Gastrointestinal endogenous proteins as a source of bioactive peptides : Doctor of Philosophy in Nutritional Sciences at Riddet Institute, Massey University, Palmerston North, New Zealand

Gastrointestinal endogenous proteins (GEP) were investigated as a source of bioactive peptides. In silico and in vitro methods were used singly or in combination to study GEP-derived peptides after simulated digestion. The presence of bioactive peptides after in vivo digestion was determined using a porcine model. Bioactivity of the peptides was assessed using selected in vitro bioactivity assays, and peptides were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In the in silico study, twenty six different GEP and seven dietary proteins were subjected to simulated in silico gastrointestinal (SIGIT) digestion. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified by screening them against an online database of bioactive peptides (BIOPEP). The predicted number of bioactive peptides released after the SIGIT digestion of GEP ranged from 1 (secretin) to 39 (mucin-5AC), while those for dietary proteins ranged from 1 (gliadin) to 55 (myosin). Angiotensin-I-converting enzyme (ACE-I) inhibitory peptide sequences were found in abundance in both GEP and dietary proteins. The GEP mucin-5AC and the dietary protein myosin were predicted to release the highest number of ACE-I inhibitory peptides (38 and 49 peptides respectively), and were found to be comparable in their potential to release ACE-I inhibitory peptides. Following SIGIT digestion of eleven representative GEP, nineteen novel GEP-derived peptide sequences were selected by applying quantitative structure-activity relationship rules, and were chemically synthesised. Two novel peptides with the amino acid sequences RPCF and MIM, showing dipeptidyl peptidase IV (DPP-IV) inhibitory activity and five novel antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH)- inhibitory and, or ferric reducing antioxidant power (FRAP) activity) peptides with amino acid sequences CCK, RPCF, CRPK, QQCP and DCR were identified. These results indicate that GEP may contain novel bioactive peptide sequences. The potential release of bioactive peptides, from four GEP (trypsin, lysozyme, mucin, and serum albumin) and a dietary protein (chicken albumin), in the gastrointestinal tract (GIT) was investigated using an in vitro digestion model. The in vitro digests were screened for ACE-I-, renin-, platelet-activating factor acetylhydrolase (PAF-AH)-, and DPP-IV-inhibition, and antioxidant activity. All four in vitro GEP digests showed ACE-I inhibition comparable to that of the positive control captopril. In comparison to the unfractionated digests, the enriched fractions (<3 and <10 kDa) of lysozyme and serum albumin showed greater renin-, PAF-AH-, and DPP-IV-inhibition, and antioxidant potential. Over 190 peptide sequences were identified from these fractions using mass spectrometry. Stomach chyme (SC) and jejunal digesta (JD) were collected from growing pigs that were fed a protein-free diet for a period of 3 days. The peptides extracted from SC and JD samples were characterized by SDS-PAGE, and their ACE-I-, DPPH-, and microsomal lipid peroxidation (MLP)- inhibition, FRAP activity determined. Potential bioactive peptides responsible for bioactivity were identified using mass spectrometry. SDS-PAGE analysis showed that all of the samples contained a heterogeneous mixture of peptides. Porcine JD samples inhibited ACE-I and DPPH, while SC samples inhibited MLP. Characterization studies identified over 180 peptide sequences from the enriched fractions of SC and JD samples that showed the highest activity. Further, a porcine serum albumin peptide sequence (FAKTCVADESAENCDKS) was found to be a sub-sequence of a larger sequence identified in the in vitro digest of human serum albumin. There was considerable inter-animal variation for the bioactivities. This may be attributed to sampling effects and, or natural variations in the gut contents, thus underlining the complexity involved in in vivo release of bioactive peptides. Together, the results indicate: 1) GEP contain abundant encrypted bioactive peptide sequences; 2) GEP-derived bioactive peptides display a range of bioactivities; 3) GEP-derived bioactive peptides are released during gastrointestinal digestion in pigs; 4) GEP may contain numerous novel bioactive peptide sequences encoded within their primary sequence. In conclusion, the evidence reported here suggests that, like the dietary proteins, GEP are also a potentially rich source of exogenously-derived bioactive peptides in the gastrointestinal tract. Beyond their primary functions, GEP may act as an important cryptomic source of bioactive peptides, given that the amount of GEP secreted into the gut is equal to or greater than the dietary protein ingested per day, and that up to 80% of GEP are known to be digested

Helicobacter suis affects the health and function of porcine gastric parietal cells

The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type

Effect of native gastric mucus on in vivo hybridization therapies directed at Helicobacter pylori

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria

Unsupervised Odometry and Depth Learning for Endoscopic Capsule Robots

In the last decade, many medical companies and research groups have tried to convert passive capsule endoscopes as an emerging and minimally invasive diagnostic technology into actively steerable endoscopic capsule robots which will provide more intuitive disease detection, targeted drug delivery and biopsy-like operations in the gastrointestinal(GI) tract. In this study, we introduce a fully unsupervised, real-time odometry and depth learner for monocular endoscopic capsule robots. We establish the supervision by warping view sequences and assigning the re-projection minimization to the loss function, which we adopt in multi-view pose estimation and single-view depth estimation network. Detailed quantitative and qualitative analyses of the proposed framework performed on non-rigidly deformable ex-vivo porcine stomach datasets proves the effectiveness of the method in terms of motion estimation and depth recovery.Comment: submitted to IROS 201

Magnetic-Visual Sensor Fusion-based Dense 3D Reconstruction and Localization for Endoscopic Capsule Robots

Reliable and real-time 3D reconstruction and localization functionality is a crucial prerequisite for the navigation of actively controlled capsule endoscopic robots as an emerging, minimally invasive diagnostic and therapeutic technology for use in the gastrointestinal (GI) tract. In this study, we propose a fully dense, non-rigidly deformable, strictly real-time, intraoperative map fusion approach for actively controlled endoscopic capsule robot applications which combines magnetic and vision-based localization, with non-rigid deformations based frame-to-model map fusion. The performance of the proposed method is demonstrated using four different ex-vivo porcine stomach models. Across different trajectories of varying speed and complexity, and four different endoscopic cameras, the root mean square surface reconstruction errors 1.58 to 2.17 cm.Comment: submitted to IROS 201

A new liver perfusion and preservation system for transplantation Research in large animals

A kidney perfusion machine, model MOX-100 (Waters Instruments, Ltd, Rochester, MN) was modified to allow continuous perfusion of the portal vein and pulsatile perfusion of the hepatic artery of the liver. Additional apparatus consists of a cooling system, a membrane oxygenator, a filter for foreign bodies, and bubble traps. This system not only allows hypothermic perfusion preservation of the liver graft, but furthermore enables investigation of ex vivo simulation of various circulatory circumstances in which physiological perfusion of the liver is studied. We have used this system to evaluate the viability of liver allografts preserved by cold storage. The liver was placed on the perfusion system and perfused with blood with a hematocrit of approximately 20% and maintained at 37°C for 3 h. The flows of the hepatic artery and portal vein were adjusted to 0.33 mL and 0.67 mL/g of liver tissue, respectively. Parameters of viability consisted of hourly bile output, oxygen consumption, liver enzymes, electrolytes, vascular resistance, and liver histology. This method of liver assessment in large animals will allow the objective evaluation of organ viability for transplantation and thereby improve the outcome of organ transplantation. Furthermore, this pump enables investigation into the pathophysiology of liver ischemia and preservation. © 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

The Role of the Mucus Barrier in Digestion

Mucus forms a protective layer across a variety of epithelial surfaces. In the gastrointestinal (GI) tract, the barrier has to permit the uptake of nutrients, while excluding potential hazards, such as pathogenic bacteria. In this short review article, we look at recent literature on the structure, location, and properties of the mammalian intestinal secreted mucins and the mucus layer they form over a wide range of length scales. In particular, we look at the structure of the gel-forming glycoprotein MUC2, the primary intestinal secreted mucin, and the influence this has on the properties of the mucus layer. We show that, even at the level of the protein backbone, MUC2 is highly heterogeneous and that this is reflected in the networks it forms. It is evident that a combination of charge and pore size determines what can diffuse through the layer to the underlying gut epithelium. This information is important for the targeted delivery of bioactive molecules, including nutrients and pharmaceuticals, and for understanding how GI health is maintained

Impact of bioactive substances on the gastrointestinal tract and performance of weaned piglets: a review

The EU ban on in-feed antibiotics has stimulated research on weaning diets as a way of reducing post-weaning gut disorders and growth check in pigs. Many bioactive components have been investigated but only few have shown to be effective. Amongst these, organic acids (OA) have been shown to exert a bactericidal action mediated by non-dissociated OA, by lowering gastric pH, increasing gut and pancreas enzyme secretion and improving gut wall morphology. It has been postulated that they may also enhance non-specific immune responses and improve disease resistance. In contrast, relatively little attention has been paid to the impact of OA on the stomach but recent data show they can differently affect gastric histology, acid secretion and gastric emptying. Butyrate and precursors of butyric acid have received special attention and although promising results have been obtained, their effects are dependent upon the dose, treatment duration, initial age of piglets, gastrointestinal site and other factors. The amino acids (AA) like glutamine, tryptophan and arginine are supportive in improving digestion, absorption and retention of nutrients by affecting tissue anabolism, stress and (or) immunity. Glutamine, cysteine and threonine are important for maintaining mucin and permeability of intestinal barrier function. Spray-dried plasma (SDP) positively affects gut morphology, inflammation and reduces acquired specific immune responses via specific and a-specific influences of immunoglobulins and other bioactive components. Effects are more pronounced in early-weaned piglets and under poorer health conditions. Little interaction between plasma protein and antibiotics has been found, suggesting distinct modes of action and additive effects. Bovine colostrum may act more or less similarly to SDP The composition of essential oils is highly variable, depending on environmental and climatic conditions and distillation methods. These oils differ widely in their antimicrobial activity in vitro and some components of weaning diets may decrease their activity Results in young pigs are highly variable depending upon the product and doses used. These studies suggest that relatively high concentrations of essential oils are needed for beneficial effects to be observed and it has been assumed that these plant extracts mimic most of the effects of antibiotics active on gut physiology, microbiology and immunology. Often, bioactive substances protective to the gut also stimulate feed intake and growth performance. New insights on the effects of selected OA and AA, protein sources (especially SDP bovine colostrum) and plant extracts with anti-bacterial activities on the gut are reported in this review

Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60-95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits