Identifying a minimal rheological configuration: a tool for effective and efficient constitutive modeling of soft tissues

We describe a modeling methodology intended as a preliminary step in the identification of appropriate constitutive frameworks for the time-dependent response of biological tissues. The modeling approach comprises a customizable rheological network of viscous and elastic elements governed by user-defined 1D constitutive relationships. The model parameters are identified by iterative nonlinear optimization, minimizing the error between experimental and model-predicted structural (load-displacement) tissue response under a specific mode of deformation. We demonstrate the use of this methodology by determining the minimal rheological arrangement, constitutive relationships, and model parameters for the structural response of various soft tissues, including ex vivo perfused porcine liver in indentation, ex vivo porcine brain cortical tissue in indentation, and ex vivo human cervical tissue in unconfined compression. Our results indicate that the identified rheological configurations provide good agreement with experimental data, including multiple constant strain rate load/unload tests and stress relaxation tests. Our experience suggests that the described modeling framework is an efficient tool for exploring a wide array of constitutive relationships and rheological arrangements, which can subsequently serve as a basis for 3D constitutive model development and finite-element implementations. The proposed approach can also be employed as a self-contained tool to obtain simplified 1D phenomenological models of the structural response of biological tissue to single-axis manipulations for applications in haptic technologies.     

Mitochondrial dysfunction and effect of antiglycolytic bromopyruvic acid in GL15 glioblastoma cells

Most cancer cells, including GL15 glioblastoma cells, rely on glycolysis for energy supply. The effect of antiglycolytic bromopyruvate on respiratory parameters and viability of GL15 cells was investigated. Bromopyruvate caused Δψ_m and MTT collapse, ATP decrease, and cell viability loss without involving apoptotic or necrotic pathways. The autophagy marker LC3-II was increased. Δψ_m decrease was accompanied by reactive oxygen species (ROS) increase and cytochrome c (cyt c) disappearance, suggesting a link between free radical generation and intramitochondrial cyt c degradation. Indeed, the free radical inducer menadione caused a decrease in cyt c that was reversed by N-acetylcysteine. Cyt c is tightly bound to the inner mitochondrial membrane in GL15 cells, which may confer protein peroxidase activity, resulting in auto-oxidation and protein targeting to degradation in the presence of ROS. This process is directed towards impairment of the apoptotic cyt c cascade, although cells are committed to die.     

Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities

Basidiomycete mushrooms are a rich source of unique substances, including lectins, that could potentially be useful in biotechnology or biomedical applications. Lectins are a group of carbohydrate-binding proteins with diverse biological activities and functions. Here, we demonstrate the presence of a number of lectins in the basidiomycete mushroom Clitocybe nebularis. Glucose-, galactose-, sucrose-, lactose-, and Sepharose-binding lectins were isolated from fruiting bodies using affinity chromatography on Sepharose-immobilized sugars or on Sepharose. The lectins were characterized biochemically and their binding specificities examined by agglutination and agglutination inhibition assays. In addition, insecticidal and anti-nutritional properties of the lectins were studied against a model organism, fruit fly (Drosophila melanogaster), and Colorado potato beetle (Leptinotarsa decemlineata). Of the several basidiomycete mushrooms screened, C. nebularis extract showed the most potent insecticidal activity. Sucrose-binding lectin showed the strongest activity against D. melanogaster, followed by lactose- and galactose-binding lectins. Feeding bioassays with Colorado potato beetle revealed that C. nebularis extract exhibited high anti-nutritional activity against the insect; and of those tested, only lactose-binding lectin, named CNL showed the effect. Mushroom C. nebularis is shown to be rich in a variety of lectins with versatile biological activities, including insecticidal and anti-nutritional effects. C. nebularis lectins could thus have potential for use as natural insecticides.     

Influence of the hydromechanical stress and temperature on growth and antibody fragment production with <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the temperature and the hydromechanical stress play a very important role for significant product formation during process development or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs of about 0.5 kW/m³ (agitation rates around 500 min⁻¹) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input of about 0.06 kW/m³ (agitation rate around 250 min⁻¹) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew at low volumetric power inputs and high cultivation temperatures.     

A method for measuring and modeling the physiological traits causing obstructive sleep apnea

There is not a clinically available technique for measuring the physiological traits causing obstructive sleep apnea (OSA). Therefore, it is often difficult to determine why an individual has OSA or to what extent the various traits contribute to the development of OSA. In this study, we present a noninvasive method for measuring four important physiological traits causing OSA: 1) pharyngeal anatomy/collapsibility, 2) ventilatory control system gain (loop gain), 3) the ability of the upper airway to dilate/stiffen in response to an increase in ventilatory drive, and 4) arousal threshold. These variables are measured using a single maneuver in which continuous positive airway pressure (CPAP) is dropped from an optimum to various suboptimum pressures for 3- to 5-min intervals during sleep. Each individual's set of traits is entered into a physiological model of OSA that graphically illustrates the relative importance of each trait in that individual. Results from 14 subjects (10 with OSA) are described. Repeatability measurements from separate nights are also presented for four subjects. The measurements and model illustrate the multifactorial nature of OSA pathogenesis and how, in some individuals, small adjustments of one or another trait (which might be achievable with non-CPAP agents) could potentially treat OSA. This technique could conceivably be used clinically to define a patient's physiology and guide therapy based on the traits.     

Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.     

Heme interacts with c1q and inhibits the classical complement pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.     

Optical measurements of vocal fold tensile properties: implications for phonatory mechanics

In voice research, in vitro tensile stretch experiments of vocal fold tissues are commonly employed to determine the tissue biomechanical properties. In the standard stretch-release protocol, tissue deformation is computed from displacements applied to sutures inserted through the thyroid and arytenoid cartilages, with the cartilages assumed to be rigid. Here, a non-contact optical method was employed to determine the actual tissue deformation of vocal fold lamina propria specimens from three excised human larynges in uniaxial tensile tests. Specimen deformation was found to consist not only of deformation of the tissue itself, but also deformation of the cartilages, as well as suture alignment and tightening. Stress-stretch curves of a representative load cycle were characterized by an incompressible Ogden model. The initial longitudinal elastic modulus was found to be considerably higher if determined based on optical displacement measurements than typical values reported in the literature. The present findings could change the understanding of the mechanics underlying vocal fold vibration. Given the high longitudinal elastic modulus the lamina propria appeared to demonstrate a substantial level of anisotropy. Consequently, transverse shear could play a significant role in vocal fold vibration, and fundamental frequencies of phonation should be predicted by beam theories accounting for such effects.