The MUC2 Gene Product: Polymerisation and Post-Secretory Organisation-Current Models.

MUC2 mucin, the primary gel-forming component of intestinal mucus, is well researched and a model of polymerisation and post-secretory organisation has been published previously. Recently, several significant developments have been made which either introduce new ideas or challenge previous theories. New ideas include an overhaul of the MUC2 C-terminal globular structure which is proposed to harbour several previously unobserved domains, and include a site for an extra intermolecular disulphide bridge dimer between the cysteine 4379 of adjacent MUC2 C-termini. MUC2 polymers are also now thought to be secreted attached to the epithelial surface of goblet cells in the small intestine and removed following secretion via a metalloprotease meprin ß-mediated cleavage of the von Willebrand D2 domain of the N-terminus. It remains unclear whether MUC2 forms intermolecular dimers, trimers, or both, at the N-termini during polymerisation, with several articles supporting either trimer or dimer formation. The presence of a firm inner mucus layer in the small intestine is similarly unclear. Considering this recent research, this review proposes an update to the previous model of MUC2 polymerisation and secretion, considers conflicting theories and data, and highlights the importance of this research to the understanding of MUC2 mucus layers in health and disease.

A Pilot Pre and Post 4 Week Intervention Evaluating the Effect of a Proprietary, Powdered, Plant Based Food on Micronutrient Status, Dietary Intake, and Markers of Health in a Healthy Adult Population.

Background: A "balanced, adequate, and varied diet" is recommended as the basis of nutritionally sound diet by the World Health Organisation and national public health agencies. Huel is a proprietary, on-the-go, powdered, plant based food, providing all 26 essential vitamins and minerals, protein, essential fats, carbohydrate, fibre, and phytonutrients. Objectives: Assessing the effect of solely consuming Huel on micronutrient status, dietary intake and markers of health was achieved through a 4-week intervention of solely Huel powder. Methods: Habitual energy intake was assessed through a one-week lead in period with healthy adult participants (aged 18 or over) logging their food intake, after which only Huel was consumed for 4 weeks. Blood samples and body composition was assessed before and after the lead in week as well the end of the intervention. Thirty participants were recruited with 20 (11 females, median age 31, range 22-44) completing the study, 19 sets of blood samples were collected. 22 blood markers were analysed along with weight, BMI, waist circumference, visceral adipose tissue (VAT), and body composition. All blood micronutrients, except for Thyroid Stimulating Hormone and choline were sent to Royal Victoria Infirmary NHS, Newcastle Laboratory (Newcastle upon Tyne, United Kingdom) for analysis. Results: Fourteen of the parameters significantly changed over the course of the study with circulating haemoglobin, iron, vitamins B12 and D as well as selenium significantly increasing (p < 0.05). HbA1c, total and non-HDL cholesterol, vitamins A and E, potassium, BMI, VAT, and waist circumference all significantly decreased (p < 0.05) post intervention. Conclusion: Although energy intake decreased during the intervention period, the adherence to recommended micronutrient intake, as quantified by the dietary Total Adherence Score, significantly increased which tallies with the preservation or improvement of micronutrient status. This study potentially demonstrates that consuming only Huel for 4 weeks does not negatively affect micronutrient status.

The effect of seaweed enriched bread on carbohydrate digestion and the release of glucose from food.

Two seaweeds; Ascophyllum nodosum and Fucus vesiculosus, were incorporated into bread at 0.5 and 2% and their effect on blood glucose in vivo and carbohydrate digestion in vitro were studied. In the five way randomised placebo controlled double blind pilot trial (n = 10) each volunteer consumed 100 g of available carbohydrate (from bread) and their blood glucose was measured over two hours. The breads were tested in a human digestion model and compared against control bread and control bread with the equivalent amount of seaweed. In the pilot human study the enriched breads did not cause any significant reductions in iAUC of blood glucose with average reductions of 0.1 ± 44.4%, 8.2 ± 19.3%, 1.0 ± 54.3% and 2.7 ± 31.9% for 0.5% F.vesiculosus, 0.5% A.nodosum, 2% F.vesiculosus, and 2% A.nodosum respectively. However, seaweed added alongside the control bread in vitro significantly reduced the level of carbohydrate digestion compared to the control bread. F.vesiculosus or A.nodosum can reduce carbohydrate digestion, however baking into bread reduces the effect.

Prominent members of the human gut microbiota express endo-acting O-glycanases to initiate mucin breakdown.

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.

Encapsulation of stevia rebaudiana Bertoni aqueous crude extracts by ionic gelation - Effects of alginate blends and gelling solutions on the polyphenolic profile.

We formulated and characterised two alginate blends for the encapsulation of stevia extract (SE) via ionic gelation through an extrusion technique. Calcium chloride in SE and calcium chloride solutions were assessed as crosslinkers to overcome phenolic losses by diffusion and increase encapsulation efficiency (EE). Regardless of the blend, all stevia-loaded beads exhibited high EE (62.7-101.0%). The size of the beads decreased as EE increased. Fourier transform infrared analysis showed increased hydrogen bonding between SE and alginates, confirming the successful incorporation of SE within the matrix. Untargeted metabolomics profiling identified 479 free and encapsulated polyphenolic compounds. Flavonoids (catechin and luteolin equivalents) were predominant in SE whereas tyrosols and 5-pentadecylresorcinol equivalents were predominant in all bead formulations. Three-common discriminant compounds were exclusive to each blend and were inversely affected by the crosslinking conditions. Both alginate blends have been shown to be feasible as carrier systems of stevia extracts independent of crosslinking conditions.

Acceptability of alginate enriched bread and its effect on fat digestion in humans.

Lifestyle interventions and physical activity remain the cornerstone of obesity management, as pharmacological therapies (orlistat) are associated with gastrointestinal (GI) side effects. Combining orlistat with fibers can reduce side effects, improving compliance. Therefore, a fiber that inhibits lipase without side effects could help treat obesity. The aims of the present work were to assess whether alginate enriched bread could inhibit fat digestion, and assess the acceptability of alginate bread and its effect on GI wellbeing. A double-blind, randomised, controlled cross-over pilot study (NCT03350958) assessed the impact of an alginate bread meal on; lipid content in ileal effluent and circulating triacylglycerol levels. This was compared against the same meal with non-enriched (control) bread. GI wellbeing and acceptability of alginate bread was compared to control bread through daily wellbeing questionnaires and food diaries (NCT03477981). Control bread followed by alginate bread were consumed for two weeks respectively. Consumption of alginate bread reduced circulating triacylglycerol compared to control (2% reduction in AUC) and significantly increased lipid content in ileal effluent (3.8 g ±â€¯1.6 after 210 min). There were no significant changes to GI wellbeing when comparing alginate bread to control bread. A significant increase in the feeling of fullness occurred with alginate bread compared to baseline and the first week of control bread consumption. This study showed that sustained consumption of alginate enriched bread does not alter GI wellbeing and can decrease lipolysis, increasing lipid leaving the small intestine. Further studies are required to demonstrate that reduced fat digestion through the action of alginate can reduce fat mass or body weight.

Efficacy and safety concerns over the use of mucus modulating agents for drug delivery using nanoscale systems.

Drug delivery to the mucus covered mucosae is fraught with difficulties and many different approaches have been developed to permeate the mucus barrier. Generally by modifying the delivery system to avoid interaction with the mucus. These modifications are reviewed here in terms of efficacy and safety. These are particular problems for oral delivery the pharmaceutical industry's favoured route for drug administration. For effective delivery through the gastrointestinal tract a drug must pass through three barriers in sufficient amounts to yield a biological effect. These barriers are the digestive barrier in the lumen, the mucus barrier, and the epithelial barrier. Other approaches involve mucolytic agents added with or prior to the delivery system or agents regulating mucus production and are reviewed here. In terms of safety, a key property of a mucus modulating delivery system is that it must not damage the protective function of the mucus layer.

ζ potential changing nanoparticles as cystic fibrosis transmembrane conductance regulator gene delivery system: an in vitro evaluation.

AIM: Aim of the study was the development of ζ potential changing nanoparticles as gene delivery system for the cystic fibrosis transmembrane conductance regulator gene. METHODS: Chitosan and carboxymethyl cellulose were modified with phosphotyrosine, a substrate for the brush border enzyme alkaline phosphatase. With these synthesized derivatives, different nanoparticle formulations, including the cystic fibrosis transmembrane conductance regulator gene were prepared by ionic gelation. RESULTS: A change from negative to positive ζ potential after enzymatic cleavage could be observed. Transfection studies with HEK-293 and Caco-2 cells showed transfection rates comparable to Lipofectamine 2000. Transfection efficiencies were significantly decreased when phosphate cleavage and thus ζ potential change was inhibited by phosphatase inhibitor. CONCLUSION: The developed nanoparticles represent a promising gene delivery system.

Inhibitory activity of extracts of Hebridean brown seaweeds on lipase activity.

The effect of three Hebridean brown seaweeds on lipase activity was assessed using a turbidimetric lipase activity assay and an in vitro simulation of the upper digestive tract. The preparations of Ascophyllum nodosum, Fucus vesiculosus, and Pelvetia canaliculata were tested; whole seaweed homogenate, sodium carbonate extract, and ethanol extracts (pellet and supernatant were tested separately). All extracts showed significant inhibition of lipase, suggesting multiple bioactive agents, potentially including alginates, fucoidans, and polyphenols. Whole homogenate extract of F. vesiculosus was the most potent inhibitor of Lipase (IC50 = 0.119 mg mL-1), followed by ethanol supernatant (IC50 = 0.159 mg mL-1) while ethanol pellet and sodium carbonate extract showed relatively weaker inhibition (IC50 = 0.360 mg mL-1 and IC50 = 0.969 mg mL-1 respectively). For A. nodosum and P. canaliculata, strongest inhibition occurred with ethanol pellet (IC50 = 0.238 and 0.228 mg mL-1, respectively). These inhibitory effects were validated in a model gut system. The data presented herein suggests the use of seaweed as a potential weight management tool is deserving of further investigation.

The properties of the mucus barrier, a unique gel--how can nanoparticles cross it?

The key criterion for a nanoparticle drug-delivery system is the ability to produce substantial bioavailability without damaging the physiological protective mechanisms. The main area for drug delivery is the aerodigestive tract. All epithelial surfaces have a membrane-bound layer and in the lung this layer is surmounted by a gel layer. In the gastrointestinal tract the membrane-bound mucin layer is covered by a mucus bilayer. The pore sizes of mucus gels are around 100 to 200 nm. Consequently, only nanoparticles in this size range could potentially penetrate without modification of these layers. To study nanoparticle permeation with results that pertain to in vivo conditions, native mucus mucin preparations must be used. Strategies to increase pores in mucus gels are discussed herein.

The role of seaweed bioactives in the control of digestion: implications for obesity treatments.

Seaweeds are an underutilised nutritional resource that could not only compliment the current western diet but potentially bring additional health benefits over and above their nutritional value. There are four groups of seaweed algae; green algae (Chlorophyceae), red algae (Rhodophycae), blue-green algae (Cyanophyceae) and brown algae (Phaeophyceae). Seaweeds are rich in bioactive components including polysaccharides and polyphenols. Polysaccharides content, such as fucoidan, laminarin, as well as alginate is generally high in brown seaweeds which are also a source of polyphenols such as phenolic acids, flavonoids, phlorotannin, stilbenes and lignans. These components have been shown to reduce the activity of digestive enzymes, modulating enzymes such as α-amylase, α-glucosidase, pepsin and lipase. This review discusses the effect of several of these components on the digestive processes within the gastrointestinal tract; focusing on the effect of alginate on pancreatic lipase activity and its potential health benefits. Concluding that there is evidence to suggest alginate has the potential to be used as an obesity treatment, however, further in vivo research is required and an effective delivery method for alginate must be designed.

Alginate as a protease inhibitor in vitro and in a model gut system; selective inhibition of pepsin but not trypsin.

Alginates are widely used in the food and medical industries, including as a Gastro-Oesophagul Reflux treatment. This work investigates the inhibitory effects of alginate on the reflux aggressors trypsin and pepsin and the role of alginate-substrate binding, pH and alginate structure on inhibition. Alginates were shown to reduce pepsin activity by up to 53.9% (±9.5SD) in vitro. Strong positive correlation between alginate mannuronate residue frequency and levels of pepsin inhibition was observed. Limited inhibition of trypsin was shown. Viscometric observations of pH dependent interactions between alginate and protein suggest a mechanism whereby pH dependent ionic interactions reduce substrate availability to enzyme at acidic pH. To understand how dietary protein digestion is affected by alginate, proteolytic digestion was investigated in an in vitro model of the upper digestive tract. Significant inhibition of proteolysis was shown in the gastric phase of digestion, but not the small intestinal phase.

Biological activity of alginate and its effect on pancreatic lipase inhibition as a potential treatment for obesity.

Alginates are classed as a dietary fibre and have been shown to inhibit digestive enzymes in vitro, and therefore could be used as an obesity treatment. The current study aims to assess whether alginate in a bread vehicle maintains its inhibition properties despite cooking and digestion, and may therefore be used as a potential treatment for obesity. After 180 min in a model gut that replicates digestion in the mouth, stomach and small intestines alginate bread (AB), control bread (CB), CB with Manucol® DM alginate, free DM alginate and model gut solution were collected. DM, LFR 5/60 and SF200 were heated at 37 °C and 200 °C, with DM also heated at 50, 100 and 150 °C. Samples from the model gut and heated alginate were assessed for molecular size and inhibition properties using viscosity, gel filtration and a lipase turbidity assay. AB does not significantly increase viscosity in the model gut. Viscosity of alginate reduces beyond 100 °C, although alginate retains its inhibition properties up to 150 °C. Cooking into the bread does not reduce the molecular size of the alginate or affect its inhibition properties. These data demonstrate the robustness of alginates lipase inhibition despite the cooking process and digestion. Therefore adding alginate to a bread vehicle may have the potential in the treatment for obesity.

Method for quantifying alginate and determining release from a food vehicle in gastrointestinal digesta.

To assess the efficacy of alginate as a modifier of enzyme activity, a suitable method to quantify its release must be developed. This paper develops and assesses the ability of the Periodic Acid Schiffs (PAS) assay to quantify alginate, and its release from bread during digestion in a model gut. Control and alginate enriched (4% w/w wet dough) bread were used. A model gut replicating the mouth, stomach and small intestines was used. Standard curves were created for alginate in deionised H2O and model gut solutions using a modified PAS to remove interference. The PAS assay quantified alginate with excellent linearity (R(2)=0.99), and optical density range (0.02-0.5). There was a significant difference in alginate release at 180 min compared to 0 and 60 min. The data indicate the modified PAS assay is a simple method for quantifying alginate release and release rate from alginate enriched products.

Bile acids in laryngopharyngeal refluxate: will they enhance or attenuate the action of pepsin?

OBJECTIVES/HYPOTHESIS: To assess if, as previously reported in the literature, bile acids inhibit pepsin activity, resulting in pepsin having a less important role in laryngopharyngeal damage in reflux disease. STUDY DESIGN: Prospective translational research study. METHODS: A total of 78 patient's fasting gastric juice samples were obtained from routine endoscopy. The total bile acid (TBA) content and pepsin activity were measured using a 3α-hydroxysteroid dehydrogenase-based kit for bile acids; and pepsin activity was measured using succinyl albumin/dimethylhaemoglobin as a substrate and the development of new N-terminals. The ability of bile acids to effect pepsin activity was assessed with three primary bile acids, two unconjugated and one taurine conjugated, using the above N-terminal assay. RESULTS: Gastric juice contained median TBA of 40 (range 10-10010) µM and pepsin activity of 408 (range 27-3892)ug/ml. We used this data to inform the relative levels of pepsin and bile acids that might occur in a reflux event, and we used concentrations of bile acids between 10-100µM. Pepsin activity was pH dependent, but 28% of the activity was retained at pH 5.5. None of the bile acids showed any significant effect on pepsin activity across the pH range 2.0-6.0. CONCLUSIONS: At the levels and pH that pepsin and bile acids might occur in an LPR event, bile acids do not attenuate pepsin activity. Pepsin could be considered a damaging factor even at high pH, and it will aggravate further any damaging effects of bile acids in the refluxate. Laryngoscope, 2012.