Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures

The airway epithelial surface liquid is generally considered to be composed of two layers, a periciliary layer and a continuous thick mucus layer moving in bulk. This view may not be appropriate for all areas of the lung. Our hypothesis, that mucus may form a discontinuous layer with dynamic attachments to the surface, is investigated using a culture system. We used live-cell confocal microscopy to investigate thin mucus layers and fluorescent beads and exogenous MUC5B to visualize mucus dynamics on ciliated human bronchial cultures. A continuous mucus layer was not observed. In sparsely ciliated cultures, mucus attached to ciliated cells; however, in highly ciliated cultures, mucus formed strands several hundred micrometers long. As with increases in ciliation, increases in bead concentration caused the appearance of mucus strands. We confirmed the involvement of mucins in the binding of mucus to cilia by adding labeled purified MUC5B to the cultures. These data suggest that mucins may have an intrinsic ability to form attachments to cilia. The significance of these findings is that aberrant modulation of such an intrinsic property may explain the initiation of highly adherent mucus in cystic fibrosis lung disease.     

Coarse-grained Monte Carlo simulations of mucus: structure, dynamics, and thermodynamics

A simple coarse-grained model of mucus structure and dynamics is proposed and evaluated. The model is based on simple cubic, face-centered lattice representation. Mucins are simulated as lattice chains in which each bead of the model chains represents a mucin domain, equivalent to its Kuhn segment. The remaining lattice sites are considered to be occupied by the solvent. Model mucins consist of three types of domains: polar (glycosylated central segments), hydrophobic, and cysteine-rich, located at the terminal part of the mucin chains. The sequence of these domains mimics the sequence of real mucins. Static and dynamic properties of the system were studied by means of Monte Carlo dynamics. It was shown that the model system undergoes sol-gel transition and that the interactions between hydrophobic domains are responsible for the transition and characteristic properties of the dynamic network in the gel phase. Cysteine-rich domains are essential for frictional properties of the system. Structural and dynamic properties of the model mucus observed in simulations are in qualitative agreement with known experimental facts and provide mechanistic explanation of complex properties of real mucus.     

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.     

Application of Enhanced Sampling Monte Carlo Methods for High-Resolution Protein-Protein Docking in Rosetta

The high-resolution refinement of docked protein-protein complexes can provide valuable structural and mechanistic insight into protein complex formation complementing experiment. Monte Carlo (MC) based approaches are frequently applied to sample putative interaction geometries of proteins including also possible conformational changes of the binding partners. In order to explore efficiency improvements of the MC sampling, several enhanced sampling techniques, including temperature or Hamiltonian replica exchange and well-tempered ensemble approaches, have been combined with the MC method and were evaluated on 20 protein complexes using unbound partner structures. The well-tempered ensemble method combined with a 2-dimensional temperature and Hamiltonian replica exchange scheme (WTE-H-REMC) was identified as the most efficient search strategy. Comparison with prolonged MC searches indicates that the WTE-H-REMC approach requires approximately 5 times fewer MC steps to identify near native docking geometries compared to conventional MC searches.     

Using Unfixed,Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight^1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract^3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins^5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms^8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods¹³. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.     

A Monte Carlo sampling method of amino acid sequences adaptable to given main-chain atoms in the proteins

We have developed a computational method of protein design to detect amino acid sequences that are adaptable to given main-chain coordinates of a protein. In this method, the selection of amino acid types employs a Metropolis Monte Carlo method with a scoring function in conjunction with the approximation of free energies computed from 3D structures. To compute the scoring function, a side-chain prediction using another Metropolis Monte Carlo method was performed to select structurally suitable side-chain conformations from a side-chain library. In total, two layers of Monte Carlo procedures were performed, first to select amino acid types (1st layer Monte Carlo) and then to predict side-chain conformations (2nd layers Monte Carlo). We applied this method to sequence design for the entire sequence on the SH3 domain, Protein G, and BPTI. The predicted sequences were similar to those of the wild-type proteins. We compared the results of the predictions with and without the 2nd layer Monte Carlo method. The results revealed that the two-layer Monte Carlo method produced better sequence similarity to the wild-type proteins than the one-layer method. Finally, we applied this method to neuraminidase of influenza virus. The results were consistent with the sequences identified from the isolated viruses.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

Extinction risk of a density-dependent population estimated from a time series of population size

Environmental threats, such as habitat size reduction or environmental pollution, may not cause immediate extinction of a population but shorten the expected time to extinction. We develop a method to estimate the mean time to extinction for a density-dependent population with environmental fluctuation. We first derive a formula for a stochastic differential equation model (canonical model) of a population with logistic growth with environmental and demographic stochasticities. We then study an approximate maximum likelihood (AML) estimate of three parameters (intrinsic growth rate r, carrying capacity K, and environmental stochasticity sigma(2)(e)) from a time series of population size. The AML estimate of r has a significant bias, but by adopting the Monte Carlo method, we can remove the bias very effectively (bias-corrected estimate). We can also determine the confidence interval of the parameter based on the Monte Carlo method. If the length of the time series is moderately long (with 40-50 data points), parameter estimation with the Monte Carlo sampling bias correction has a relatively small variance. However, if the time series is short (less than or equal to 10 data points), the estimate has a large variance and is not reliable. If we know the intrinsic growth rate r, however, the estimate of K and sigma(2)(e)and the mean extinction time T are reliable even if only a short time series is available. We illustrate the method using data for a freshwater fish, Japanese crucian carp (Carassius auratus subsp.) in Lake Biwa, in which the growth rate and environmental noise of crucian carp are estimated using fishery records.     

Microbial adhesins to gastrointestinal mucus

The gastrointestinal tract (GIT) is lined by a layer of mucus formed by mucin glycoproteins. This layer constitutes a physical and chemical barrier between the intestinal contents and the underlying epithelia. In addition to this protective role, mucins harbor glycan-rich domains that provide preferential binding sites for pathogens and commensal bacteria. Although mucus-microbial interactions in the GIT play a crucial role in determining the outcome of relationships of both commensal and pathogens with the host, the adhesins and ligands involved in the interaction are poorly delineated. This review focuses on the current knowledge of microbial adhesins to gastrointestinal mucus and mucus components.     

The gastric mucus layers: constituents and regulation of accumulation

The mucus layer continuously covering the gastric mucosa consists of a loosely adherent layer that can be easily removed by suction, leaving a firmly adherent mucus layer attached to the epithelium. These two layers exhibit different gastroprotective roles; therefore, individual regulation of thickness and mucin composition were studied. Mucus thickness was measured in vivo with micropipettes in anesthetized mice [isoflurane; C57BL/6, Muc1-/-, inducible nitric oxide synthase (iNOS)-/-, and neuronal NOS (nNOS)-/-] and rats (inactin) after surgical exposure of the gastric mucosa. The two mucus layers covering the gastric mucosa were differently regulated. Luminal administration of PGE(2) increased the thickness of both layers, whereas luminal NO stimulated only firmly adherent mucus accumulation. A new gastroprotective role for iNOS was indicated since iNOS-deficient mice had thinner firmly adherent mucus layers and a lower mucus accumulation rate, whereas nNOS did not appear to be involved in mucus secretion. Downregulation of gastric mucus accumulation was observed in Muc1-/- mice. Both the firmly and loosely adherent mucus layers consisted of Muc5ac mucins. In conclusion, this study showed that, even though both the two mucus layers covering the gastric mucosa consist of Muc5ac, they are differently regulated by luminal PGE(2) and NO. A new gastroprotective role for iNOS was indicated since iNOS-/- mice had a thinner firmly adherent mucus layer. In addition, a regulatory role of Muc1 was demonstrated since downregulation of gastric mucus accumulation was observed in Muc1-/- mice.     

Monte Carlo replica‐exchange based ensemble docking of protein conformations

A replica‐exchange Monte Carlo (REMC) ensemble docking approach has been developed that allows efficient exploration of protein–protein docking geometries. In addition to Monte Carlo steps in translation and orientation of binding partners, possible conformational changes upon binding are included based on Monte Carlo selection of protein conformations stored as ordered pregenerated conformational ensembles. The conformational ensembles of each binding partner protein were generated by three different approaches starting from the unbound partner protein structure with a range spanning a root mean square deviation of 1–2.5 Å with respect to the unbound structure. Because MC sampling is performed to select appropriate partner conformations on the fly the approach is not limited by the number of conformations in the ensemble compared to ensemble docking of each conformer pair in ensemble cross docking. Although only a fraction of generated conformers was in closer agreement with the bound structure the REMC ensemble docking approach achieved improved docking results compared to REMC docking with only the unbound partner structures or using docking energy minimization methods. The approach has significant potential for further improvement in combination with more realistic structural ensembles and better docking scoring functions. Proteins 2017; 85:924–937. © 2016 Wiley Periodicals, Inc.     

Exploring the energy landscape of a beta hairpin in explicit solvent

We studied the energy landscape of the peptide Ace-GEWTYDDATKTFTVTE-Nme, taken from the C-terminal fragment (41-56) of protein G, in explicit aqueous solution by a highly parallel replica-exchange approach that combines molecular dynamics trajectories with a temperature exchange Monte Carlo process. The combined trajectories in T and configurational space allow a replica to overcome a free energy barrier present at one temperature by increasing T, changing configurations, and cooling in a self-regulated manner, thus allowing sampling of broad regions of configurational space in short (nanoseconds) time scales. The free energy landscape of this system over a wide range of temperatures shows that the system preferentially adopts a beta hairpin structure. However, the peptide also samples other stable ensembles where the peptide adopts helices and helix-turn-helix states, among others. The helical states become increasingly stable at low temperatures, but are slightly less stable than the beta turn ensemble. The energy landscape is rugged at low T, where substates are separated by large energy barriers. These barriers disappear at higher T (approximately 330 K), where the system preferentially adopts a "molten globule" state with structures similar to the beta hairpin.     

Butyrate enemas upregulate Muc genes expression but decrease adherent mucus thickness in mice colon

Colonic mucosal protection is provided by the mucus gel, mainly composed of mucins. Several factors can modulate the formation and the secretion of mucins, and among them butyrate, an end-product of carbohydrate fermentation. However, the specific effect of butyrate on the various colonic mucins, and the consequences in terms of the mucus layer thickness are not known. Our aim was to determine whether butyrate modulates colonic MUC genes expression in vivo and whether this results in changes in mucus synthesis and mucus layer thickness. Mice received daily for 7 days rectal enemas of butyrate (100 mM) versus saline. We demonstrated that butyrate stimulated the gene expression of both secreted (Muc2) and membrane-linked (Muc1, Muc3, Muc4) mucins. Butyrate especially induced a 6-fold increase in Muc2 gene expression in proximal colon. However, butyrate enemas did not modify the number of epithelial cells containing the protein Muc2, and caused a 2-fold decrease in the thickness of adherent mucus layer. Further studies should help understanding whether this last phenomenon, i.e. the decrease in adherent mucus gel thickness, results in a diminished protective function or not.     

Monte Carlo folding of trans-membrane helical peptides in an implicit generalized Born membrane

An efficient Monte Carlo (MC) algorithm using concerted backbone rotations is combined with a recently developed implicit membrane model to simulate the folding of the hydrophobic transmembrane domain M2TM of the M2 protein from influenza A virus and Sarcolipin at atomic resolution. The implicit membrane environment is based on generalized Born theory and has been calibrated against experimental data. The MC sampling has previously been used to fold several small polypeptides and been shown to be equivalent to molecular dynamics (MD). In combination with a replica exchange algorithm, M2TM is found to form continuous membrane spanning helical conformations for low temperature replicas. Sarcolipin is only partially helical, in agreement with the experimental NMR structures in lipid bilayers and detergent micelles. Higher temperature replicas exhibit a rapidly decreasing helicity, in agreement with expected thermodynamic behavior. To exclude the possibility of an erroneous helical bias in the simulations, the model is tested by sampling a synthetic Alanine-rich polypeptide of known helicity. The results demonstrate there is no overstabilization of helical conformations, indicating that the implicit model captures the essential components of the native membrane environment for M2TM and Sarcolipin.     

Gastrointestinal Cell Lines Form Polarized Epithelia with an Adherent Mucus Layer when Cultured in Semi-Wet Interfaces with Mechanical Stimulation

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.     

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.     

Differential Binding of IgG and IgA to Mucus of the Female Reproductive Tract

Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.     

Enhanced sampling of peptide and protein conformations using replica exchange simulations with a peptide backbone biasing-potential

During replica exchange molecular dynamics (RexMD) simulations, several replicas of a system are simulated at different temperatures in parallel allowing for exchange between replicas at frequent intervals. This technique allows significantly improved sampling of conformational space and is increasingly being used for structure prediction of peptides and proteins. A drawback of the standard temperature RexMD is the rapid increase of the replica number with increasing system size to cover a desired temperature range. In an effort to limit the number of replicas, a new Hamiltonian-RexMD method has been developed that is specifically designed to enhance the sampling of peptide and protein conformations by applying various levels of a backbone biasing potential for each replica run. The biasing potential lowers the barrier for backbone dihedral transitions and promotes enhanced peptide backbone transitions along the replica coordinate. The application on several peptide cases including in all cases explicit solvent indicates significantly improved conformational sampling when compared with standard MD simulations. This was achieved with a very modest number of 5-7 replicas for each simulation system making it ideally suited for peptide and protein folding simulations as well as refinement of protein model structures in the presence of explicit solvent.