The Hippo Pathway Regulates Caveolae Expression and Mediates Flow Response via Caveolae

1. Download high-res image (131KB)
2. Download full-size image

     

Effect of repeated microwave disinfections on bonding of different commercial teeth to resin denture base

doi: 10.1111/j.1741‐2358.2011.00516.x Effect of repeated microwave disinfections on bonding of different commercial teeth to resin denture base Objective: To verify the influence of repeated microwave disinfections on the shear bond strength of two commercial types of teeth to acrylic resin, when the ridge lap surfaces were unmodified, bur abraded, bur grooved or etched by monomer. Material and methods: Eighty specimens (n = 10) were adhered to the tooth ridge lap surface, polymerised in a water bath at 74°C for 9 h. Microwaved specimens were individually immersed in 150 ml of water and submitted to five simulated disinfections in a microwave oven calibrated at 650 W for 3 min. Control specimens were not microwave treated. Shear bond strength tests were performed in an Instron machine with a cross‐speed of 1 mm/min. The fracture load values were transformed into shear bond strength as a function of the bonding area (0.28 cm²). Data were submitted to anova and Tukey’s test (α = 0.05). Fractured areas were classified as adhesive, cohesive (resin or tooth) or mixed failures. Results: Repeated microwave disinfections increased the shear strength of the tooth/resin bond. Mechanical retention in microwaved and non‐microwaved procedures improved the shear bond strength. Conclusions: The different commercial types of teeth influenced shear bond strength values, with Biotone teeth showing the lower values.     

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

Exposure of Bacillus subtilis to a shear rate of 1,482/s leads to a rapid loss of cell viability after 10 h of growth. Biochemical and molecular evidences provided below strongly suggest that cell death under high shear results from an apoptosis-like process similar to that described in eukaryotes, with activation of a caspase-3-like protease (C(3)LP) followed by DNA fragmentation. Shear stress leads to an increase in specific intracellular reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS precedes the activation of C(3)LP and DNA fragmentation, thus establishing siROS as the molecular link between shear stress and apoptosis-like cell death. A model is proposed in which NOX is viewed as being strategically placed on the plasma membrane of B. subtilis that senses and converts a mechanical force arising from shear stress into a chemical signal leading to activation of C(3)LP, DNA fragmentation, and thus, apoptosis-like cell death.     

Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.     

Ultra scale-down studies of the effect of flow and impact conditions during E. coli cell processing

The ability to recover cells from a fermentation broth in an intact form can be an important criterion for determining the overall performance of a recovery and purification sequence. Disruption of the cells can lead to undesired contamination of an extracellular product with intracellular components and vice versa loss of intracellular products may occur. In particular, the value of directed location of a product in the periplasmic space of say Escherichia coli (E. coli) would be diminished by such premature non-selective cell disruption. Several options exist for cell recovery/removal; namely centrifugation, in batch or continuous configuration, filtration or membrane operations, and in selected cases expanded beds. The choice of operation is dependant on many variables including the impact on the overall process sequence. In all cases, the cells are exposed to shear stresses of varying levels and times and additionally such environments exist in ancillary operations such as pumping, pipe flow, and control valves. In this study, a small-scale device has been designed to expose cells to controlled levels of shear, time and impact in a way that seeks to mimic those effects that may occur during full-scale processes. The extent of cell breakage was found to be proportional to shear stress. An additional level of breakage occurred due to the jet impacting on the collecting surface. Here it was possible to correlate the additional breakage with the impact velocity, which is a function of the distance that the jet travels before meeting the collection surface and the initial jet velocity.     

Modeling fluid flow through irregular scaffolds for perfusion bioreactors

Direct perfusion of 3D tissue engineered constructs is known to enhance osteogenesis, which can be partly attributed to enhanced nutrient and waste transport. In addition flow mediated shear stresses are known to upregulate osteogenic differentiation and mineralization. A quantification of the hydrodynamic environment is therefore crucial to interpret and compare results of in vitro bioreactor experiments. This study aims to deal with the pitfalls of numerical model preparation of highly complex 3D bone scaffold structures and aims to provide more accurate wall shear stress (WSS) estimates. µCT imaging techniques were used to reconstruct the geometry of both a titanium (Ti) and a hydroxyapatite scaffold, starting from 430 images with a resolution of 8 µm. To tackle the tradeoff between model size and mesh resolution we selected two concentric regions of interest (cubes with a volume of 1 and 3.375 mm³, respectively) for both scaffolds. A flow guidance in front of the real inlet surface of the scaffold was designed to mimic realistic inlet conditions. With a flow rate of 0.04 mL/min perfused through a 5 mm diameter scaffold at an inlet velocity of 33.95 µm/s we obtained average WSSs of 1.10 and 1.46 mPa for the 1 mm³ and the 3.375 mm³ model of the hydroxyapatite scaffold compared to 1.40 and 1.95 mPa for the 1 mm³ model and the 3.375 mm³ model of the Ti scaffold, showing the important influence of the scaffold micro‐architecture heterogeneity and the proximity of boundaries. To assess that influence we selected cubic portions, of which the WSS data were analyzed, with the same size and the same location within both 1 and 3.375 mm³ cubic models. Varying the size of the inner portions simultaneously in both model selections gives a quantification of the sensitivity to boundary neighborhood. This methodology allows to get more insight in the complex concept of tissue engineering and will likely help to understand and eventually improve the fluid‐mechanical aspects. Biotechnol. Bioeng. 2009;103: 621–630. © 2009 Wiley Periodicals, Inc.     

Regenerative medicine bioprocessing: Concentration and behavior of adherent cell suspensions and pastes

Regenerative medicines based on human cells demand their harvesting, culture, and processing. Manufacturing processes are likely to include cell concentration and subsequent controlled dosing of concentrates, for example, to the patient or tissue construct. The integrity and functionality of the cells must be maintained during these processing stages. In this study the performance of two different cell concentration protocols (involving centrifugation and resuspension) are compared and consideration given to possible causes of cell loss. Further studies examine cell size and rheological behavior of anchorage‐dependent mammalian cell suspensions, and the effect of capillary flow stress (0.5–15 Pa, laminar flow regime) on cell number and membrane integrity as quantified by flow cytometry. The cell concentration protocols achieved maximum cell volume fraction of around 0.3 and the improved protocol exhibited intact cell yield of 80 ± 13%, demonstrating proof‐of principle for achieving tissue‐like cell concentrations by a process of centrifugation and orbital shaking. Volume mean cell diameter (cell diameter at the mean cell volume) for the rat aortic smooth muscle cells (CRL‐1444) used in this study was 22.4 µm. Concentrated cell suspension rheology approximated to power law behavior and exhibited similar trends to reports for plant and yeast cells. Capillary transfer at 2–15 Pa (wall shear stress) did not significantly affect cell number or membrane integrity while losses observed at low shear (0.5, 1.0 Pa) were probably due to surface attachment of cells in the apparatus. Biotechnol. Bioeng. 2009;103: 1236–1247. © 2009 Wiley Periodicals, Inc.     

Factors influencing antibody stability at solid–liquid interfaces in a high shear environment

A rotating disk shear device was used to study the effect of interfacial shear on the structural integrity of human monoclonal antibodies of IgG4 isotype. Factors associated with the solution conditions (pH, ionic strength, surfactant concentration, temperature) and the interface (surface roughness) were studied for their effect on the rate of IgG4 monomer loss under high shear conditions. The structural integrity of the IgG4 was probed after exposure to interfacial shear effects by SDS‐PAGE, IEF, dynamic light scattering, and peptide mapping by LC‐MS. This analysis revealed that the main denaturation pathway of IgG4 exposed to these effects was the formation of large insoluble aggregates. Soluble aggregation, breakdown in primary structure, and chemical modifications were not detected. The dominant factors found to affect the rate of IgG4 monomer loss under interfacial shear conditions were found to be pH and the nanometer‐scale surface roughness associated with the solid‐liquid interface. Interestingly, temperature was not found to be a significant factor in the range tested (15–45°C). The addition of surfactant was found to have a significant stabilizing effect at concentrations up to 0.02% (w/v). Implications of these findings for the bioprocessing of this class of therapeutic protein are briefly discussed. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effects of aquatic macrophytes on organic matter deposition,resuspension and phosphorus entrainment in a lowland river

1. Although macrophytes play a key role in the structure and functioning of lowland rivers, most of the basic plant, hydrodynamic and sediment‐water interactions have only been described qualitatively. We therefore studied quantitatively, the seasonal dynamics of matter deposition and mobilisation inside and outside (free path) a representative patch of arrowhead, Sagittaria sagittifolia, in the lowland River Spree, NE Germany, in August 2006. Our in situ study combined resuspension experiments, a hydrodynamically calibrated erosion chamber and concurrent measurements of the prevailing flow characteristics and bed load. 2. Increasing entrainment rates (E) of particles (E_SPM) and total P (E_TP), with increments of shear velocity (U_*) from 0.53 to 2.42 cm s^?1, were significantly higher inside the plant patch than outside. Indeed, E_SPM and E_TP at the lowest U_* were 8‐ and 12‐fold higher inside than outside the patch, reflecting the resuspension potential of the upper nutrient‐enriched layer and the extent of pulsed P inputs even at small increases in U_*. 3. Vertical distribution of velocity (u) revealed a flow pattern of a mixing layer inside the S. sagittifolia patch, and that of a boundary layer in the free path. The highest gradient of u in the mixing layer was located in the water column at about 0.5 m depth, whereas the highest gradient of u for the boundary layer was found near the riverbed. The maximum of U_* (1.65 cm s^?1) was only 4 mm above the sediment. 4. A plant mosaic provides a low‐energetic environment promoting extensive particle trapping and the accumulation of a fine‐grained, nutrient‐enriched sediment, and forming a large resuspension potential. Consequently, after plant decay and the concomitant increase of U_* this material is preferentially entrained at higher rates. Hence, the key role of submerged macrophytes in lowland rivers is more directly related to modifying the dynamic equilibria between vegetation trapping and resuspension, than to the retention of nutrients, particularly P, and the reduction of P loads downstream to other waters.     

Effect of spatial gradient in fluid shear stress on morphological changes in endothelial cells in response to flow

Arterial bifurcations are common sites for development of cerebral aneurysms. Although this localization of aneurysms suggests that high shear stress (SS) and high spatial SS gradient (SSG) occurring at the bifurcations may be crucial factors for endothelial dysfunction involved in aneurysm formation, the details of the relationship between the hemodynamic environment and endothelial cell (EC) responses remain unclear. In the present study, we sought morphological responses of ECs under high-SS and high-SSG conditions using a T-shaped flow chamber. Confluent ECs were exposed to SS of 2-10 Pa with SSG of up to 34 Pa/mm for 24 and 72 h. ECs exposed to SS without spatial gradient elongated and oriented to the direction of flow at 72 h through different processes depending on the magnitude of SS. In contrast, cells did not exhibit preferred orientation and elongation under the combination of SS and SSG. Unlike cells aligned to the flow by exposure to only SS, development of actin stress fibers was not observed in ECs exposed to SS with SSG. These results indicate that SSG suppresses morphological changes of ECs in response to flow.