The structure of the second CysD domain of MUC2 and role in mucin organization by transglutaminase-based cross-linking.

The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Gut microbiota shape the inflammatory response in mice with an epithelial defect.

Intestinal epithelial cell endoplasmic reticulum (ER) stress has been implicated in intestinal inflammation. It remains unclear whether ER stress is an initiator of or a response to inflammation. Winnie mice, carrying a Muc2 gene mutation resulting in intestinal goblet cell ER stress, develop spontaneous colitis with a depleted mucus barrier and increased bacterial translocation. This study aims to determine whether the microbiota was required for the development of Winnie colitis, and whether protein misfolding itself can initiate inflammation directly in absence of the microbiota. To assess the role of microbiota in driving Winnie colitis, WT and Winnie mice on the same background were rederived into the germ-free facility and housed in the Trexler-type soft-sided isolators. The colitis phenotype of these mice was assessed and compared to WT and Winnie mice housed within a specific pathogen-free facility. We found that Winnie colitis was substantially reduced but not abolished under germ-free conditions. Expression of inflammatory cytokine genes was reduced but several chemokines remained elevated in absence of microbiota. Concomitantly, ER stress was also diminished, although mucin misfolding persisted. RNA-Seq revealed that Winnie differentiated colon organoids have decreased expression of the negative regulators of the inflammatory response compared to WT. This data along with the increase in Mip2a chemokine expression, suggests that the epithelial cells in the Winnie mice are more responsive to stimuli. Moreover, the data demonstrate that intestinal epithelial intrinsic protein misfolding can prime an inflammatory response without initiating the unfolded protein response in the absence of the microbiota. However, the microbiota is necessary for the amplification of colitis in Winnie mice. Genetic predisposition to mucin misfolding in secretory cells initiates mild inflammatory signals. However, the inflammatory signal sets a forward-feeding cycle establishing progressive inflammation in the presence of microbiota.Abbreviations: Endoplasmic Reticulum: ER; Mucin-2: Muc-2; GF: Germ-Free; Inflammatory Bowel Disease: IBD.

Accurate and cost-effective NMR chemical shift predictions for proteins using a molecules-in-molecules fragmentation-based method.

We have developed an efficient protocol using our two-layer Molecules-in-Molecules (MIM2) fragmentation-based quantum chemical method for the prediction of NMR chemical shifts of large biomolecules. To investigate the performance of our fragmentation approach and demonstrate its applicability, MIM-NMR calculations are first calibrated on a test set of six proteins. The MIM2-NMR method yields a mean absolute deviation (MAD) from unfragmented full molecule calculations of 0.01 ppm for 1H and 0.06 ppm for 13C chemical shifts. Thus, the errors from fragmentation are only about 3% of our target accuracy of ∼0.3 ppm for 1H and 2-3 ppm for 13C chemical shifts. To compare with experimental chemical shifts, a standard protocol is first derived using two smaller proteins 2LHY (176 atoms) and 2LI1 (146 atoms) for obtaining an appropriate protein structure for NMR chemical shift calculations. The effect of the solvent environment on the calculated NMR chemical shifts is incorporated through implicit, explicit, or explicit-implicit solvation models. The expensive first solvation shell calculations are replaced by a micro-solvation model in which only the immediate interaction between the protein and the explicit solvation environment is considered. A single explicit water molecule for each amine and amide proton is found to be sufficient to yield accurate results for 1H chemical shifts. The 1H and 13C NMR chemical shifts calculated using our protocol give excellent agreement with experiments for two larger proteins, 2MC5 (the helical part with 265 atoms) and 3UMK (33 residue slice with 547 atoms). Overall, our target accuracy of ∼0.3 ppm for 1H and ∼2-3 ppm for 13C has been achieved for the larger proteins. The proposed MIM-NMR method is accurate and computationally cost-effective and should be applicable to study a wide range of large proteins.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps.

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

Quantifying and Engineering Mucus Adhesion of Probiotics.

Mucus in the gastrointestinal (GI) tract is the primary point-of-interaction between humans and their gut microbiota. This intimates that mucus not only ensures protection against endogenous and exogenous opportunists but also provisions for the human microbiota to reside and flourish. With the emergence of living therapeutics, engineered microbes can deliver and produce increasingly complex medicine, and controlling the mucoadhesive properties of different microbial chassis can dictate dose-response in a patient. Here we present a redesigned, in vitro, plate-based assay to measure the mucus adhesion of various probiotics. Cell-mucus interactions were isolated by immobilizing mucus to the plate surface. Binding parameters were derived for each probiotic strain by measuring cell adhesion over a wide range of cell concentrations, providing dose-dependent adhesion metrics. Surface proteins and cell components known to influence mucoadhesion were then heterologously expressed or altered in Lactococcus lactis MG1363 and Escherichia coli Nissle 1917 to control mucus-binding capacity, avidity, and cooperativity.

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals.

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

High MUC2 Mucin Expression and Misfolding Induce Cellular Stress, Reactive Oxygen Production, and Apoptosis in Goblet Cells.

MUC2 mucin is a large glycoprotein produced by goblet cells that forms the protective mucus blanket overlying the intestinal epithelium as the first line of innate host defense. High MUC2 production in inflammatory bowel disease and infectious colitis depletes goblet cells and the mucus layer by an unknown mechanism. Herein, we analyzed the effect of high MUC2 biosynthesis on endoplasmic reticulum (ER) stress and apoptosis in goblet cells using a high MUC2-producing human goblet cell line (HT29-H) and an HT29-H clone (HT29-L) silenced for MUC2 expression by lentivirus-mediated shRNA. Goblet cell ER stress and apoptosis were quantified during early onset of dextran sulfate sodium-induced colitis in C57BL/6 and Math1M1GFP mice. Compared with HT29-L and MUC2 nonproducing Caco-2 cells, high MUC2-producing HT29-H cells had significantly increased ER stress and apoptosis after treatment with ER stress-inducing agents. Apoptosis was driven by increased misfolded MUC2 that triggered elevated levels of reactive oxygen species. Correcting MUC2 folding and inhibiting reactive oxygen species alleviated ER stress and rescued cells from apoptosis. During early-onset colitis, mucus hypersecretion caused severe ER stress and apoptosis of goblet cells that preceded absorptive epithelial cell damage. Thus, in gastrointestinal inflammation, high MUC2 biosynthesis and goblet cell apoptosis lead to a dysfunctional epithelial barrier. Enhancing MUC2 folding may help alleviate goblet cell depletion and maintain mucosal integrity.

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein-carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly- N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule-grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.-Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins.

Application of recent advances in hydrodynamic methods for characterising mucins in solution.

Mucins are the primary macromolecular component of mucus--nature's natural lubricant--although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 10(6) g mol(-1), in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 10(6) g mol(-1). Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length L p ~10 nm and mass per unit length, M L ~2380 g mol(-1) nm(-1), consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient.

Titanium Surface Roughing Treatments contribute to Higher Interaction with Salivary Proteins MG2 and Lactoferrin.

UNLABELLED: Some surface treatments performed on titanium can alter the composition of salivary pellicle formed on this abiotic surface. Such treatments modify the titanium's surface properties and can promote higher adsorption of proteins, which allow better integration of titanium to the biotic system. PURPOSE: This study aimed to evaluate the interactions between salivary proteins and titanium disks with different surface treatments. MATERIALS AND METHODS: Machined titanium disks (n = 48) were divided into four experimental groups (n = 12), according to their surface treatments: surface polishing (SP); acid etching (A); spot-blasting plus acid etching (SB-A); spot-blasting followed by acid etching and nano-functionalization (SB-A-NF). Titanium surfaces were characterized by surface roughness and scanning electron microscopy (SEM). Specimens were incubated with human saliva extracted from submandibular and sublingual glands. Total salivary protein adsorbed to titanium was quantified and samples were submitted to western blotting for mucin glycoprotein 2 (MG2) and lactoferrin identification. RESULTS: Surface roughness was statistically higher for SB-A and SB-A-NF groups. Scanning electron microscopy images confirmed that titanium surface treatments increased surface roughness with higher number of porous and scratches for SB-A and SB-A-NF groups. Total protein adsorption was significantly higher for SB-A and SB-A-NF groups (p < 0.05), which also presented higher interactions with MG2 and lactoferrin proteins. CONCLUSION: The roughing of titanium surface by spot-blasting plus acid etching treatments contribute to higher interaction with salivary proteins, such as MG2 and lactoferrin. CLINICAL SIGNIFICANCE: Titanium surface roughing increases the interactions of the substratum with salivary proteins, which can influence the integration of dental implants and their components to the oral environment. However, those treatments should be used carefully intraorally, avoiding increase biofilm formation.

High-resolution structure of a new Tn antigen-binding lectin from Vatairea macrocarpa and a comparative analysis of Tn-binding legume lectins.

Plant lectins have been studied as histological markers and promising antineoplastic molecules for a long time, and structural characterization of different lectins bound to specific cancer epitopes has been carried out successfully. The crystal structures of Vatairea macrocarpa (VML) seed lectin in complex with GalNAc-α-O-Ser (Tn antigen) and GalNAc have been determined at the resolution of 1.4Å and 1.7Å, respectively. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest VML as a candidate tool for cancer research.

Multiple enzyme approach for the characterization of glycan modifications on the C-terminus of the intestinal MUC2mucin.

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein's central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 N-glycan consensus sites were identified as glycosylated. Fifty-six N-glycopeptides covering 10 N-glycan sites, and 14 O-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Microbial-induced meprin ß cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus.

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin ß-deficient mice was now found to be attached. Meprin ß is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin ß was added to the attached mucus of meprin ß-deficient mice, the mucus was detached from the epithelium. Similar to meprin ß-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin ß into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin ß to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin ß cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin ß in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

A combined small-angle X-ray and neutron scattering study of the structure of purified soluble gastrointestinal mucins.

The structures of purified soluble porcine gastric (Muc5ac) and duodenal (Muc2) mucin solutions at neutral and acidic pH were examined using small-angle X-ray scattering and small-angle neutron scattering experiments. We provide evidence for the morphology of the network above the semidilute overlap concentration and above the entanglement concentration. Furthermore, we investigated the gelation of both types of mucin solutions in response to a reduction in pH, where we observed the formation of large-scale heterogeneities within the polymer solutions, typical of microphase-separated gels. The concentration dependence of the inhomogeneity length scale (Ξ) and the amplitude of the excess scattering intensity [I(ex) (0)] are consistent with previously studied gelled synthetic polymeric systems. The persistence lengths of the chains were found to be similar for both Muc5ac and Muc2 from Kratky plots of the neutron data (8 ± 2 nm).

Intestinal MUC2 mucin supramolecular topology by packing and release resting on D3 domain assembly.

MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

Cloning, annotation and developmental expression of the chicken intestinal MUC2 gene.

Intestinal mucin 2 (MUC2) encodes a heavily glycosylated, gel-forming mucin, which creates an important protective mucosal layer along the gastrointestinal tract in humans and other species. This first line of defense guards against attacks from microorganisms and is integral to the innate immune system. As a first step towards characterizing the innate immune response of MUC2 in different species, we report the cloning of a full-length, 11,359 bp chicken MUC2 cDNA, and describe the genomic organization and functional annotation of this complex, 74.5 kb locus. MUC2 contains 64 exons and demonstrates distinct spatiotemporal expression profiles throughout development in the gastrointestinal tract; expression increases with gestational age and from anterior to posterior along the gut. The chicken protein has a similar domain organization as the human orthologue, with a signal peptide and several von Willebrand domains in the N-terminus and the characteristic cystine knot at the C-terminus. The PTS domain of the chicken MUC2 protein spans ∼1600 amino acids and is interspersed with four CysD motifs. However, the PTS domain in the chicken diverges significantly from the human orthologue; although the chicken domain is shorter, the repetitive unit is 69 amino acids in length, which is three times longer than the human. The amino acid composition shows very little similarity to the human motif, which potentially contributes to differences in the innate immune response between species, as glycosylation across this rapidly evolving domain provides much of the musical barrier. Future studies of the function of MUC2 in the innate immune response system in chicken could provide an important model organism to increase our understanding of the biological significance of MUC2 in host defense and highlight the potential of the chicken for creating new immune-based therapies.

Mucoadhesive chitosan-coated cationic microemulsion of dexamethasone for ocular delivery: in vitro and in vivo evaluation.

PURPOSE: Dexamethasone (DXN) is an effective anti-inflammatory drug in the treatment of acute and chronic eye disease such as uveitis. However, its low aqueous solubility limits its clinical usefulness. The purpose of this study was to investigate the mucoadhesive chitosan-coated cationic microemulsions (CH-MEs) for ophthalmic delivery of DXN to treat uveitis. MATERIALS AND METHODS: The pseudo-ternary phase diagrams were developed and various MEs were prepared using isopropyl myristate as oil, Tween 80 as a surfactant, propylene glycol as a co-surfactant and distilled water. MEs were prepared and coated with chitosan by the dropwise addition of chitosan solution in the ME dispersion. Physicochemical parameters (globule size, zetapotential, drug content, viscosity, refractive index and pH), mucoadhesive properties and the in vitro release of MEs were studied. The in vivo efficacy of prepared formulations and the marketed drug solution were studied by administering them topically to endotoxin-induced uveitis rabbit model. RESULTS: All formulations displayed an average globule size less than 200 nm and a positive surface charge. The developed CH-MEs showed acceptable physico-chemical behavior, good mucoadhesive properties, good stability for three months and exhibited sustained drug release. In vivo studies in rabbit eye showed a marked improvement in the anti-inflammatory activity of mucoadhesive CH-ME-treated eye compared with a marketed suspension formulation in a uveitis-induced rabbit eye model. CONCLUSION: The developed CH-MEs are a viable alternative to conventional eye drops for its ability to enhance bioavailability through its longer precorneal residence time and its ability to sustain the release of the drug.