Search

The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.

Cows produce saliva in very large quantities to lubricate and facilitate food processing. Estimates indicate an amount of 50-150 L per day. Human saliva has previously been found to contain numerous antibacterial components, such as lysozyme, histatins, members of the S-100 family and lactoferrin, to limit pathogen colonization. Cows depend on a complex microbial community in their digestive system for food digestion. Our aim here was to analyze how this would influence the content of their saliva. We therefore sampled saliva from five humans and both nose secretions and saliva from six cows and separated the saliva on SDS-PAGE gradient gels and analyzed the major protein bands with LC-MS/MS. The cow saliva was found to be dominated by a few major proteins only, carbonic anhydrase 6, a pH-stabilizing enzyme and the short palate, lung and nasal epithelium carcinoma-associated protein 2A (SPLUNC2A), also named bovine salivary protein 30 kDa (BSP30) or BPIFA2B. This latter protein has been proposed to play a role in local antibacterial response by binding bacterial lipopolysaccharides (LPSs) and inhibiting bacterial growth but may instead, according to more recent data, primarily have surfactant activity. Numerous peptide fragments of mucin-5B were also detected in different regions of the gel in the MS analysis. Interestingly, no major band on gel was detected representing any of the antibacterial proteins, indicating that cows may produce them at very low levels that do not harm the microbial flora of their digestive system. The nose secretions of the cows primarily contained the odorant protein, a protein thought to be involved in enhancing the sense of smell of the olfactory receptors and the possibility of quickly sensing potential poisonous food components. High levels of secretory IgA were also found in one sample of cow mouth drippings, indicating a strong upregulation during an infection. The human saliva was more complex, containing secretory IgA, amylase, ca

The inflammatory process plays a crucial role in frailty syndrome, which can appear in middle age and is associated with a poor health outcome. Consequently, gerontologists recommend screening inflammatory biomarkers in middle-aged adults to detect frailty and, therefore, prevent chronic diseases and mortality. External factors could be a risk factor for frailty because they can generate and extend the inflammatory process. For these reasons, we analysed the effect of long-term contact lens wear on mRNA level of genes linked to inflammation (IL-6, NLRP3, NK1R, CD73, MUC16 and TRPV1 genes) in conjunctival cells of middle-aged individuals, by quantitative PCR. Middle-aged contact lens wearers presented a significant increase of NLRP3 and MUC16 mRNA level as well as a decrease of CD73 mRNA level, in comparison with non-contact lens wearers. Additionally, we checked for a potential correlation between these transcript levels and clinical changes of the participants’ ocular surface. Unlike molecular analysis, clinical examination fails to detect inflammation in contact lens wearers. These data suggest that long-term contact lens wear could trigger an inflammatory response in middle age orchestrated by NLRP3 inflammasome and modulated by CD73 and MUC16 proteins. Further studies are needed to confirm our gene expression findings at the protein level as well as to investigate the potential role of long-term CL wear in the onset of ocular frailty., Unión Europea, Depto. de Optometría y Visión, Unidad Docente de Bioquímica y Biología Molecular, Fac. de Óptica y Optometría, TRUE, inpress

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

Mucus reduces friction between epithelial surfaces by providing lubrication in the boundary and mixed regime. Mucins, the main macromolecule, are heavily glycosylated proteins that polymerise and retain water molecules, resulting in a hydrated biogel. It is assumed that positively charged ions can influence mucin film structure by screening the electrostatic repulsions between the negatively charged glycans on mucin moieties and draw in water molecules via hydration shells. The ionic concentration can vary significantly in different mucus systems and here we show that increasing the ionic concentration in mucin films leads to an increase in lubrication between two polydimethylsiloxane surfaces at sliding contact in a compliant oral mimic. Mucins were found to bind sodium ions in a concentration-dependent manner and increased ionic concentration appears to cause mucin films to swell when assessed by Quartz Crystal hiMicrobalance with Dissipation (QCM-D) analysis. Furthermore, we determined that the removal of negatively charged sialic acid moieties by sialidase digestion resulted in reduced adsorption to hydrophilic surfaces but did not affect the swelling of mucin films with increasing ionic concentrations. Moreover, the coefficient of friction was increased with sialic acid removal, but lubrication was still increased with increasing ionic concentrations. Taken together this suggests that sialic acids are important for lubrication and may exert this through the sacrificial layer mechanism. Ionic concentration appears to influence mucin films and their lubrication, and sialic acids, at least partly, may be important for ion binding., QC 20230713

Aberrant mucus secretion is a hallmark of chronic obstructive pulmonary disease (COPD). Expression of the membrane-tethered mucins 3A and 3B (MUC3A, MUC3B) in human lung is largely unknown. In this observational cross-sectional study, we recruited subjects 45–65 years old from the general population of Stockholm, Sweden, during the years 2007–2011. Bronchial mucosal biopsies, bronchial brushings, and bronchoalveolar lavage fluid (BALF) were retrieved from COPD patients (n = 38), healthy never-smokers (n = 40), and smokers with normal lung function (n = 40). Protein expression of MUC3A and MUC3B in bronchial mucosal biopsies was assessed by immunohistochemical staining. In a subgroup of subjects (n = 28), MUC3A and MUC3B mRNAs were quantified in bronchial brushings using microarray. Non-parametric tests were used to perform correlation and group comparison analyses. A value of p %lt; 0.05 was considered statistically significant. MUC3A and MUC3B immunohistochemical expression was localized to ciliated cells. MUC3B was also expressed in basal cells. MUC3A and MUC3B immunohistochemical expression was equal in all study groups but subjects with emphysema had higher MUC3A expression, compared to those without emphysema. Smokers had higher mRNA levels of MUC3A and MUC3B than non-smokers. MUC3A and MUC3B mRNA were higher in male subjects and correlated negatively with expiratory air flows. MUC3B mRNA correlated positively with total cell concentration and macrophage percentage, and negatively with CD4/CD8 T cell ratio in BALF. We concluded that MUC3A and MUC3B in large airways may be a marker of disease or may play a role in the pathophysiology of airway obstruction.

Despite the promise of antimicrobial peptides (AMPs) as treatments for antibiotic-resistant infections, their therapeutic efficacy is limited due to the rapid degradation and low bioavailability of AMPs. To address this, we have developed and characterized a synthetic mucus (SM) biomaterial capable of delivering LL37 AMPs and enhancing their therapeutic effect. LL37 is an AMP that exhibits a wide range of antimicrobial activity against bacteria, including Pseudomonas aeruginosa. LL37 loaded SM hydrogels demonstrated controlled release with 70%–95% of loaded LL37 over 8 h due to charge-mediated interactions between mucins and LL37 AMPs. Compared to treatment with LL37 alone where antimicrobial activity was reduced after 3 h, LL37-SM hydrogels inhibited P. aeruginosa (PAO1) growth over 12 h. LL37-SM hydrogel treatment reduced PAO1 viability over 6 h whereas a rebound in bacterial growth was observed when treated with LL37 only. These data demonstrate LL37-SM hydrogels enhance antimicrobial activity by preserving LL37 AMP activity and bioavailability. Overall, this work establishes SM biomaterials as a platform for enhanced AMP delivery for antimicrobial applications.

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. However, the molecular and cellular mechanisms that regulate mucin production and movement are unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), which is generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. Using genetic mouse models, we found that loss of epithelial TNF promoted differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, co-treatment of intestinal organoids with recombinant TNF and a CFTR inhibitor demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF-CFTR signaling axis in the adult intestine and identify epithelial-derived TNF as an upstream regulator of mucin homeostasis.

Gastrointestinal (GI) mucus is continuously secreted and lines the entire length of the GI tract. Essential for health, it keeps the noxious luminal content away from the epithelium. Our aim was to characterize the composition and structure of mucus throughout the various GI segments in dog. Mucus was collected from the stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, proximal and distal colon) from dogs. Composition was determined by multi-omics. Structural properties were investigated using cryoSEM and rheology. GI mucus contained 74-95% water and maintained a pH around 6.5. The proteome was similar across the different GI segments. The highest abundant secreted gel-forming mucin in the gastric mucus was mucin 5AC, whether mucin 2 had highest abundance in the intestinal mucus. Lipid and metabolite abundance was generally higher in the jejunal mucus than the colonic mucus. CryoSEM microscopy revealed smaller pore size in small intestinal mucus, which increased in the large intestine. All mucus samples showed shear-thinning behavior and characteristics of gel-like structure. In conclusion, the mucus is a highly viscous and hydrated material. These data provide an important baseline for future studies on human and canine intestinal diseases and the dog model in drug absorption.

The gastrointestinal (GI) mucus layer is a protective and lubricating hydrogel of polymer-forming glycoproteins that covers our intestinal epithelium. This mucus layer serves as an interface between the intestinal epithelium and environment as well as a as first line of defense against the potentially harmful microorganisms. While the GI mucus layer closer to the gut epithelium is highly condensed and acts as a physical barrier for invading microorganisms, further away from the epithelium, proteolytic degradation makes it loose. This looser part of the mucus layer serves as an attachment site and a nutrient source for some commensal gut bacteria. The molecular mechanisms that drive the mucus–microbe interactions are emerging and are important to understand the functional role of the gut microbiome in health and disease. Previous work by my research group showed that a dietary fiber-deprived gut microbiota erodes the colonic mucus barrier and enhances susceptibility to a mucosal pathogen Citrobacter rodentium, a mouse model for human Escherichia coli infections. In this PhD thesis, I studied role of the gut mucus layer in the context of various other infectious and autoimmune diseases by inducing the natural erosion of the mucus layer by dietary fiber deprivation. In order to unravel the mechanistic details in the intricate interactions between diet, mucus layer and gut microbiome, I leveraged our previously established gnotobiotic mouse model hosting a synthetic human gut microbiota of fully characterized 14 commensal bacteria (14SM). I employed three different types of infectious diseases for the following reasons: 1) attaching and effacing (A/E) pathogen (C. rodentium), to better understand which commensal bacteria aid in enhancing the pathogen susceptibility when a fiber-deprived gut microbiota erodes the mucus barrier; 2) human intracellular pathogens (Listeria monocytogenes and Salmonella Tyhimurium) to investigate, whether like the A/E pathogen, erosion of the

The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth., QC 20220615

This is the peer reviewed version of the following article: Nakahata, M., Tominaga, N., Saito, K., Nishiyama, K., Tanino, Y., Saiki, K., Kojima, M. and Sakai, S. (2022), A Bio-synthetic Hybrid Hydrogel Formed under Physiological Conditions Consisting of Mucin and a Synthetic Polymer Carrying Boronic Acid. Macromol. Biosci., 22: 2200055, which has been published in final form at https://doi.org/10.1002/mabi.202200055. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited., Mucin-containing bio-synthetic hybrid hydrogel is successfully formed under physiological conditions upon mixing aqueous solutions of native mucin and synthetic polymers carrying boronic acids. The mechanical properties and stability of the hydrogel in physiological solutions, e.g., cell culture media, are tunable depending on the boronic acid content of polymers. The hydrogel dissolved in the physiological solutions releases native mucin and boronic acid-containing polymer, which can control the adhesion of mammalian cells to the surface.

Primary cutaneous mucinous carcinoma is a rare malignant neoplasm, that belong to a group of adnexal tumors of the skin. They are slow growing, mucin producer and commonly arises in the head and neck region. Some of its differential diagnoses are sebaceous cyst, hemangioma, melanoma, neurofibroma, pilomatrixoma and metastatic deposits. The main challenge in these lesions is to differentiate it from more frequent mucinous secondary deposits. Immunohistochemistry is mandatory to differentiate these lesions from metastases. Surgical excision with wide margins of at least 10 millimeters or Mohs surgery is the main stay of treatment due to the tendency of local recurrence., El carcinoma mucinoso cutáneo primario es una rara neoplasia maligna, que pertenece al grupo de tumores anexiales de la piel. Son de crecimiento lento, productores de mucina y se presentan principalmente en cabeza y cuello. Algunos de sus diagnósticos diferenciales son quiste sebáceo, hemangioma, melanoma, neurofibroma, pilomatrixoma y depósitos metastásicos. El desafío principal en estas lesiones es diferenciarlas de los depósitos secundarios mucinosos más frecuentes. La inmunohistoquímica es obligatoria para diferenciar estas lesiones de metástasis. El tratamiento principal es la resección con márgenes amplios de al menos 10 milímetros o cirugía de Mohs, debido a la tendencia de recurrencia local.

Hydrogels of cross-linked mucin glycoproteins (Muc-gel) have shown strong immune-modulating properties toward macrophages in vitro, which are translated in vivo by the dampening of the foreign body response to implantation in mice. Beyond mucin hydrogels, other biomaterials such as sensors, electrodes, and other long-term implants would also benefit from such immune-modulating properties. In this work, we aimed to transfer the bioactivity observed for three-dimensional Muc-gels to the surface of two model materials by immobilizing mucin into thin films (Muc-film) using covalent layer-by-layer assembly. We tested how the surface immobilization of mucins affects macrophage responses compared to Muc-gels. We showed that Muc-films on soft polyacrylamide gels mimic Muc-gel in their modulation of macrophage responses with activated gene expression of inflammatory cytokines on day 1 and then dampening them on day 3. Also, the markers of polarized macrophages, M1 and M2, were expressed at the same level for macrophages on Muc-film-coated soft polyacrylamide gels and Muc-gel. In contrast, Muc-film-coated hard polystyrene led to a different macrophage response compared to Muc-gel, having no activated expression of inflammatory cytokines and a different M1 marker expression. This suggested that the substrate mechanical properties and mucin molecular configuration determined by substrate-mucin interactions affect mucin immune-modulating properties. We conclude that mucin immune-modulating properties can be transferred to materials by mucin surface immobilization but will be dependent on the substrate chemical and mechanical properties., QC 20230523

Familial colorectal cancer (CRC) is only partially explained by known germline predisposing genes. We performed whole-genome sequencing in 15 Polish families of many affected individuals, without mutations in known CRC predisposing genes. We focused on loss-of-function variants and functionally characterized them. We identified a frameshift variant in the CYBA gene (c.246delC) in one family and a splice site variant in the TRPM4 gene (c.25–1 G > T) in another family. While both variants were absent or extremely rare in gene variant databases, we identified four additional Polish familial CRC cases and two healthy elderly individuals with the CYBA variant (odds ratio 2.46, 95% confidence interval 0.48–12.69). Both variants led to a premature stop codon and to a truncated protein. Functional characterization of the variants showed that knockdown of CYBA or TRPM4 depressed generation of reactive oxygen species (ROS) in LS174T and HT-29 cell lines. Knockdown of TRPM4 resulted in decreased MUC2 protein production. CYBA encodes a component in the NADPH oxidase system which generates ROS and controls, e.g., bacterial colonization in the gut. Germline CYBA variants are associated with early onset inflammatory bowel disease, supported with experimental evidence on loss of intestinal mucus barrier function due to ROS deficiency. TRPM4 encodes a calcium-activated ion channel, which, in a human colonic cancer cell line, controls calcium-mediated secretion of MUC2, a major component of intestinal mucus barrier. We suggest that the gene defects in CYBA and TRPM4 mechanistically involve intestinal barrier integrity through ROS and mucus biology, which converges in chronic bowel inflammation.

The intestinal epithelium is protected from direct contact with gut microbes by a mucus layer. This mucus layer consists of secreted mucin glycoproteins. The outer mucus layer in the large intestine forms a niche that attracts specific gut microbiota members of which several gut commensals can degrade mucin. Mucin glycan degradation is a complex process that requires a broad range of glycan degrading enzymes, as mucin glycans are intricate and diverse molecules. Consequently, it is hypothesized that microbial mucin breakdown requires concerted action of various enzymes in a network of multiple resident microbes in the gut mucosa. This review investigates the evolutionary relationships of microbial carbohydrate-active enzymes that are potentially involved in mucin glycan degradation and focuses on the role that microbial enzymes play in the degradation of gut mucin glycans in microbial cross-feeding and syntrophic interactions.

Many aspects of the mechanisms underlying the symbiosis between humans and gut microbes remain unknown and encompass some of the most intriguing questions in microbiome research. An important factor in this symbiosis is the interplay between microbes and human-produced glycans in mucin and breast milk. In this Opinion paper, I propose a synergy between the structural diversity of human mucin glycans and the enzymatic repertoire of the gut microbiome. The contribution of microbes to mucosal health is discussed, and the role of breast milk glycans in mucosal colonization by microbes is explained. The use of prebiotic mucin glycans in general, and specialized infant and medical nutrition in particular, should be considered as the field of interest to modulate the microbiota and improve mucosal health.

Hypothesis: Salivary pellicles i.e., thin films formed upon selective adsorption of saliva, protect oral surfaces against chemical and mechanical insults. Pellicles are also excellent aqueous lubricants. It is generally accepted that reconstituted pellicles have a two-layer structure, where the outer layer is mainly composed of MUC5B mucins. We hypothesized that by comparing the effect of ionic strength on reconstituted pellicles and MUC5B films we could gain further insight into the pellicle structure. Experiments: Salivary pellicles and MUC5B films reconstituted on solid surfaces were investigated at different ionic strengths by Force Spectroscopy, Quartz Crystal Microbalance with Dissipation, Null Ellipsometry and Neutron Reflectometry. Findings: Our results support the two-layer structure for reconstituted salivary pellicles. The outer layer swelled when ionic strength decreased, indicating a weak polyelectrolyte behavior. While initially the MUC5B films exhibited a similar tendency, this was followed by a drastic collapse indicating an interaction between exposed hydrophobic domains. This suggests that mucins in the pellicle outer layer form complexes with other salivary components that prevent this interaction. Lowering ionic strength below physiological values also led to a partial removal of the pellicle inner layer. Overall, our results highlight the importance that the interactions of mucins with other pellicle components play on their structure.

Hydrogels made of crosslinked macromolecules used in regenerative medicine technologies can be designed to affect the fate of surrounding cells and tissues in defined ways. Their function typically depends on the type and number of bioactive moieties such as receptor ligands present in the hydrogel. However, the detail in how such moieties are presented to cells can also be instrumental. In this work, how the crosslinking architecture of a hydrogel can affect its bioactivity is explored. It is shown that bovine submaxillary mucins, a highly glycosylated and immune-modulating protein, exhibit strikingly different bioactivities whether they are crosslinked through their glycans or their protein domains. Both the susceptibility to enzymatic degradation and macrophage response are affected, while rheological properties and barrier to diffusion are mostly unaffected. The results suggest that crosslinking architecture affects the accessibility of the substrate to proteases and the pattern of sialic acid residues exposed to the macrophages. Thus, modulating the accessibility of binding sites through the choice of the crosslinking strategy appears as a useful parameter to tune the bioactivity of hydrogel-based systems., QC 20211110

Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as nonproductive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined because of a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBCs) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with the current view of mucins as providing a protective shield against pathogens. Counterintuitively, increasing the density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.

A major barrier to the emergence of distant metastases is the survival of circulating tumor cells (CTCs) within the vasculature. Lethal stressors, including shear forces from blood flow, anoikis arising from cellular detachment, and exposure to natural killer cells, combine to subvert the ability of primary tumor cells to survive and ultimately seed distant lesions. Further attenuation of this rate-limiting process via therapeutic intervention offers a very attractive opportunity for improving cancer patient outcomes, in turn prompting the need for a deeper understanding of the molecular and cellular mechanisms underlying CTC viability. MUC4 is a very large and heavily glycosylated protein expressed at the apical surfaces of the epithelia of a variety of tissues, is involved in cellular growth signaling and adhesiveness, and contributes to the protection and lubrication of cellular linings. Analysis of patient-matched breast tumor specimens has demonstrated that MUC4 protein levels are upregulated in metastatic lesions relative to primary tumor among all breast tumor subtypes, pointing to a possible selective advantage for MUC4 overexpression in metastasis. Analysis of a genetically engineered mouse model of HER2-positive breast cancer has demonstrated that metastatic efficiency is markedly suppressed with Muc4 deletion and Muc4-knockout tumor cells are poorly associated with platelets and white blood cells known to support CTC viability. In this review, we discuss the diverse roles of MUC4 in tumor progression and metastasis and propose that intervening in MUC4 intercellular interactions with binding partners on blood-borne aggregating cells could potentially thwart breast cancer metastatic efficiency.

Background: Most respiratory viruses show pronounced seasonality, but for SARS-CoV-2, this still needs to be documented. Methods: We examined the disease progression of COVID-19 in 6,914 patients admitted to hospitals in Europe and China. In addition, we evaluated progress of disease symptoms in 37,187 individuals reporting symptoms into the COVID Symptom Study application. Findings: Meta-analysis of the mortality risk in seven European hospitals estimated odds ratios per 1-day increase in the admission date to be 0.981 (0.973–0.988, p < 0.001) and per increase in ambient temperature of 1°C to be 0.854 (0.773–0.944, p = 0.007). Statistically significant decreases of comparable magnitude in median hospital stay, probability of transfer to the intensive care unit, and need for mechanical ventilation were also observed in most, but not all hospitals. The analysis of individually reported symptoms of 37,187 individuals in the UK also showed the decrease in symptom duration and disease severity with time. Interpretation: Severity of COVID-19 in Europe decreased significantly between March and May and the seasonality of COVID-19 is the most likely explanation.

Mucus plays a pivotal role in protecting the respiratory tract against microbial infections. It acts as a primary contact site to entrap microbes and facilitates their removal from the respiratory tract via the coordinated beating of motile cilia. The major components of airway mucus are heavily O-glycosylated mucin glycoproteins, divided into gel-forming mucins and transmembrane mucins. The gel-forming mucins MUC5AC and MUC5B are the primary structural components of airway mucus, and they enable efficient clearance of pathogens by mucociliary clearance. MUC5B is constitutively expressed in the healthy airway, whereas MUC5AC is upregulated in response to inflammatory challenge. MUC1, MUC4, and MUC16 are the three major transmembrane mucins of the respiratory tracts which prevent microbial invasion, can act as releasable decoy receptors, and activate intracellular signal transduction pathways. Pathogens have evolved virulence factors such as adhesins that facilitate interaction with specific mucins and mucin glycans, for example, terminal sialic acids. Mucin expression and glycosylation are dependent on the inflammatory state of the respiratory tract and are directly regulated by proinflammatory cytokines and microbial ligands. Gender and age also impact mucin glycosylation and expression through the female sex hormone estradiol and age-related downregulation of mucin production. Here, we discuss what is currently known about the role of respiratory mucins and their glycans during bacterial and viral infections of the airways and their relevance for the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Understanding the impact of microbe-mucin interaction in the respiratory tract could inspire the development of novel therapies to boost mucosal defense and combat respiratory infections.

The enteropathogenic bacterium Campylobacter jejuni was considered to be non-saccharolytic, but recently it emerged that l-fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l-fucose utilization. In the intestinal tract, l-fucose is abundantly available in mucin O-linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l-fucose. We set out to determine how C. jejuni can gain access to these intestinal l-fucosides. Growth of the fuc + C. jejuni strains 129,108 and NCTC 11168 increased in the presence of l-fucose while fucose permease knockout strains did not benefit from additional l-fucose. With fucosidase assays and an activity-based probe we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. C. jejuni invaded Caco-2 intestinal cells that express complex O-linked glycan structures that contain l-fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion. This article is protected by copyright. All rights reserved.

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.

Enzymes active on glycosidic bonds are defined according to the stereochemistry of both substrates and products of the reactions they catalyse. The CAZy classification further assigns these enzymes into sequence-based families sharing a common stereochemistry for substrates (either α- or β-) and products, i.e. inverting or retaining mechanism. Here we describe the N-acetylgalactosaminidases AmGH109A and AmGH109B from the human gut symbiont Akkermansia muciniphila. Notably, AmGH109A displays α-retaining and β-inverting N-acetylgalactosaminidase activities with comparable efficiencies on natural disaccharides. This dual specificity could provide an advantage in targeting a broader range of host-derived glycans. We rationalise this discovery through bioinformatics, structural, mutational, and computational studies, unveiling a histidine residing in a conserved GGHGG motif as the elusive catalytic acidbase of the GH109 family.

Growing evidence supports the importance of gut microbiota in the control of tumor growth and response to therapy. Here, we select prebiotics that can enrich bacterial taxa that promote anti-tumor immunity. Addition of the prebiotics inulin or mucin to the diet of C57BL/6 mice induces anti-tumor immune responses and inhibition of BRAF mutant melanoma growth in a subcutaneously implanted syngeneic mouse model. Mucin fails to inhibit tumor growth in germ-free mice, indicating that the gut microbiota is required for the activation of the anti-tumor immune response. Inulin and mucin drive distinct changes in the microbiota, as inulin, but not mucin, limits tumor growth in syngeneic mouse models of colon cancer and NRAS mutant melanoma and enhances the efficacy of a MEK inhibitor against melanoma while delaying the emergence of drug resistance. We highlight the importance of gut microbiota in anti-tumor immunity and the potential therapeutic role for prebiotics in this process.

Rationale: Smoking-related chronic obstructive pulmonary disease (COPD) is associated with dysregulated production of mucus. Mucins (MUC) are important both for mucus secretion and epithelial defense. We have examined the distribution of MUC1 and MUC4 in the airway epithelial cells of never-smokers and smokers with and without COPD. Methods: Mucosal biopsies and bronchial wash samples were obtained by bronchoscopy from age- and sex-matched COPD-patients (n = 38; GOLD I-II/A-B), healthy never-smokers (n = 40) and current smokers with normal lung function (n = 40) from the Karolinska COSMIC cohort (NCT02627872). Cell-specific expressions of MUC1, MUC4 and regulating factors, i.e., epithelial growth factor receptor (EGFR) 1 and 2, were analyzed by immunohistochemistry. Soluble MUC1 was measured by quantitative immunodetection on slot blot. Results: The levels of cell-bound MUC1 expression in basal cells and in soluble MUC1 in bronchial wash were increased in smokers, regardless of airway obstruction. Patients with chronic bronchitis had higher MUC1 expression. The expression of MUC4 in cells with goblet cell phenotype was increased in smokers. The expression of EGFR2, but not that of EGFR1, was higher in never-smokers than in smokers. Conclusions: Smoking history and the presence of chronic bronchitis, regardless of airway obstruction, affect both cellular and soluble MUC1 in human airways. Therefore, MUC1 may be a novel marker for smoking- associated airway disease.

Intestinal adenomyoma is a rare benign tumour which can occur in infancy. We report a case of a 1-year-old boy who presented with neurological symptoms who was found to have intussusception with an anatomic lead point. Histopathology demonstrates a nodule within the submucosa, characterised by variably dilated glands lined by mucin-secreting columnar epithelium which are accompanied by smooth muscle bands within the stroma. Awareness of this benign entity is useful as it may be mistaken for clinically relevant entities such as a hamartomatous polyp or well differentiated adenocarcinoma.Copyright © 2020

The ability to adhere to the intestinal mucus layer is an important property of probiotic bacteria. Lactobacillus reuteri strains ZJ615 and ZJ617 show low and high adhesion, respectively, to intestinal epithelial cells. In this study, we quantified bacterial cell wall-associated glyceraldehyde-3-phosphate dehydrogenases (cw-GAPDH) and bacterial cell membrane permeability in both strains using immunoblotting and flow cytometry, respectively. Highly adhesive L. reuteri ZJ617 possessed significantly more cw-GAPDH, higher cell membrane permeability, and significantly higher adhesive ability toward mucin compared with low-adhesive L. reuteri ZJ615. In vitro adhesion studies and analysis of interaction kinetics using the Octet, the system revealed significantly decreased interaction between L. reuteri and mucin when mucin was oxidized when bacterial surface proteins were removed when bacteria were heat-inactivated at 80 °C for 30 min, and when the interaction was blocked with an anti-GAPDH antibody. SWISS-MODEL analysis suggested intensive interactions between mucin glycans (GalNAcα1-O-Ser, GalNAcαSer, and Galβ3GalNAc) and GAPDH. Furthermore, in vivo studies revealed significantly higher numbers of bacteria adhering to the jejunum, ileum, and colon of piglets orally inoculated with L. reuteri ZJ617 compared with those inoculated with L. reuteri ZJ615; this led to a significantly decreased rate of diarrhea in piglets inoculated with L. reuteri ZJ617. In conclusion, there are strong correlations among the abundance of cw-GAPDH in L. reuteri, the ability of the bacterium to adhere to the host, and the health benefits of this probiotic.

Immune-orchestrating biomaterials that precisely modulate the immune reac-tion to the host could lead the way for improving the implantation outcomein the transplantation field, in comparison to passive biomaterials. The lab-oratory of Dr. Thomas Crouzier has shown hydrogels derived from mucinsare capable of orchestrating the immune response mediated by foreign bodyresponse (FBR), as a result of evading fibrosis. Further, a recent study fromhis group showed sialic acid on mucin hydrogels is essential for the immuno-logical activity of those materials. Mucin glycans transiently activated thendampened macrophages, important orchestrators for material-mediated FBR,in a sialic acid-dependent manner for the majority of cytokines followed. Thematerial properties such as rheological properties, self-healing capacity, andstability, can be governed by the crosslinking chemistry used and have a drasticimpact on the functionalities of the materials. In this project, various cross-linking strategies are applied to tune the hydrogel properties. We show thatthe robust cross-linking formed mucin hydrogels having a 1.5% (wt/v) bettersupported insulin-secreting cells form islet-like organoids, compared to 2.5%mucin hydrogels. We then investigate the self-healing properties of the newmucin hydrogels and their interactions with various cell systems., Immunologiska biomaterial som specifikt modulerar en immunologisk reak-tion hos värden kan vara vägen till att förbättra resultatet av en transplantationi jämförelse med att använda passiva biomaterial. Inom laboratoriet lett av Dr.Thomas Crouzier har det kunnat påvisas att hydrogels erhållna från mucinerär kapabla till att förändra en immunlogisk respons, Foreign Body Respon-se (FBR) vilket resulterat i att kringgå fibrosis. Utöver detta har ytterligareen studie utförts av hans grupp vilken kunde påvisa att sialinsyra på mucinhydrogel är essentiellt för den immunologiska aktiviteten för dessa biomateri-al. Mucin glykaner aktiveras tillfälligt för att dämpa bildandet av makrofager,vilket är viktigt vid biomaterial FBR i ett sialinsyra-beroende för bildandet avmajoriteten av cytokiner. Materialegenskaperna såsom reologiska egenskaper,självläkande kapacitet, och stabilitet kan regleras genom en krosslänkad kemisom har en drastisk impakt för funktionen för biomaterialen. I detta projekt, ärflera krosslänkande strategier tillämpade för att förstå egenskaperna för hyd-rogelerna. Vi har kunnat påvisa att robusta krosslänkande system av mucinhydrogel bestående av 1.5% (wt/v) ger bättre support till insulin sekreterandeceller att bilda islet liknande organider, att jämföra med 2.5% mucin hydrogel.Vi undersöker sedan den självläkande egenskaperna av nya mucin hydrogeleroch deras interaktioner med ett flertal olika cellsystem.

The ability to adhere to the intestinal mucus layer is an important property of probiotic bacteria. Lactobacillus reuteri strains ZJ615 and ZJ617 show low and high adhesion, respectively, to intestinal epithelial cells. In this study, we quantified bacterial cell wall-associated glyceraldehyde-3-phosphate dehydrogenases (cw-GAPDH) and bacterial cell membrane permeability in both strains using immunoblotting and flow cytometry, respectively. Highly adhesive L. reuteri ZJ617 possessed significantly more cw-GAPDH, higher cell membrane permeability, and significantly higher adhesive ability toward mucin compared with low-adhesive L. reuteri ZJ615. In vitro adhesion studies and analysis of interaction kinetics using the Octet, the system revealed significantly decreased interaction between L. reuteri and mucin when mucin was oxidized when bacterial surface proteins were removed when bacteria were heat-inactivated at 80 °C for 30 min, and when the interaction was blocked with an anti-GAPDH antibody. SWISS-MODEL analysis suggested intensive interactions between mucin glycans (GalNAcα1-O-Ser, GalNAcαSer, and Galβ3GalNAc) and GAPDH. Furthermore, in vivo studies revealed significantly higher numbers of bacteria adhering to the jejunum, ileum, and colon of piglets orally inoculated with L. reuteri ZJ617 compared with those inoculated with L. reuteri ZJ615; this led to a significantly decreased rate of diarrhea in piglets inoculated with L. reuteri ZJ617. In conclusion, there are strong correlations among the abundance of cw-GAPDH in L. reuteri, the ability of the bacterium to adhere to the host, and the health benefits of this probiotic.

The Colonic Mucosal Barrier (CMB) is the site of interaction between the human body and the colonic microbiota. The mucus is the outer part of the CMB and is considered as the front-line defense of the colon. It separates the host epithelial lining from the colonic content, and it has previously been linked to health and diseases. In this study, we assessed the relationship between red meat and whole-grain intake and (1) the thickness of the colonic mucus (2) the expression of the predominant mucin gene in the human colon (MUC2). Patients referred to colonoscopy at the University Hospital of Southern Denmark-Sonderjylland were enrolled between June 2017 and December 2018, and lifestyle data was collected in a cross-sectional study design. Colonic biopsies, blood, urine, and fecal samples were collected. The colonic mucus and bacteria were visualized by immunostaining and fluorescence in situ hybridization techniques. We found a thinner mucus was associated with high red meat intake. Similarly, the results suggested a thinner mucus was associated with high whole-grain intake, albeit to a lesser extent than red meat. This is the first study assessing the association between red meat and whole-grain intake and the colonic mucus in humans. This study is approved by the Danish Ethics Committee (S-20160124) and the Danish Data Protecting Agency (2008-58-035). A study protocol was registered at clinical trials.gov under NCT04235348.

In this study, insulin-loaded nanoparticles (NPs) were prepared via self-gelation method using chitosan and aqueous soluble snail mucin as natural polymers. Herein, mucins were ionically interacted with chitosan at different concentrations to obtained insulin-loaded NPs, labelled as A1 (1:1) (i.e., chitosan 2 % w/v + mucin 2 % w/v) and A2 (2:1) (chitosan 4 % w/v + mucin 2 % w/v), using poloxamer and poly vinyl alcohol as solid surfactant. Such formulation was selected to provide the necessary dynamics for the formation of the nanoparticles while maintaining the surface properties that will favor the encapsulation of insulin. Each system was characterized in terms of their particle size distribution, morphology, zeta potential, and polydispersity index. In vitro release of insulin was evaluated in acidic solution (pH 1.2) and phosphate buffer solution (pH 7.4), and the hypoglycaemic activity was evaluated in diabetes rats. The prepared insulin-loaded NPs displayed particles with relatively smooth surfaces and an average particle size of 479.6 and 504.1 nm for A1 and A2, respectively. Zeta potential and polydispersity index, ranged from 22.1 to 31.2 mV and 0.155–0.185, respectively. The encapsulating efficiency for the systems A1 and A2 were 88.6 and 92.5, respectively, and a self-sustained release of encapsulated insulin was observed for over a period of 8 h. In vivo studies revealed a pronounced hypoglycaemic effect in diabetic rats after peroral administration of the insulin-loaded NPs compared to the effect caused by free oral insulin solution. In addition, both the pharmacokinetic and toxicity results showed low plasma clearance of insulin and no signs of toxicity on the liver enzyme and cell viability, which suggested good biocompatibility of the NPs formulations. Overall, the formation of NPs of insulin with chitosan and snail mucin represents a potentially safe and promising approach to protect insulin and enhance its peroral delivery.

The cellular invasion machinery of the enteric pathogen Salmonella consists of a type III secretion system (T3SS) with injectable virulence factors that induce uptake by macropinocytosis. Salmonella invasion at the apical surface of intestinal epithelial cells is inefficient, presumably because of a glycosylated barrier formed by transmembrane mucins that prevents T3SS contact with host cells. We observed that Salmonella is capable of apical invasion of intestinal epithelial cells that express the transmembrane mucin MUC1. Knockout of MUC1 in HT29-MTX cells or removal of MUC1 sialic acids by neuraminidase treatment reduced Salmonella apical invasion but did not affect lateral invasion that is not hampered by a defensive barrier. A Salmonella deletion strain lacking the SiiE giant adhesin was unable to invade intestinal epithelial cells through MUC1. SiiE-positive Salmonella closely associated with the MUC1 layer at the apical surface, but invaded Salmonella were negative for the adhesin. Our findings uncover that the transmembrane mucin MUC1 is required for Salmonella SiiE-mediated entry of enterocytes via the apical route.

Human saliva, a seemingly simple aqueous fluid, is, in fact, an extraordinarily complex biocolloid that is not fully understood, despite many decades of study. Salivary lubrication is widely believed to be a signature of good oral health and is also crucial for speech, food oral processing and swallowing. However, saliva has been often neglected in food colloid research, primarily due to its high intra- to inter-individual variability and altering material properties upon collection and storage, when used as an ex vivo research material. In the last few decades, colloid scientists have attempted designing model (i.e. ‘saliva mimicking fluid’) salivary formulations to understand saliva-food colloid interactions in an in vitro set up and its contribution on microstructural aspects, lubrication properties and sensory perception. In this Review, we critically examine the current state of knowledge on bulk and interfacial properties of model saliva in comparison to real human saliva and highlight how far such model salivary formulations can match the properties of real human saliva. Many, if not most, of these model saliva formulations share similarities with real human saliva in terms of biochemical compositions, including electrolytes, pH and concentrations of salivary proteins, such as α-amylase and highly glycosylated mucins. This, together with similarities between model and real saliva in terms of surface charge, has led to significant advancement in decoding various colloidal interactions (bridging, depletion) of charged emulsion droplets and associated sensory perception in the oral phase. However, model saliva represents significant dissimilarity to real saliva in terms of lubricating properties. Based on in-depth examination of properties of mucins derived from animal sources (e.g. pig gastric mucins (PGM) or bovine submaxillary mucin (BSM)), we can recommend that BSM is currently the most optimal commercially available mucin source when attempting to replicate

In the human body, mucin glycoproteins efficiently reduce friction between tissues and thereby protect the mucosa from mechanical damage. Mucin lubricity is closely related to their molecular structure: it has been demonstrated previously that the hydrophobic termini of mucins critically contribute to their lubricity. If and how intrinsic sources of negative charge in mucins, e.g., sulfated glycans and sialic acid residues, are relevant for the tribological behavior of mucin solutions has, however, not been addressed yet. In this manuscript, we show that the removal of either sialic acid or sulfate groups, which comprise only a minor amount of the total molecular weight, from MUC5B drastically reduces its lubricity. For MUC5AC solutions, however, this effect only occurs once mucin-associated DNA is removed as well. We find that neither the hydration state nor the average conformation of mucins adsorbed onto hydrophilic or hydrophobic surfaces is affected by the removal of anionic sugars. Instead, our data suggests that a loss of anionic sugars mainly influences the dynamic adsorption process of mucins onto both hydrophilic and hydrophobic surfaces., QC 20200505