Surface Of Intestinal Cells Research Articles

Screening and verification of outer membrane proteins of Cronobacter sakazakii with potential adhesion capability to human ileocecal adenocarcinoma cells

Cronobacter sakazakii (C. sakazakii), a food-borne pathogen, can cause severe intestinal diseases, such as intestinal damage and necrotizing enterocolitis, and even death in neonates and infants through its adhesion to intestinal epithelium cells by protein-protein interaction and translocation across the intestinal barrier. Nevertheless, the mechanisms of adhesion, translocation, and pathogenesis remain unclear. This study aimed to screen and validate the outer membrane proteins of C. sakazakii capable of adhering to the surface of intestinal cells through biotinylated protein adhesion and LC-MS/MS identification. A total of 319 proteins were identified. Gene Ontology and KEGG analysis revealed significant enrichment of ligand proteins in metabolism-related processes and catalytic activity. The identified 37 outer membrane proteins and 41 proteins of unknown subcellular localization or function mainly enriched in five protein-protein interaction clusters. Ten of these outer membrane/unknown proteins were selected to test their roles on C. sakazakii adhesion and invasion of intestinal cells by silencing the encoding gene expression and nine of the ten proteins, Protein_26/30/34/38/42/98/104/194/282, were shown to be critical in this process. This study contributes to the understanding of proteins involved in the host adhesion process by C. sakazakii as well as the mechanisms for its translocation across the intestinal barrier.

Integrated Evaluation of the Multifunctional DPP-IV and ACE Inhibitory Effect of Soybean and Pea Protein Hydrolysates.

Nowadays, notwithstanding their nutritional and technological properties, food bioactive peptides from plant sources garner increasing attention for their ability to impart more than one beneficial effect on human health. Legumes, which stand out thanks to their high protein content, represent valuable sources of bioactive peptides. In this context, this study focused on the characterization of the potential pleotropic activity of two commercially available soybean (SH) and pea (PH) protein hydrolysates, respectively. Since the biological activity of a specific protein hydrolysate is strictly correlated with its chemical composition, the first aim of the study was to identify the compositions of the SH and PH peptides. Peptidomic analysis revealed that most of the identified peptides within both mixtures belong to storage proteins. Interestingly, according to the BIOPEP-UWM database, all the peptides contain more than one active motive with known inhibitory angiotensin converting enzyme (ACE) and dipeptidyl-dipeptidases (DPP)-IV sequences. Indeed, the results indicated that both SH and PH inhibit DPP-IV and ACE activity with a dose-response trend and IC50 values equal to 1.15 ± 0.004 and 1.33 ± 0.004 mg/mL, and 0.33 ± 0.01 and 0.61 ± 0.05 mg/mL, respectively. In addition, both hydrolysates reduced the activity of DPP-IV and ACE enzymes which are expressed on the surface of human intestinal Caco-2 cells. These findings clearly support that notion that SH and PH may represent new ingredients with anti-diabetic and hypotensive effects for the development of innovative multifunctional foods and/or nutraceuticals for the prevention of metabolic syndrome.

Importance of the Precautionary Principle With Regard to the Risk of Exposure to Aerosols Containing Viral Loads of SARS-CoV-2 Present in Feces: In Perspective.

In COVID-19 infection, the emissions of droplets and aerosols produced by the respiratory tract of contaminated subjects may represent a high risk of spreading the SARS-COV-2 virus in the environment. Thus, studies have shown that there is, at least, another source of droplets and aerosols in which viral particles of SARS-COV-2 can be found. It happens after flushing of toilet to dispose of the stools of a patient who has contracted COVID-19. The presence of viral particles of SARS-COV-2 in the stool could be linked to the concentration of angiotensin-converting enzyme 2 (ACE2) found on the surface of intestinal cells. Therefore, there is a reason to wonder whether the emission of viral particles by activating a toilet flush could represent an important potential risk of contamination for health care workers. To investigate this hypothesis, we have correlated different studies on the production of droplets and aerosols as well as the presence of viral particles following flush of toilet. This pooling of these studies led to the following conclusion: the precautionary principle should be applied with regard to the potential risk represented by viral particles of SARV-COV-2 in the stool when flushing the toilet.

Integrin αEβ7 is involved in the intestinal barrier injury of sepsis.

Background: IL-9-producing CD4(+) T (Th9) cell was related to acute intestinal barrier injury in sepsis. Integrin αEβ7 was an important lymphocyte homing receptor on the surface of intestinal Th9 cells. However, the roles of αEβ7 in the intestinal injury caused by Th9 cells were not clear in sepsis.Methods: To investigate the roles of αEβ7 in the intestinal injury caused by Th9 cells in sepsis model, the Th9 cells percentages, αEβ7, E-cadherin, IL-9, and D-lactate levels in both serum and intestinal tissue were measured. The intestinal histopathology, epithelium apoptosis, and mucosal permeability measurement were also performed. The survival rate of septic rats was recorded daily for 14 days.Results: Rats were assigned to four cohorts: control cohort, sepsis cohort, sepsis+αEβ7i (αEβ7 inhibition) cohort, and sepsis+αEβ7e (αEβ7 overexpression) cohort. The Th9 cells percentages, αEβ7, IL-9, and D-lactate levels of the sepsis cohort were significantly higher than those of the control cohort. The levels of these variables were also elevated progressively in the sepsis+αEβ7i cohort, sepsis cohort, and sepsis+αEβ7e cohort. The E-cadherin levels were decreased progressively in the control cohort, sepsis+αEβ7i cohort, sepsis cohort, and sepsis+αEβ7e cohort. Moreover, αEβ7 overexpression could decrease the 14-day survival rate. The findings of histopathology staining, apoptosis detection, and intestinal permeability test also confirmed that the barrier injury was deteriorated or relieved by elevating or decreasing the αEβ7 expression levels, respectively.Conclusions: Integrin αEβ7 was closely associated with the intestinal barrier injury caused by Th9 lymphocytes in sepsis.

The Caenorhabditis elegans homolog of human mitochondrial pyrimidine nucleotide transporter regulates glucose transport

Caenorhabditis elegans T09F3.2 is a homolog of the human mitochondrial pyrimidine nucleotide transporter. We isolated a T09F3.2 mutant (TOG2) with a 0.7 kb deletion in T09F3.2, which exhibited low growth and movement. TOG2 worms exhibited high glucose content and low lipid content in intestinal cells and oocytes, suggesting glucose leakage from these cells. The glucose transport inhibitor phloretin improved the growth of TOG2 worms, suggesting that T09F3.2 regulates the phloretin-sensitive glucose transporter FGT-1. The localization of T09F3.2 was examined to assess the regulation of FGT-1 by T09F3.2. Distinct expression of T09F3.2 fused with DsRed-Monomer (T09F3.2:DsRed-Monomer) was observed in the basal domain of intestinal cells and was weakly expressed in many cells. Colocalization of FGT-1 and T09F3.2 was observed in the intestinal cell surface and body wall muscle. This colocalization supports the regulation of FGT-1 by T09F3.2. These results reveal new aspects of glucose transporter regulation.

COVID-19 Diarrhea

In 2019, a novel virus emerged in Wuhan, China that caused a febrile respiratory illness, sometimes rapidly fatal and thought to be transmitted by droplet contact. It likely originated from another species (possibly the bat), but later, spread occurred from human-to-human. Over a period of months, this disease disseminated to more than 100 nations as a highly infectious pandemic, reminiscent of disease associated with other coronavirus agents in 2002 (SARS) and 2012 (MERS). This COVID-19 virus also caused other forms of clinical illness, albeit in the minority, and most infected patients (over 50%) had few symptoms or remained completely asymptomatic. Prominent in some patients was watery diarrhea, often lasting less than a week, but sometimes without any respiratory features, such as fever, cough or shortness of breath. It is believed that fecal-oral transmission may result. The virus attaches to the surface of human respiratory and intestinal cells, gains entry into these cells, ultimately leading to production of new virions intraluminally. Fecal shedding may occur for extended periods, even after respiratory secretions become negative. Apart from preventive measures, vaccine development remains the most important strategy.

THE PRESENCE OF A POTENTIAL PATIENT OF COLIBACTERISIS IN THE POPULATION OF PIGS OF LOCAL SELECTION OF UKRAINIAN MEAT BREED

Each year about 10 million piglets die all over the world because of oodema disease and postweaning diarrhea. These diseases associated with the adhesion of enterotoxic Escherichia coli on the surface of pig intestinal cells are called colibacteriosis. The disease exciter is enteropathogenic hemolytic strains of Eschenchia (E. coli). The enterotoxic E. coli O157 is able to produce specific adhesins in piglet colibacteriosis, the most important role being played by F18 and F4 (K 88). The genetic resistance of piglets to diarrhea is based on the lack of appropriative receptors on the surface of the intestinal cells of animals. It is practical interest in pigs breading to study using polymorphism of E. coli F18 and F4 (ECR F18/FUT1 and ECR F4/MUC4) receptor genes in the selection process associated with the occurrence of colibacteriosis in piglets. The studies were apropiate on the basis of a breeder reproducer of the Ukrainian meat breed pigs of Dnipropetrovsk state agricultural institute selection at "Lugovskoye" Ltd. of firm "Avias 2000" of Solonyansky district, Dnipropetrovsk region. Microbiological studies of the presence of E. coli O157 in the animals faeces were performed at the Department of Biotechnology of the Ukrainian Chemical Technology University, using a ready-made selective Compact Dry EC medium. Animal genotype estimation for FUT1 and MUC4 genes was carried out at the genetic laboratory of the Institute of Pig Breeding andAgro Industrial Production, National Academy of Agrarian Sciences of Ukraine, by the PCR-RFLP method. Studies have been conducted to detect enterotoxigenic E. coli O157 in a closed population of pigs of Ukrainian meat breed type of selection of Dnepropetrovsk state agricultural institute. 58.3% of clinically healthy sows were found to contain E. coli O157, while in the 5–12 days group of clinically healthy pigs the detection rate was 20.0% and in piglets with signs of diarrhea 44.5%. On 12th day the average weight of sick piglets was lower on 30.45% compared to the average weight of healthy individuals was found, and the losses among sick pigs at weaning on day 21 reached to 56%. The relationship between colibacteriosis piglets susceptibility with genotypes of FUT1 and MUC4 genes was identified. The studied population of pigs is unreliable for colibacteriosis, so a gene pool study for the presence of the desired colibacteriosis resistance genotypes of FUT1 and MUC4 marker genes, followed by selection of parental cross-breeding forms, should be included in the livestock reproduction program.

Thiol/disulfide exchange occurs in rotavirus structural proteins during contact with intestinal villus cell surface.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.

Topographic Lectin Mapping of the Epithelial Cell Surface in Normal Intestine and Celiac Disease

The etiology and pathogenesis of celiac disease remains obscure but remains the focus of intense research investigation. It is generally believed to be an immune-mediated small intestinal mucosal disorder that can cause diarrhea, impaired nutrient assimilation and weight loss. A key component in this process occurs at the intestinal epithelial cell surface that is closely associated with the luminal intestinal microbiome. Here, epithelial membrane glycoproteins and glycolipids are present along with adsorbed molecules that permit interaction with the intestinal microbiome. In recent years, use of specific sugar residue seeking proteins, lectins, that can be found in the diet have been employed topographically to map the small intestinal cell surface and goblet cell secretory mucins to further elucidate the structure and function of this tissue. Evidence has accumulated to indicate that this microenvironment may be critically important in further understanding the etiology and pathogenesis of celiac disease and other sprue-like intestinal disorders.

Identifying Efficient Clostridium difficile Toxin A Binders with a Multivalent Neo-Glycoprotein Glycan Library.

Clostridium difficile infections cause gastrointestinal disorders and can lead to life-threatening conditions. The symptoms can vary from severe diarrhea to the formation of pseudomembranous colitis and therefore trigger a need for new therapies. The initial step of disease is the binding of the bacterial enterotoxins toxin A and B to the cell surface of epithelial intestinal cells. Scavenging of the toxins is crucial to inhibit their fatal effect in the human body and circumvent the administration of antibiotics. Cell surface glycans are common as ligands for TcdA. Although crucial for carbohydrate-protein interactions, a multivalent presentation of glycans for binding has been hardly considered. Here, we establish a neo-glycoprotein-based glycan library to identify an effective multivalent glycan ligand for TcdA. It comprises 40 different glycan epitopes based on N-acetyllactosamine precursors. Nine structures exhibit strong binding of the receptor domain. Among them, the Lewisy-Lewisx-epitope shows the best performance for binding both the receptor domain and the holotoxin. Therefore, the glycan was synthesized de novo and coupled to BSA as a scaffold for multivalent presentation. The corresponding neo-glycoprotein facilitates the proper scavenging of TcdA in vitro and effectively protects HT29 cells from TcdA-induced cell damage.

Binding activity to intestinal cells and transient colonization in mice of two Lactobacillus paracasei subsp. paracasei strains with high aggregation potential.

Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.

Bovine colostrum-driven modulation of intestinal epithelial cells for increased commensal colonisation.

Nutritional intake may influence the intestinal epithelial glycome and in turn the available attachment sites for bacteria. In this study, we tested the hypothesis that bovine colostrum may influence the intestinal cell surface and in turn the attachment of commensal organisms. Human HT-29 intestinal cells were exposed to a bovine colostrum fraction (BCF) rich in free oligosaccharides. The adherence of several commensal bacteria, comprising mainly bifidobacteria, to the intestinal cells was significantly enhanced (up to 52-fold) for all strains tested which spanned species that are found across the human lifespan. Importantly, the changes to the HT-29 cell surface did not support enhanced adhesion of the enteric pathogens tested. The gene expression profile of the HT-29 cells following treatment with the BCF was evaluated by microarray analysis. Many so called "glyco-genes" (glycosyltransferases and genes involved in the complex biosynthetic pathways of glycans) were found to be differentially regulated suggesting modulation of the enzymatic addition of sugars to glycoconjugate proteins. The microarray data was further validated by means of real-time PCR. The current findings provide an insight into how commensal microorganisms colonise the human gut and highlight the potential of colostrum and milk components as functional ingredients that can potentially increase commensal numbers in individuals with lower counts of health-promoting bacteria.

Strong Inhibition of Cholera Toxin B Subunit by Affordable, Polymer-Based Multivalent Inhibitors.

Cholera is a potentially fatal bacterial infection that affects a large number of people in developing countries. It is caused by the cholera toxin (CT), an AB5 toxin secreted by Vibrio cholera. The toxin comprises a toxic A-subunit and a pentameric B-subunit that bind to the intestinal cell surface. Several monovalent and multivalent inhibitors of the toxin have been synthesized but are too complicated and expensive for practical use in developing countries. Meta-nitrophenyl α-galactoside (MNPG) is a known promising ligand for CT, and here mono- and multivalent compounds based on MNPG were synthesized. We present the synthesis of MNPG in greatly improved yields and its use while linked to a multivalent scaffold. We used economical polymers as multivalent scaffolds, namely, polyacrylamide, dextran, and hyperbranched polyglycerols (hPGs). Copper-catalyzed alkyne azide cycloaddition reaction (CuAAC) produced the inhibitors that were tested in an ELISA-type assay and an intestinal organoid swelling inhibition assay. The inhibitory properties varied widely depending on the type of polymer, and the most potent conjugates showed IC50 values in the nanomolar range.

Adhesion Ability and Cytotoxic Evaluation of Lactobacillus Strains Isolated from Malaysian Fermented Fish (Pekasam) on HT-29 and CCD-18CO Intestinal Cells

Bacterial adhesion to host cells is the most important probiotic character. However, the adhesion of probiotic should not affect the viability of the host cells. In this study, Lactobacillus plantarum strain L8, Lactobacillus plantarum strain L20 and Lactobacillus pentosus strain S1 were tested for their cytotoxic effects through MTT assay and their ability to adhere and colonize on HT-29 and CCD-18Co intestinal cells as detected microscopically using light microscopy and Scanning Electron Microscopy (SEM). No cytotoxicity effects were observed on both intestinal cells following 24 h treatment with all Lactobacillus strains. Additionally, all strains demonstrated strong adhesive activity where more than 100 bacteria adhered to both intestinal cells although differences in the adhesion scores observed among different strains. The adhesion as observed via SEM showed an autoagreggative pattern and adhered as clusters on the surface of both intestinal cells. In conclusion, all three Lactobacillus strains are non-cytotoxic to both cells with strong adhesion ability on intestinal cells and this study also proved that Malaysian fermented fish are good source of probiotic bacteria.

Escherichia coli O157:H7 suppresses host autophagy and promotes epithelial adhesion via Tir-mediated and cAMP-independent activation of protein kinase A

Autophagy is a pivotal innate immune response that not only degrades cytosolic components, but also serves as one of the critical antimicrobial mechanisms eliminating intracellular pathogens. However, its role in host defense against extracellular pathogens is largely unknown. Here we showed that E. coli O157:H7 altered autophagy to evade host defense and facilitate adhesion. Enhancing host cell autophagy with tumor necrosis factor (TNF), host starvation or rapamycin reduced the adherence of E. coli O157:H7 to HT-29 cells. As a key regulator of autophagy, protein kinase A (PKA) was activated by E. coli O157:H7 infection. PKA inhibition by H89 abrogated E. coli O157:H7 inhibition of autophagy and prevented bacterial epithelial adhesion. Thus, PKA had a mediatory role in blocking autophagy and E. coli O157:H7 epithelial adhesion. Furthermore, deletion of translocated intimin receptor (tir) prevented PKA activation, whereas ectopic tir expression in a Δtir mutant strain restored its ability to activate PKA and inhibited autophagy in host cells. This indicated that Tir and PKA played pivotal roles in manipulating host autophagy during infection. Consistent with autophagy inhibition, E. coli O157:H7 infection inhibited endoplasmic reticulum (ER) stress in HT-29 cells, which was reversed by TNF, starvation, or H89 treatment. Additionally, E. coli O157:H7-induced PKA activation suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activation and enhanced phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling, thereby repressing autophagic signaling. Conversely, PKA inhibition prevented downregulation of ERK1/2 signaling due to E. coli O157:H7 infection. In summary, E. coli O157:H7 inhibited host autophagy via Tir-mediated PKA activation that favored bacterial persistence on intestinal epithelial cell surfaces.

C3a Enhances the Formation of Intestinal Organoids through C3aR1.

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using Lgr5.egfp mice we showed significant expression of C3 in Lgr5+ intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration.

Manano oligosacaridos como prebioticos en acuicultura de crustaceos

Probiotics have the potential to increase the efficiency and sustainability of aquaculture production. The mannan-oligosaccharides (MOS) are complex carbohydrates derived from the cell wall of the yeast Saccharomyces cerevisiae , which act by blocking adherence of bacterial lectins to carbohydrates on the surface of intestinal cells; this action prevents colonization and infection processes carried out by bacterial pathogens. In this work, we review the current evidence about the use of MOS as prebiotics in shellfish aquaculture, enlightening the positive responses in most of the experiences, such as improvement in growth and survival, modulating the immune response and modifying the morphology of the digestive tract. The identification of strains that are promoted or excluded, and the characterization of the resulting modulating action in gut´s microbiota, remains open as one of the fields to be developed in conjunction with the extension of these results to the productive practice.

Identification of Glycoproteins on the Surface of Bovine Milk Exosomes and Intestinal Cells that Facilitate Exosome Uptake in Human Colon Carcinoma Caco‐2 Cells

BackgroundExosomes are small, cargo‐containing vesicles that are secreted by donor cells to elicit changes in gene expression and metabolism in recipient cells. In previous studies, we provided evidence that, in addition to endogenous synthesis, exosomes may also be obtained from dietary sources such as bovine milk, and intestinal transport is facilitated by endocytosis.HypothesisGlycoproteins on the surface of bovine milk exosomes and human intestinal cells play essential roles in exosome transport.MethodsExosomes were isolated from bovine milk by differential centrifugation and authenticated as recommended by International Society for Extracellular Vesicles (Fig. 1). Surface proteins or glycan modifications in exosomes or Caco‐2 cells were removed using proteases (Glu‐C, trypsin, Arg‐C, Asp‐N, or proteinase K) or glycosidases [N‐glycosidase F (PNGase F), galactosidase, O‐glycosidase, neuraminidase, N‐acetyl glucosaminidase, or a combination of all]. Controls were incubated with solvent. Exosomes were labeled with FM4–64, and unlabeled fluorophore was removed. Surface peptides released after the treatment were identified using LC/MS‐MS, Mascot and Sequest databases. Glycoproteins on the external surface were identified using a series of tools based on neuronal networks including NetNglyc, NetOglyc, NetCglyc 1.0 and GlycoEP. Transport kinetics were modelled using the Michaelis Menten equation. One‐way ANOVA and Bonferroni's multiple comparison were used for statistical analyses.ResultsWhen exosomes or cells were treated with individual proteases (Fig. 2) or glycosidases (Fig. 3), exosome uptake decreased up to 80% when compared to controls even at prolonged incubation times. Treatment with galactosidase, PNGase F and O‐glycosidase had a stronger effect than other glycosidases. When exosomes or cells were treated with the glycosidase mixture, no uptake was detected or uptake was decreased by 80%, respectively, in comparison to controls (Fig. 4). 149 total proteins were identified in bovine exosome, including 4 (N), 2 (O) and 2 (C) glycan binding sites; 298 proteins were identified in Caco‐2 cells, including 46 membrane proteins that included 29 (N), 33 (O) and 6 (C) glycan binding sites.ConclusionsGlycoproteins on the surface of bovine milk exosomes and intestinal cells facilitate the uptake of exosomes in human intestinal cells. The β‐galactoside, Core 1 & Core 3‐O‐linked disaccharide and N‐acetylglucosamine modifications appear to be of greater importance than other glycan features present on complex glycoproteins.Future DirectionsThe transport of bovine exosomes will be studied in conditional glycan knockout mice.Support or Funding InformationNIFA 2015‐67017‐23181, NIFA 2016‐67001‐25301/NIH DK107264, NIH 1P20GM104320, the Gerber Foundation, and USDA Hatch Act and W3002.

Mucus interactions with liposomes encapsulating bioactives: Interfacial tensiometry and cellular uptake on Caco-2 and cocultures of Caco-2/HT29-MTX

Structuring of delivery matrices in foods aquires careful designing for optimal delivery and subsiquent absorption of the beneficial compounds in the gut. There has been quite improvement in mimicking digestion and absorption in vitro but as of yet little is understood on mucus interference in nutrient absorption Therefore in this study interactions of human intestinal mucus with milk and soy phospholipids liposomes carring hydrophilic (epigallocatechin-3-gallate) or hydrophobic (β-carotene) bioactive molecules were investigated. Liposomes of about 100nm were obtained using microfluidization and their behaviour with the human intestinal mucus were evaluated using drop shape tensiometry. The chemistry of the liposomes (milk or soy) and the encapsulated bioactive structure can affect the viscoelastic behaviour of the complex itself. Empty or loaded liposomes were differently interacting with the mucus at the interface. Mucus-liposomes interactions were also studied using cell cultures, Caco-2 (without mucus) and cocultures Caco-2/HT29-MTX (mucus producing). The interaction of mucus layer with liposomes was at some extent aligned with rheological studies. This work demonstrated that delivery systems may interact with the mucosal surface of intestinal cells, and in vitro approaches allow for screening of such interactions. These highlights could help us in carefully designing the delivery systems and moreover choosing the right carrier and/or bioactive that does not jeopardize the optimal delivery of the bioactive structure.

Dimerization of the fungal defense lectin CCL2 is essential for its toxicity against nematodes.

Lectins are used as defense effector proteins against predators, parasites and pathogens by animal, plant and fungal innate defense systems. These proteins bind to specific glycoepitopes on the cell surfaces and thereby interfere with the proper cellular functions of the various antagonists. The exact cellular toxicity mechanism is in many cases unclear. Lectin CCL2 of the mushroom Coprinopsis cinerea was previously shown to be toxic for Caenorhabditis elegans and Drosophila melanogaster. This toxicity is dependent on a single, high-affinity binding site for the trisaccharide GlcNAc(Fucα1,3)β1,4GlcNAc, which is a hallmark of nematode and insect N-glycan cores. The carbohydrate-binding site is located at an unusual position on the protein surface when compared to other β-trefoil lectins. Here, we show that CCL2 forms a compact dimer in solution and in crystals. Substitution of two amino acid residues at the dimer interface, R18A and F133A, interfered with dimerization of CCL2 and reduced toxicity but left carbohydrate-binding unaffected. These results, together with the positioning of the two carbohydrate-binding sites on the surface of the protein dimer, suggest that crosslinking of N-glycoproteins on the surface of intestinal cells of invertebrates is a crucial step in the mechanism of CCL2-mediated toxicity. Comparisons of the number and positioning of carbohydrate-binding sites among different dimerizing fungal β-trefoil lectins revealed a considerable variability in the carbohydrate-binding patterns of these proteins, which are likely to correlate with their respective functions.