The gel matrix of gastric mucus is maintained by a complex interplay of transient and nontransient associations

The gel nature of mucus is fundamental to its physiological functions; however, the structure of the mucus gel matrix is unclear. Here, small and large deformation rheology has been used to investigate the physical nature of the gel matrix and the forces that maintain this matrix in pig gastric mucus. The gelation process in mucus has been shown to be comparable with that of other polymer gel systems. Nongelling portions of mucin have been identified within the gel network, and the importance of transient, relaxable interactions to the maintenance of the mucus gel matrix has been demonstrated. The structure of the mucus gel matrix is considered in relation to the functional properties of mucus gels.     

Development of an in vitro model of primate cervical goblet cells

Mucins, are densely packed in secretory granules of goblet cells. Upon exocytosis they undergo massive hydration that results in the formation of the mucus gel. Because the mucin polymer network is held together by tangles and low energy bonds, the rheological properties of this gel are mainly determined by the degree of postexocytotic hydration. Hydration in mucus is governed by a Donnan equilibrium as it is driven by electrostatic interaction among the polyionic charges of the mucins and other fixed polyions. Although, variations of charge density of the mucin polymer could be an efficient physiologic mechanism to control the rate of mucus hydration and rheology, this subject has not been investigated. In here we describe a primary tissue culture system of cervical goblet cells of the monkey uterus. This preparation allows to measure directly the kinetic of hydration of exocytosed mucins. Because the physicochemical parameters of the bathing medium can be effectively controlled, variations in the kinetic of mucins swelling upon exocytosis, can be used as a convenient indicator of fluctuations of charge density in secretory products. Since the cervical mucosa readily respond to endocrine influences, this preparation can provide a useful model to study the effect of hormones or other transmitters on polyionic charge density of secretory product.     

Effect of Ca++ on the structure and rheology of canine tracheal mucin

Mucin glycoproteins are known to be the principal determinants of epithelial mucus rheology and hence of mucociliary transport rates. We are studying the structure of such glycoproteins using a model mucin purified from canine tracheal pouch secretions. Of particular interest is the effect on mucin structure of increased Ca++ such as occurs in certain disease states. Quasielastic laser light scattering was used to study the effect of Ca++ on the hydrodynamic radius of the mucin molecules. Scattering data from 0.3mg/ml mucin solutions in physiological phosphate buffer containing 0, 5 X 10(-5)M, and 5 X 10(-4)M Ca++ were analyzed to obtain an average translational diffusion coefficient and the distribution of molecular radii for the dispersion. The effect of Ca++ was to decrease the average Stokes radius. The light scattering results are supported by rheologic measures of mucin gel viscoelasticity.     

Rheology of gastric mucin exhibits a pH-dependent sol-gel transition

Gastric mucin, a high molecular weight glycoprotein, is responsible for providing the gel-forming properties and protective function of the gastric mucus layer. Bulk rheology measurements in the linear viscoelastic regime show that gastric mucin undergoes a pH-dependent sol-gel transition from a viscoelastic solution at neutral pH to a soft viscoelastic gel in acidic conditions, with the transition occurring near pH 4. In addition to pH-dependent gelation behavior in this system, further rheological studies under nonlinear deformations reveal shear thinning and an apparent yield stress in this material which are also highly influenced by pH.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Rheological properties controlling mucociliary frequency and respiratory mucus transport

Respiratory mucus and mucosa possess highly hydrophilic structures which are difficult to preserve using standard fixative methods. The close interaction between cilia and mucus can be observed after instantaneously interrupting the ciliary movement using ultra rapid and cryosubstitution fixation methods. Mucus possess several rheological properties such as pseudoplasticity, elastothixotropy, spinability and adhesiveness. Rheological properties of mucus may control, per se, the ciliary beating frequency. By measuring the mucociliary frequency on the excised mucus-depleted frog palate of native mucus and xanthan gum using a simulant of mucus, we observed that beyond an optimal value of viscosity (close to 12 Pa.s) the mucociliary frequency and transport rate decrease in parallel. Other rheological factors such as adhesion and spinability of mucus can also be implicated in the regulation of the mucociliary transport rate.     

Oligosaccharides as modulators of rheology in complex mucous systems

Mucus rheology is integral to physiological function with the exact secretion rheology resulting from the macromolecular components, both mucin and nonmucin, and the interactions between these macromolecules. Here we present data demonstrating that low-molecular-weight guluronic acid oligomers extracted from alginate are able to disrupt intermolecular interactions in both purified and native mucous systems, resulting in rheological changes that are compatible with a lower cross-link density and thus reduced resistance to deformation. Additionally, these changes are associated with altered physiological function, raising the possibility of the use of such oligomers in biomedical applications.     

Mucin secretion induced by titanium dioxide nanoparticles

Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO(2)), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO(2) NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO(2) NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO(2) NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca(2+) signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca(2+) concentration were monitored with Rhod-2, a fluorescent Ca(2+) dye. We found that TiO(2) NP-evoked mucin secretion was a function of increasing intracellular Ca(2+) concentration resulting from an extracellular Ca(2+) influx via membrane Ca(2+) channels and cytosolic ER Ca(2+) release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca(2+) signal and in sustaining a cytosolic Ca(2+) increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.     

Tracheal mucus rheology and epithelial potential difference in two day old puppies

The transepithelial potential difference (PD) value represents an integral of ion fluxes across the epithelium, and relates to the regulation of airway fluid. We studied six healthy two day old husky puppies for their tracheal mucus rheology and bioelectric properties, since this data in newborns are still unknown. PD (-mV, epithelium vs. subcutaneous space) was measured using the agar bridge technique in two locations - lower trachea and subglottic region. For the rheological analysis, the magnetic microrheometer was employed; data are presented as mechanical impedance log G* and loss tangent tan delta (1 rad/s). The mucus collection rate (mg/min) and the solid content (%) were determined by gravimetry. Mucociliary clearability, normalized to frog mucus, (MCFP) was determined directly by the frog palate method; a cough clearability index (CCI) was computed from simulated cough machine data obtained with mucus-like gels. The mucus collection rates and PD values were considerably lower than those observed in adult dogs; the mechanical impedance values were also reduced in comparison with adult data. The PD profile within the trachea (-13.9 +/- 1.2 mV lower trachea vs. -18.4 +/- 1.4 mV subglottical, i.e. more negative subglottically), however, is similar to that observed in adult dogs. Intratracheal profiles in mucus collection rate and mucus rheology were also comparable between puppies and adult dogs. The low collection rates in puppies, particularly in lower trachea, could indicate either reduced mucus volume or slower clearance. PD and collection rate correlated very strongly (r = 0.82, p = 0.0003). PD also correlated negatively with log G* (r = 0.73, p = 0.003) and positively with tan delta (r = 0.58, p = 0.03). MCFP and % solids correlated positively (r = 0.84, p = 0.0012), in contradistinction to the usual relationship, perhaps due to the presence of non-glycoprotein components that do not contribute to crosslink formation. The apparent maturation of airway bioelectric properties, mucus collection rate and mucus viscoelasticity are all consistent with the maturation of mucociliary clearance, which has previously been reported.     

Biodegradable nanoparticles meet the bronchial airway barrier: how surface properties affect their interaction with mucus and epithelial cells

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.     

Diffusion of charged ions in mucus gel: effect of net charge

The interposition of a neutral starch gel greatly retarded bulk ionic movement by free flow. A mucus (charged) gel preparation of identical concentration and thickness further retarded ionic diffusion. The findings suggest that the charges in the mucus matrix may exert an ionic exclusion effect (Donnan Exclusion), thus retarding other ionic diffusion. We speculate that a mucus layer may physiologically behave as an ion exchange gel column and modify the traffic of charged ions through it.     

Two rheologically different gastric mucus secretions with different putative functions

Previous work has shown the presence of different mucin gene products and glycosylated species in gastric mucus secretions, however, the functional relevance of these differences is unclear. This study aimed to investigate rheologically, differences in the gel behaviour within gastric mucus samples using a pig model. Rheological measurements were made on a Bohlin CVO50 rheometer. Mucins were characterised by antigenicity, lectin reactivity and proteolytic fragmentation patterns. Two distinct mucus gel secretions, one compliant with and the other resistant to shear stress, were removed from the gastric mucosa. The two gels had different rheological behaviour profiles and exhibited structural differences in their constituent mucins. The shear-compliant mucus was located superficially to the adherent shear-resistant mucus layer and was shown not to be a proteolytic product of the latter. This study has demonstrated that there are two rheologically distinct mucus gel secretions with structural/compositional differences in the stomach. Rheological properties suggest that the adherent, shear-resistant gel could provide the mucus barrier in vivo while the shear-compliant gel could act primarily as a lubricant.     

Numerical and experimental investigation of mucociliary clearance breakdown in cystic fibrosis

The human tracheobronchial tree surface is covered with mucus. A healthy mucus is a heterogeneous material flowing toward the esophagus and a major defense actor against local pathogen proliferation and pollutant deposition. An alteration of mucus or its environment such as in cystic fibrosis dramatically impacts the mucociliary clearance. In the present study, we investigate the mechanical organization and the physics of such mucus in human lungs by means of a joint experimental and numerical work. In particular, we focus on the influence of the shear-thinning mucus mobilized by a ciliated epithelium for mucociliary clearance. The proposed robust numerical method is able to manage variations of more than 5 orders of magnitude in the shear rate and viscosity. It leads to a cartography that allows to discuss major issues on defective mucociliary clearance in cystic fibrosis. Furthermore, the computational rheological analysis based on measurements shows that cystic fibrosis shear-thinning mucus tends to aggregate in regions of lower clearance. Yet, a rarefaction of periciliary fluid has a greater impact than the mucus shear-thinning effects.     

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ～75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.     

Glycan profiling of gel forming mucus layer from the scleractinian symbiotic coral Oculina arbuscula

The gel forming mucus layer surrounding scleractinian corals play fundamental functions in the maintenance of a favorable microenvironment required for the survival of these organisms. In particular, it harbors a rich partially species-specific symbiotic community through yet poorly understood molecular interactions. However, removal or contamination of this community by exogenous bacteria is closely linked to the worldwide bleaching events that are presently devastating coral colonies. The present study investigates the structure of major high molecular weight glycoconjugates that are responsible for both rheological properties of mucus and sugar-protein interactions with microbial communities. We demonstrated that it is composed by two distinct types of sulfated macromolecules: mucin type glycoproteins densely substituted by short unusual O-linked glycans and repetitive polysaccharides.     

Monte Carlo simulation of gold nanoparticles for X-ray enhancement application

BackgroundGold nanoparticles (Au NPs) are regarded as potential agents that enhance the radiosensitivity of tumor cells for theranostic applications. To elucidate the biological mechanisms of radiation dose enhancement effects of Au NPs as well as DNA damage attributable to the inclusion of Au NPs, Monte Carlo (MC) simulations have been deployed in a number of studies.Scope of ReviewThis review paper concisely collates and reviews the information reported in the simulation research in terms of MC simulation of radiosensitization and dose enhancement effects caused by the inclusion of Au NPs in tumor cells, simulation mechanisms, benefits and limitations.Major conclusionsIn this review, we first explore the recent advances in MC simulation on Au NPs radiosensitization. The MC methods, physical dose enhancement and enhanced chemical and biological effects is discussed, followed by some results regarding the prediction of dose enhancement. We then review Multi-scale MC simulations of Au NP-induced DNA damages for X-ray irradiation. Moreover, we explain and look at Multi-scale MC simulations of Au NP-induced DNA damages for X-ray irradiation.General significanceUsing advanced chemical module-implemented MC simulations, there is a need to assess the radiation-induced chemical radicals that contribute to the dose-enhancing and biological effects of multiple Au NPs.     

Viscoelastic properties of human tracheobronchial mucin in aqueous solution

Human tracheobronchial mucin isolated from cystic fibrosis patients (CF HTBM) was purified using a combination of gel filtration and density gradient centrifugation. The resulting mucin was fractionated to reduce polydispersity and to facilitate studies of the molecular weight dependence of mucin viscoelasticity in concentrated solution. The viscoelastic properties of CF HTBM were examined in distilled water, 0.1M salt solutions and chaotropic solvents. In controlled strain experiments (strain ≥ 5%) with increasing mucin concentration, a crossover from sol to gel behavior is observed. The gel strength, as measured by the magnitude of the storage modulus at comparable mucin concentrations, is greatest for distilled water, intermediate for 0.1M NaCl, and lowest far 6M GdnHCl. In distilled water, high molecular weight mucin undergoes a sol-gel transition at ～ 12 mg/mL, and shows evidence of a plateau modulus at higher concentrations. The storage and loss moduli of concentrated high molecular weight fractions in 6M GdnHCl exhibit a power law dependence on frequency typical of weak gels near the sol–gel transition at 20 mg/mL. Similar rheology is observed in 0.1M NaCl and 0.091M NaCl/3 mM CaCl₂, but with evidence for additional weak associations at low frequency. The power law exponent in these systems is 0.70 ± 0.02, in good agreement with prediction for networks formed by a percolation mechanism. Low molecular weight fractions in these solvents exhibit a fluid-like viscoelastic response. However, low molecular weight mucin in distilled water shows a strain-dependent increase in elasticity at low frequency indicative of weak intermolecular associations. Comparison of the rheological behavior of CF HTBM with our earlier studies of ovine submaxillary mucin lends support to the idea that carbohydrate side-chain interactions are important in the gelation mechanism of mucins. © 1995 John Wiley & Sons, Inc.     

Factors That Influence the Extensional Rheological Property of Saliva

The spinnbarkeit of saliva reflects the ability of saliva to adhere to surfaces within the mouth, thereby serving as a protective role and aiding in lubrication. Therefore, alterations in the extensional rheology of saliva may result in the loss in adhesiveness or the ability to bind onto surfaces. Mucin glycoproteins and their structures are known to be important factors for the extensional rheological properties of saliva. The conformation of mucin depends on factors such as pH and ionic strength. Chewing is one of the main stimuli for salivary secretion but creates significant sheer stress on the salivary film which could influence mouthfeel perceptions. The current study investigates the possible factors which affect the extensional rheological properties of saliva by comparing submandibular/sublingual saliva with different oral stimuli within the same group of subjects. Unstimulated and stimulated saliva (chew, smell and taste) salivas were collected primarily from submandibular/sublingual glands. The saliva samples were measured for Spinnbarkeit followed by the measuring mucin, total protein, total calcium and bicarbonate concentrations. The results indicated correlations between rheological properties and mucin/ion concentrations. However, chewing stimulated submandibular/sublingual saliva is shown to have significantly lower Spinnbarkeit, but factors such as mucin, protein and calcium concentrations did not account for this variation. Analysis of the concentration of bicarbonate and pH appears to suggest that it has a prominent effect on extensional rheology of saliva.