactivation partially recovered when NOS inhibitor or NO scavenger was added into cell cultures during shear stress. Treatment with reactive nitrogen species (RNS) also caused inactivation of GST in cells. The results indicate that NO plays a crucial role in GST inactivation in Taxus cuspidata cells under shear stress.     

Metabolic response of biofilm to shear stress in fixed-film culture

AIMS: In a biofilm reactor, detachment force resulting from hydraulic shear is a major factor that determines the formation and structure of steady state biofilm. The metabolic response of biofilm to change in shear stress was therefore investigated. METHODS AND RESULTS: A conventional annular reactor made of PVC was used, in which shearing over the rotating disc surface was strictly defined. Results from the steady state aerobic biofilm reactor showed that the biofilm structure (density and thickness) and metabolic behaviour (growth yield and dehydrogenase activity) were closely related to the shear stress exerted on the biofilm. Smooth, dense and stable biofilm formed at relatively high shear stress. Higher dehydrogenase activity and lower growth yield were obtained when the shear stress was raised. Growth yield was inversely correlated with the catabolic activity of biofilm. The reduced growth yield, together with the enhanced catabolic activity, suggests that a dissociation of catabolism from anabolism would occur at high shear stress. CONCLUSION: Biofilms may respond to shear stress by regulating metabolic pathways associated with the substrate flux flowing between catabolism and anabolism. A biological phenomenon, besides a simple physical effect, is underlying the observed relation between the shear stress and resulting biofilm structure. SIGNIFICANCE AND IMPACT OF THE STUDY: A hypothesis is proposed that the shear-induced energy spilling would be associated with a stimulated proton translocation across the cell membrane, which favours formation of a stronger biofilm. This research may provide a basis for experimental data on biofilm obtained at different shear stresses to be interpreted in relation to energy.     

The effect of shear on the desorption of liposomes adsorbed to bacterial biofilms

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,beta[N-(N1,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immobilized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome-biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen-antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear-induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (ED). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break-up of doublets (two-cell agglutinates) is produced at a critical shear stress (tauC) reflecting the work done to breaking-up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r-log tau] to the backscattered signal (r0) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Gamma) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, ED and Gamma, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.     

Shear deformation effects in enzyme catalysis. Metal ion effect in the shear inactivation of urease.

The mechanism of the inactivation of the enzyme urease produced by subjecting its dilute solutions to hydrodynamic shear stresses in the range 0.5-2.5 Pa has been determined. By studying the kinetics of urease-catalyzed urea hydrolysis during application of hydrodynamic shear under varying chemical environments, we demonstrate that micromolar quantities of metal ions, in this case adventitious Fe, can accelerate the oxidation of thiol groups on urease and thus inactivate it when the protein is subjected to a shearing stress of order 1.0 Pa. In the absence of metal ion this stress level is ineffectual. It is proposed that this type of synergy between deformation and chemical environment may be crucial in many situations where biological macromolecules are subjected to mechanical stress.     

The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Adaptive regulation of wall shear stress optimizing vascular tree function

The branching structure of the mammalian arterial tree has been known to be close to that of an optimal conduit system of the minimum work model characterized as the branch system of constant wall shear rate. The physiological mechanism producing such construction was considered to be based on the local response of arterial caliber induced by the wall shear stress (shear rate × blood viscosity) and thereby maintaining this stress constant, which was previously observed at the canine common carotid artery shunted to the external jugular vein. The stress levels at various parts of the arterial system estimated from available data fell within ±50% of the mean (15 dyn/cm²), which was consistent with the value predicted from the model. Theoretical analyses on the cost function of the model indicated that the suspected variation of shear rate levels in the arterial tree due to the anomalous changes in blood viscosity which might bring about 3- to 4-fold differences between the minimum and maximum shear rates would cause less than 10% increase in the total energy cost. It was concluded that a local adaptive response to wall shear stress is the mechanism which effectively optimizes the design of the arterial tree.     

Theory to Predict Shear Stress on Cells in Turbulent Blood Flow

Shear stress on blood cells and platelets transported in a turbulent flow dictates the fate and biological activity of these cells. We present a theoretical link between energy dissipation in turbulent flows to the shear stress that cells experience and show that for the case of physiological turbulent blood flow: (a) the Newtonian assumption is valid, (b) turbulent eddies are universal for the most complex of blood flow problems, and (c) shear stress distribution on turbulent blood flows is possibly universal. Further we resolve a long standing inconsistency in hemolysis between laminar and turbulent flow using the theoretical framework. This work demonstrates that energy dissipation as opposed to bulk shear stress in laminar or turbulent blood flow dictates local mechanical environment of blood cells and platelets universally.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.     

An optimization principle for vascular radius including the effects of smooth muscle tone.

An optimization principle is proposed for the regulation of vascular morphology. This principle, which extends Murray's law, is based on the hypothesis that blood vessel diameter is controlled by a mechanism that minimizes the total energy required to drive the blood flow, to maintain the blood supply, and to support smooth muscle tone. A theoretical analysis reveals that the proposed principle predicts that the optimum shear stress on the vessel wall due to blood flow increases with blood pressure. This result agrees qualitatively with published findings that the fluid shear stress in veins is significantly smaller than it is in arteries.     

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Experimental measurement of dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm² at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.     

A physical derivation of nutrient-uptake rates in coral reefs: effects of roughness and waves

Mass-transfer rates between water and benthos are derived based on the dissipation of energy by the benthic communities of coral reefs. Roughness of the benthic communities causes currents and waves to dissipate energy on reef flats at rates which far exceed ocean values of energy dissipation. The derivation here shows that first-order rate constants for nutrient uptake are (1) proportional to energy dissipation to the 0.25 root, (2) proportional to the bottom shear stress to the 0.4 root, and (3) proportional to current speed to the 0.75 root (decreasing to the 0.4 root under extreme wave activity). The shear stress, thus nutrient uptake, is positively correlated to the large-scale roughness, and to excess wave height (above the breaking height) of incoming waves. These causal relationships between nutrient-uptake rates and dissipation of energy support the general observations of reef zonation and reef metabolic rates, and are the paramount reason that coral reefs can maintain high productivity in low-nutrient tropical waters.     

Experimental study of microorganism disruption using shear stress

There has been a broad spectrum of theoretical and experimental works on microorganism disruption methods undertaken in the past. However, there is a lack of understanding regarding the actual reasons for microorganism disruption using ultrasound and whether it is caused by shock or shear. In the case of shear stress, which is the focus of this paper, analysis of the intense turbulent flow region of an in-house built shear apparatus combined with the experimental results demonstrated that when the energy dissipation rate in the turbulence region is high, and the size of the eddy is smaller than the size of the cell, the likelihood of yeast disruption is high. The mechanical properties of yeast cells combined with the calculated energy dissipation rate were used to evaluate the yeast disruption efficiency (log reduction). The results show that the shear apparatus can efficiently and effectively disrupt S. cerevisiae at different treatment times, suspension temperatures and rotor speeds. The experimental work suggests that maximum yeast log reduction was achieved when the maximum power dissipation of 2.095 kW was recorded at 10,000 RPM, while suspension temperature was controlled below 35 °C. The corresponding shear stress at 10,000 RPM was 2586.2 Pa.     

Accurate prediction of wall shear stress in a stented artery: newtonian versus non-newtonian models

A significant amount of evidence linking wall shear stress to neointimal hyperplasia has been reported in the literature. As a result, numerical and experimental models have been created to study the influence of stent design on wall shear stress. Traditionally, blood has been assumed to behave as a Newtonian fluid, but recently that assumption has been challenged. The use of a linear model; however, can reduce computational cost, and allow the use of Newtonian fluids (e.g., glycerine and water) instead of a blood analog fluid in an experimental setup. Therefore, it is of interest whether a linear model can be used to accurately predict the wall shear stress caused by a non-Newtonian fluid such as blood within a stented arterial segment. The present work compares the resulting wall shear stress obtained using two linear and one nonlinear model under the same flow waveform. All numerical models are fully three-dimensional, transient, and incorporate a realistic stent geometry. It is shown that traditional linear models (based on blood's lowest viscosity limit, 3.5 Pa s) underestimate the wall shear stress within a stented arterial segment, which can lead to an overestimation of the risk of restenosis. The second linear model, which uses a characteristic viscosity (based on an average strain rate, 4.7 Pa s), results in higher wall shear stress levels, but which are still substantially below those of the nonlinear model. It is therefore shown that nonlinear models result in more accurate predictions of wall shear stress within a stented arterial segment.     

Hyperactivated Wnt Signaling Induces Synthetic Lethal Interaction with Rb Inactivation by Elevating TORC1 Activities

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.     

Non-Newtonian blood flow in human right coronary arteries: steady state simulations

This study looks at blood flow through four different right coronary arteries, which have been reconstructed from bi-plane angiograms. Five non-Newtonian blood models, as well as the usual Newtonian model of blood viscosity, are used to study the wall shear stress in each of these arteries at a particular point in the cardiac cycle. It was found that in the case of steady flow in a given artery, the pattern of wall shear stress is consistent across all models. The magnitude of wall shear stress, however, is influenced by the model used and correlates with graphs of shear stress versus strain for each model. For mid-range velocities of around 0.2 m s(-1) the models are virtually indistinguishable. Local and global non-Newtonian importance factors are introduced, in an attempt to quantify the types of flows where non-Newtonian behaviour is significant. It is concluded that, while the Newtonian model of blood viscosity is a good approximation in regions of mid-range to high shear, it is advisable to use the Generalised Power Law model (which tends to the Newtonian model in those shear ranges in any case) in order to achieve better approximation of wall shear stress at low shear.     

Rheology and ultrasound scattering from aggregated red cell suspensions in shear flow

The shear flow dynamics of reversible red cell aggregates in dense suspensions were investigated by ultrasound scattering, to study the shear disruption processes of Rayleigh clusters and examine the effective mean field approximation used in microrheological models. In a first section, a rheo-acoustical model, in the Rayleigh scattering regime, is proposed to describe the shear stress dependence of the low frequency scattered power in relation to structural parameters. The fractal scattering regime characterizing the anisotropic scattering from flocs of size larger than the ultrasound wavelength is further discussed. In the second section, we report flow-dependent changes in the low-frequency scattering coefficient in a plane-plane flow geometry to analyze the shear disruption processes of hardened or deformable red cell aggregates in neutral dextran polymer solution. Rheo-acoustical experiments are examined on the basis of the rheo-acoustical model and the effective medium approximation. The ability of ultrasound scattering technique to determine the critical disaggregation shear stress and to give quantitative information on particle surface adhesive energy is analyzed. Lastly, the shear-thinning behavior of weakly aggregated hardened or deformable red cells is described.