Mucin Synthesis Research Articles

Hystometric evaluation of nickel chronic exposure effects on large instestine of adult Wistar male rats

The ingestion of considerable amounts of water or food contaminated with nickel can be very toxic. The present work was conducted aiming to evaluate the effects of nickel exposures on ascending colon of adult Wistar male rats at hystometric level. We used 12 animals that were divided in a control (ingested uncontaminated water) and a nickel-contaminated (i.e., 25 mg de nickel/L of water) groups. Nickel chloride was offered in declorinated water and the experiment had a 56 days exposure period. A portion of the ascending colon was removed of the animals and subjected to hystological labelling processes using blue toluidin (for general hystometric description), Alcian Blue (AB, for acid mucins) and periodic acid-Schiff (PAS) technique (for neutral mucins). The potential differences between groups were desgined by applying the Whitney test and t test (p < 0.05). The crypts were smaller for the nickel-contaminated group, even though these organism exhibited broader and higher crypts. Nickel-contaminated animals exhibited a smaller amount of calyceform cells with AB and PAS positive reactions as well as a less mucus quantities when compared with nickel-uncontaminated animals. Such reductions on the amount of calyceform cells with AB and PAS positive reactions may be related wiht the shallower crypts, which possibly reduced the synthesis and secretion of mucins, compromissing the functional aspects (e.g., lubrification and intestinal mucosa protection) of the nickel-contaminated large intestines. Interestingly, the wider and higher crypts and higher epithelium collumn on the nickel-contaminated animals may represent a relevant trade-off for the intestinal mucosa protection.

Effects of low protein diets with amino acids supplementation on biochemical and faeces parameters in weaned piglets

Effects of low protein diets with amino acids supplementation on biochemical and faeces parameters in weaned piglets

Dietary threonine prevented stress-related mucosal diseases in rats.

Stress-related mucosal disease (SRMD), or stress ulceration, is a group of conditions ranging from stress-related superficial gastric mucosal damage to deep gastric ulcers that are primarily correlated with mucosal ischemia, and pharmacologic interventions that optimize tissue perfusion or preserve defensive mucus aim to decrease the occurrence of conditions, such as gastric acidity, or enhance gastric defenses. However, the identification of multifactorial pathogenesis may be effective in preventing SMRD, and the use of stress prophylaxis is generally preferred. Since threonine is a component in the polymerization and synthesis of gastric mucin and possibly enhanced defense actions and lignin may provide structural support for defense and antioxidative function, we hypothesized that dietary intake of threonine and/or lignin can enhance defense against SRMD. The water immersion-restraint stress (WIRS) was used in rats and additional groups were pretreated with threonine alone or the combination of threonine and lignin. Based on gross and microscopic evaluations, threonine alone or the combination of threonine and lignin, a natural antioxidant, significantly reduced the development of SRMD (P < 0.05). According to molecular explorations, the levels of inflammatory mediators, such as interleukin (IL)-8, IL-6, IL-1β, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), all of which are mediators that play a significant role in controlling WIRS, significantly decreased in the groups pretreated with either threonine alone or the combination of threonine and lignin (P < 0.01). WIRS significantly increased apoptosis in the stomach. However, the apoptotic index significantly decreased with threonine pretreatment. According to periodic acid Schiff staining results, the expression of gastric mucin was significantly preserved in groups pretreated with threonine but remarkedly decreased in the WIRS group. The gastric heme oxygenase-1 levels significantly increased in the group treated with threonine. In conclusion, the dietary intake of threonine or the combination of threonine and lignin is effective in preventing SRMD.

Docosahexaenoic and Eicosapentaenoic Acids Prevent Altered-Muc2 Secretion Induced by Palmitic Acid by Alleviating Endoplasmic Reticulum Stress in LS174T Goblet Cells.

Diets high in saturated fatty acids (FA) represent a risk factor for the development of obesity and associated metabolic disorders, partly through their impact on the epithelial cell barrier integrity. We hypothesized that unsaturated FA could alleviate saturated FA-induced endoplasmic reticulum (ER) stress occurring in intestinal secretory goblet cells, and consequently the reduced synthesis and secretion of mucins that form the protective mucus barrier. To investigate this hypothesis, we treated well-differentiated human colonic LS174T goblet cells with palmitic acid (PAL)—the most commonly used inducer of lipotoxicity in in vitro systems—or n-9, n-6, or n-3 unsaturated fatty acids alone or in co-treatment with PAL, and measured the impact of such treatments on ER stress and Muc2 production. Our results showed that only eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids protect goblet cells against ER stress-mediated altered Muc2 secretion induced by PAL, whereas neither linolenic acid nor n-9 and n-6 FA are able to provide such protection. We conclude that EPA and DHA could represent potential therapeutic nutrients against the detrimental lipotoxicity of saturated fatty acids, associated with type 2 diabetes and obesity or inflammatory bowel disease. These in vitro data remain to be explored in vivo in a context of dietary obesity.

Physiological Effects of Dietary Amino Acids on Gut Health and Functions of Swine.

Gut health has significant implications for swine overall health status and nutrient utilization, due to its various functions including digestion and absorption of nutrients, secretion of mucins and immunoglobulins, and selective barrier protection against harmful antigens and pathogens. Both the basic anatomical structure of the gut (such as epithelial cells) and its luminal microbiota play important roles for maintaining gut health and functions. The interactions between epithelial cells and luminal microbiota have significant impact on host nutrition and health through the metabolism of dietary components. Amino acids, which are major nutrients for pigs, are not only obligatory for maintaining the intestinal mucosal mass and integrity, but also for supporting the growth of microorganisms in the gut. Dietary amino acids are the major fuel of the small intestinal mucosa. Particularly, glutamate, glutamine, and aspartate are the major oxidative fuel of the intestine. Emerging evidence shows that arginine activates the mTOR signaling pathway in the small intestine. Utilization of glycine by the small intestinal mucosa to synthesize glutathione is a very important physiological pathway, and the role of glycine as a powerful cytoprotectant has also been recognized. The major end products of methionine and cysteine metabolism are glutathione, homocysteine and taurine, which play important roles in the intestinal immune and anti-oxidative responses. Threonine is highly utilized by the gut and is particularly important for mucin synthesis and maintenance of gut barrier integrity. Moreover, either a deficiency or an excess of dietary threonine can reduce the synthesis of intestinal mucosal proteins and mucins in young pigs. Various new functions of amino acids on gut health and functions have been discovered in recent years. Thus, this review is to provide some up-to-date knowledge for industry application of dietary amino acids in order to enhance swine gut health and functions, and also it is to provide a comprehensive reference for further scientific research in this regard.

Fibroblasts Impact Goblet Cell Responses to Lactic Acid Bacteria After Exposure to Inflammatory Cytokines and Mucus Disruptors.

ScopeMucus produced by goblet cells contributes to gut barrier function. Lactic acid bacteria (LAB) have been shown to impact mucus production. It is not completely known whether mucus production is influenced by the abundantly present fibroblasts in the intestine.Methods and resultsThe influence of fibroblasts on mucus‐related genes including mucin‐2 (MUC2), trefoil factor 3 (TFF3), resistin‐like molecule β (RETNLB), carbohydrate sulfotransferase 5 (CHST5), and galactose‐3‐O‐sulfotransferase 2 (GAL3ST2) is examined after co‐culture of LS174T‐goblet cells and CCD‐18Co colonic fibroblasts in the presence and absence of LAB‐strains known to impact mucus function. This is also tested after exposure to TNF‐α, IL‐13, or the mucin synthesis inhibitor tunicamycin (Tm). Effects of fibroblasts are treatment duration‐ and bacterial species‐dependent under homeostatic conditions. During TNF‐α challenge, fibroblasts reverse Lactobacillus (L.) rhamnosus CCFM237‐elicited declined TFF3 expression. After IL‐13 exposure, L. rhamnosus CCFM237 and L. fermentum CCFM787 attenuate enhanced TFF3 and RETNLB expression, respectively, only in the presence of fibroblasts. LAB has no effects on Tm‐induced decreased expression of goblet cell‐related genes regardless of the presence of fibroblasts.ConclusionIt is demonstrated that goblet cell–fibroblast crosstalk impacts mucus synthesis and influences the effects of LAB on goblet cell‐related genes. Effects are LAB‐species and stressor dependent.

Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5–9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

HSP90 inhibitor geldanamycin reverts IL-13- and IL-17-induced airway goblet cell metaplasia.

Goblet cell metaplasia, a disabling hallmark of chronic lung disease, lacks curative treatments at present. To identify novel therapeutic targets for goblet cell metaplasia, we studied the transcriptional response profile of IL-13-exposed primary human airway epithelia in vitro and asthmatic airway epithelia in vivo. A perturbation-response profile connectivity approach identified geldanamycin, an inhibitor of heat shock protein 90 (HSP90) as a candidate therapeutic target. Our experiments confirmed that geldanamycin and other HSP90 inhibitors prevented IL-13-induced goblet cell metaplasia in vitro and in vivo. Geldanamycin also reverted established goblet cell metaplasia. Geldanamycin did not induce goblet cell death, nor did it solely block mucin synthesis or IL-13 receptor-proximal signaling. Geldanamycin affected the transcriptome of airway cells when exposed to IL-13, but not when exposed to vehicle. We hypothesized that the mechanism of action probably involves TGF-β, ERBB, or EHF, which would predict that geldanamycin would also revert IL-17-induced goblet cell metaplasia, a prediction confirmed by our experiments. Our findings suggest that persistent airway goblet cell metaplasia requires HSP90 activity and that HSP90 inhibitors will revert goblet cell metaplasia, despite active upstream inflammatory signaling. Moreover, HSP90 inhibitors may be a therapeutic option for airway diseases with goblet cell metaplasia of unknown mechanism.

Bisphenol A inhibits mucin 2 secretion in intestinal goblet cells through mitochondrial dysfunction and oxidative stress

AimsBisphenol A (BPA) can induce intestinal epithelial cell barrier dysfunction; however, its effects on the intestinal mucus barrier remain unclear. We used LS174T cells as a model to investigate the effects of BPA on the functions of intestinal goblet cells. Main methodsThis study used CCK-8, flow cytometry, ELISA and real-time PCR to investigate the effects of BPA on mitochondrial dynamics, oxidative stress and apoptosis in goblet cells. In addition, mucin synthesis and secretion were evaluated using PAS staining and a PAS assay, respectively. Key findingsOur results indicate that BPA reduced cell viability in a time- and concentration-dependent manner. BPA induced mitochondrial dysfunction, as indicated by the depolarization of the mitochondrial membrane potential, inhibition of mitochondrial respiratory chain complex enzyme activity and reduction of ATP production. Moreover, BPA caused oxidative stress by significantly increasing the accumulation of ROS, as well as oxidative stress products, and reducing the antioxidant capacity. Furthermore, BPA induced intestinal goblet cell apoptosis, accompanied by increased DNA fragmentation, caspase-3, -8, -9,-10 gene expression and enzyme activity. Additionally, BPA inhibited the synthesis and secretion of mucin 2. SignificanceOur data suggest that BPA affected the secretory function of intestinal goblet cells by inducing mitochondrial dysfunction, oxidative stress, and apoptosis.

Effects of dietary supplementation of the lignocelluloses FibreCell and OptiCell on performance, expression of inflammation-related genes and the gut microbiome of broilers

This study investigated the hypothesis that dietary supplementation of lignocellulose in broilers influences the gut bacterial population and bacterial fermentation, has anti-inflammatory effects, and increases mucin synthesis in the intestine, and, through these changes, influences broiler performance positively. Day-old male Cobb 500 broilers (n = 96) were allotted to 3 experimental groups and fed 3 different maize-wheat-soybean meal-based basal diets during days 1 to 10, 11 to 21, and 22 to 35. The basal diets were fed to the control group, and were supplemented with 0.8% of a standard lignocellulose (LCS) or a fermentable lignocellulose (LCF). Body weight and feed consumption were determined, and at slaughter (day 35), carcass and gizzard weights and gizzard content pH were recorded, and samples of jejunum, cecum, and colon mucosa and of cecum digesta were collected from 15 birds/group. Growth performance and feed intake were not influenced, but dressing percentage was higher in group LCF compared to the other groups. In group LCS and the control group, performance, gizzard weight and gizzard content pH, intestinal gene expression of pro-inflammatory cytokines and of the mucins 2, 5ac and 13, the cecal short-chain fatty acid (SCFA) profile, and bacterial diversity were similar, and relative abundance of bacterial groups (16S DNA sequencing) differed. Supplementation of LCF decreased the expression of the pro-inflammatory genes encoding interleukins 1ß and 17 (P < 0.05) and those of 2 and 8 (P < 0.10) in the jejunum only. The bacterial population differed, and the SCFA profile shifted toward acetate at the expense of butyrate in group LCF compared to the control group. For example, the abundance of Firmicutes and of Ruminococcaceae and Lactobacillaceae decreased, whereas those of Peptostreptococcaceae, Erysipelotrichaceae, and Enterobacteriaceae and that of members of the phylum Proteobacteria increased in group LCF compared to the control group. These data indicate that the susceptibility of lignocellulose to fermentation is crucial for mediating its effects on intestinal gene expression and the bacterial population in the cecum, which may also affect dressing percentage.

Caspase‐1‐dependent mechanism mediating the harmful impacts of the quorum‐sensing molecule N‐(3‐oxo‐dodecanoyl)‐l‐homoserine lactone on the intestinal cells

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

Reconsidering the central role of mucins in dry eye and ocular surface diseases.

Mucins are key actors in tear film quality and tear film stability. Alteration of membrane-bound mucin expression on corneal and conjunctival epithelial cells and/or gel-forming mucin secretion by goblet cells (GCs) promotes in ocular surface diseases and dry eye disease (DED). Changes in the mucin layer may lead to enhanced tear evaporation eventually contributing to tear hyperosmolarity which has been associated with ocular surface inflammation. Inflammatory mediators in turn may have a negative impact on GCs differentiation, proliferation, and mucin secretion. This sheds new light on the position of GCs in the vicious circle of DED. As contributor to ocular surface immune homeostasis, GC loss may contribute to impaired ocular surface immune tolerance observed in DED. In spite of this, there are no tools in routine clinical practice for exploring ocular surface mucin deficiency/dysregulation. Therefore, when selecting the most appropriate treatment options, there is a clear unmet need for a better understanding of the importance of mucins and options for their replacement. Here, we comprehensively revisited the current knowledge on ocular surface mucin biology, including functions, synthesis, and secretion as well as the available diagnostic tools and treatment options to improve mucin-associated homeostasis. In particular, we detailed the potential link between mucin dysfunction and inflammation as part of the uncontrolled chronic inflammation which perpetuates the vicious circle in DED.

L-Threonine improves intestinal mucin synthesis and immune function of intrauterine growth-retarded weanling piglets.

The aim of this study was to investigate the effects of dietary l-threonine supplementation on the growth performance, intestinal immune function, mucin synthesis, and goblet cell differentiation in weanling piglets with intrauterine growth retardation (IUGR). Eighteen litters of newborn piglets were selected at birth, with one normal birthweight (NBW) and two IUGR piglets in each litter. At weaning, the NBW piglet and one of the IUGR piglets were assigned to groups fed a basal diet (i.e., the NBW-CON and IUGR-CON groups). The other IUGR piglet was assigned to a group fed the basal diet supplemented with 2g l-threonine per kg of diet (i.e., IUGR-Thr group). Therefore, all piglets were distributed across three groups for a 3-wk feeding trial. Compared with NBW, IUGR decreased growth performance, increased ileal proinflammatory cytokine levels, and reduced ileal mucin 2 (Muc2) content and goblet cell density of weanling piglets. Supplementation of l-threonine increased the feed efficiency of the IUGR-Thr group compared with the IUGR-CON group. The l-threonine-supplemented diet attenuated ileal inflammatory responses of the IUGR-Thr piglets and increased production of Muc2 and secretory immunoglobulin A and density of goblet cells. In addition, L-threonine supplementation downregulated δ-like 1 and hes family bHLH transcription factor 1, whereas growth factor independence 1 and Kruppel-like factor 4 expression levels were upregulated. Dietary l-threonine supplementation attenuates inflammatory responses, facilitates Muc2 synthesis, and promotes goblet cell differentiation in the ileum of IUGR piglets.

Low Dose Carbon Black Nanoparticle Exposure Does Not Aggravate Allergic Airway Inflammation in Mice Irrespective of the Presence of Surface Polycyclic Aromatic Hydrocarbons.

Exposure to exogenous noxae, such as particulate matter, can trigger acute aggravations of allergic asthma—a chronic inflammatory airway disease. We tested whether Carbon Black nanoparticles (CBNP) with or without surface polycyclic aromatic hydrocarbons (PAH) aggravate an established allergic airway inflammation in mice. In an ovalbumin mouse model, Printex®90 (P90), P90 coated with benzo[a]pyrene (P90-BaP) or 9-nitroanthracene (P90-9NA), or acetylene soot exhibiting a mixture of surface PAH (AS-PAH) was administered twice (70 µL, 100 µg/mL) during an established allergic airway inflammation. We analyzed the immune cell numbers and chemokine/cytokine profiles in bronchoalveolar lavages, the mRNA expressions of markers for PAH metabolism (Cyp1a1, 1b1), oxidative stress (HO-1, Gr, Gpx-3), inflammation (KC, Mcp-1, IL-6, IL-13, IL-17a), mucin synthesis (Muc5ac, Muc5b), the histology of mucus-producing goblet cells, ciliary beat frequency (CBF), and the particle transport speed. CBNP had a comparable primary particle size, hydrodynamic diameter, and ζ-potential, but differed in the specific surface area (P90 > P90-BaP = P90-9NA = AS-PAH) and surface chemistry. None of the CBNP tested increased any parameter related to inflammation. The unmodified P90, however, decreased the tracheal CBF, decreased the Muc5b in intrapulmonary airways, but increased the tracheal Muc5ac. Our results demonstrated that irrespective of the surface PAH, a low dose of CBNP does not acutely aggravate an established allergic airway inflammation in mice.

Altered goblet cell function in Hirschsprung's disease.

Hirschsprung's disease-associated enterocolitis (HAEC) is the most serious complication of Hirschsprung's disease (HSCR). HAEC occurs in 17-50% of patients with HSCR and may occur before or after a properly performed pull-through operation. The pathogenesis of HAEC is poorly understood. It is well recognized that a complex mucosal barrier protects, as the first line of defense, the surface of healthy intestinal tract from adhesion and invasion by luminal micro-organisms. Within the intestinal epithelium, goblet cells secrete gel-forming mucins, the major component of mucus, which block the direct attachment of commensal bacteria to the epithelial layer. Mucin 2 (MUC2) is the predominant mucin expressed in humans. Trefoil factor 3 (TFF3) synergizes with mucin and enhances the protective barrier properties of the mucus layer. SAM pointed domain-containing ETS transcription factor (SPDEF) drives terminal differentiation and maturation of secretory progenitors into goblet cells. Krueppel-like factor 4 (KLF4) is a goblet cell-specific differentiation factor in the colon and controls goblet cell differentiation and activates mucin synthesis. We hypothesized that the goblet cell function in the ganglionic pulled-through bowel in HSCR is abnormal and, therefore, we investigated the changes in goblet cell differentiation and functional expression of mucin in the bowel specimens from patients with HSCR. We investigated MUC2, TFF3, SPDEF and KLF4 expression, and the goblet cell population in the ganglionic and aganglionic bowel of HSCR patients (n = 10) and controls (n = 10) by qPCR, Western blotting, confocal immunofluorescence, and alcian blue staining. The qPCR and Western blotting analysis revealed that TFF3, SPDEF and KLF4 expressions were significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR as compared to controls (p < 0.05). Alcian blue staining revealed that the goblet cell population was significantly decreased in aganglionic and ganglionic colon as compared to controls (p < 0.05). Confocal microscopy revealed a markedly decreased expression of TFF3, SPDEF and KLF4 in colonic epithelium of patients with HSCR as compared to controls. This is, to our knowledge, the first report of decreased expression of TFF3, SPDEF, KLF4, and goblet cell population in the colon of patients with HSCR. Altered goblet cell function may result in intestinal barrier dysfunction contributing to the development of HAEC.

Abstract 1159: Targeting novel mucin synthesis pathway for pancreatic cancer treatment

Abstract Pancreatic cancer (PC) is most fatal and is the fourth leading cause of cancer related deaths in USA with a least five year survival of &amp;lt;7%. Although several overexpressed mucin subtypes (MUC1, MUC4, MUC5AC, and MUC16) that impede drug delivery to PC have been targeted with limited results, core enzymes involved in mucin biosynthesis have yet to be evaluated as potential targets. The core 2 β 1,6 N-acetylglucosaminyltransferase (GCNT3/C2GNT) plays a significant role in mucin glycan biosynthesis. We analyzed GCNT3 expression on 180 specimens from individuals with PC (90) or matched controls (90). Patients with low GCNT3 expression survived longer than patients with high GCNT3 expression (median survival: 17.5 vs. 10.5 months, p=0.036). We observed a significant increase in GCNT3 mRNA expression (103.16 folds; p&amp;lt;0.0001) in correlation with increased mucins subtypes (p&amp;lt;0.05) in the pancreatic tumors from genetically engineered mouse (GEM) compared with pancreata from wild-type mice, as determined by Next-generation Sequencing/RNAseq analysis. In in silico studies, blind docking simulations revealed that a novel drug talniflumate binds to GCNT3 with a better docking affinity of -8.3 kcal/mol. We synthesized talniflumate and determined quality/purity using NMR, HRMS, and HPLC for in vitro and in vivo studies. Pancreata from 6-week-old Kras GEM (N=6/group) treated with talniflumate showed a significant decrease in GCNT3 and mucin expression in PanIN lesions. In human PC MiaPaCa cell line, talniflumate reduced GCNT3 expression and the number of mucin-positive cells (38% p&amp;lt;0.0001). Further, PC cells (BxPc3 and Panc1) were treated with talniflumate for 24 hrs for mucin disruption followed by radioactive 3H gemcitabine treatment for 2 hrs. The cells treated with talniflumate showed 40% (p&amp;lt;0.03) and 50% (p&amp;lt;0.025) increased 3H gemcitabine uptake in BxPc3 and Panc1 cells compared to respective controls. GCNT3 CRISPR knock out PC cells and talniflumate treated PC cells showed a reduction in cell proliferation and significantly reduced spheroid formation (p&amp;lt;0.05). In in vivo, we did not observe any overt toxicity upon talniflumate treatment as determined by liver enzyme toxicity and body weight gain (N=5 mice/group). BxPC3 cells implanted nude mice (N=5 mice/group) treated with talniflumate or GCNT3 siRNA showed a marked reduction in xenograft tumors compared with untreated controls. Both talniflumate and GCNT3 siRNA significantly inhibited xenograft tumor weight (48% p&amp;lt;0.002 with siRNA, 63% p&amp;lt;0.0001 with talniflumate) and tumor volume (75% p&amp;lt;0.0001 with both siRNA and talniflumate) and GCNT3 enzyme activity (28% p&amp;lt;0.03). Transcriptome analysis of xenograft tumors (N=3/group) treated with GCNT3 SiRNA or talniflumate showed reduced GCNT3 and top hits related to mucin expression. These results justify the use of talniflumate for mucin synthesis disruption, thereby enhancing the uptake of gemcitabine and thereby efficacy for PC treatment. Citation Format: Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Gaurav Kumar, Scott Edgar, Gopal Pathuri, Taylor Bryant, Yuting Zhang, Hariprasad Gali, Yan Daniel Zhao, Stan Lightfoot, Chinthalapally V. Rao. Targeting novel mucin synthesis pathway for pancreatic cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1159. doi:10.1158/1538-7445.AM2017-1159

Abstract 4499: The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway

Abstract Cancer cells co-opt mechanisms of adaptation to hypoxia to minimize energy expenditure and halt proliferation thereby increasing hypoxic tolerance, a major factor contributing to chemo- and radioresistance in patients. The endoplasmic reticulum (ER) kinase PERK is one of the three signaling transducers of the unfolded protein response (UPR) that inhibits mRNA translation via eIF2α phosphorylation to alleviate hypoxia-induced ER stress. We have previously shown that accumulation of fumarate, resulting from loss-of-function (LOF) of the mitochondrial enzyme fumarate hydratase (FH), augments the antioxidant capacity of hypoxic cancer cells through upregulation of the pentose phosphate pathway (PPP). Here, we report for the first time that the fumarate-dependent increase of antioxidants generated by the PPP prevents hypoxic activation of the PERK→eIF2α axis enabling protein synthesis and proliferation leading to an impairment of hypoxic tolerance. HeLa (cervix), HCT-116 and LS174T (colon) adenocarcinoma cells were transfected with lentiviral vectors encoding for shRNAs targeting FH. Immunoblot assays showed that FH LOF impaired PERK activity under hypoxia thus inactivating eIF2α. Moreover, the ability of fumarate to prevent PERK activation was specific to hypoxia since fumarate did not affect the pharmacological activation of the UPR by thapsigargin, DTT or tunicamycin. A comprehensive analysis utilizing a RT-qPCR array to profile the mRNA expression of 84 UPR genes, showed that FH LOF inhibited the UPR in HCT-116 cells. A similar response was observed in patient-derived UOK262 kidney cancer cells whereby bi-allelic FH mutations resulted in downregulation of 72% of the UPR genes whereas FH re-introduction restored UPR signaling. We employed LS174T mucinous adenocarcinoma cells as a model to assess protein synthesis. Mucin synthesis was not affected in hypoxic FH deficient cells whereas it was abrogated in wild-type cells. Consistent with these results, FH LOF increased 3D spheroid growth and decreased clonogenic potential in HCT-116 and HeLa cells. Pharmacological inhibition of the PPP by 6-aminonicotinamide activated the PERK→eIF2α axis in FH silenced cells thereby suggesting that PPP activation by fumarate is implicated in PERK inhibition. RNAseq data analysis in melanoma, prostate, colon, breast, lung and renal cancers from TCGA cohorts shows a negative correlation between PERK and the PPP enzyme PGLS suggesting that the mechanism hereby delineated occurs in cancer patients. In conclusion, we show that fumarate impairs PERK→eIF2α UPR signaling in hypoxic cancer cells. Clinical and molecular data show that UPR activation is one of the mechanisms responsible for chemo- and radioresistance in hypoxic tumors. This study suggests that fumarate accumulation (either through extrinsic or FH LOF) represents a novel approach to target hypoxic cancer cells and improve patient prognosis. Citation Format: Luana Schito, Sergio Rey, Bradly G. Wouters, Marianne Koritzinsky. The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2017-4499

Grape Seed Proanthocyanidin Inhibits Mucin Synthesis and Viral Replication by Suppression of AP-1 and NF-κB via p38 MAPKs/JNK Signaling Pathways in Respiratory Syncytial Virus-Infected A549 Cells.

Airway epithelial cells are often infected by respiratory syncytial virus (RSV), one of the most common causes of asthma, bronchiolitis, chronic obstructive pulmonary disease, and pneumonia. During the infection process, excessive mucins instigate airway inflammation. However, the mechanism underlying RSV-induced airway hyper-responsiveness and inflammation is poorly understood. Furthermore, no reliable vaccines or drugs for antiviral therapy are available. In this study, the effect of the natural compound grape seed proanthocyanidin (GSP) on RSV-infected human airway epithelial cells A549 was evaluated. After pretreatment of the cells with or without exposure to RSV with 5-10 μg GSP/mL, the expression of various mucins (MUC1, MUC2, MUC5AC, MUC5B, and MUC8) was evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting, as well as confocal microscopy. We found that GSP significantly decreased RSV-induced mucin synthesis at the mRNA and protein levels. In addition, GSP suppressed the RSV-induced signaling pathways, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, together with nuclear factor kappa B (NF-κB) and activating protein-1 family members (c-Jun and c-Fos). Concomitantly, GSP inhibited the replication of RSV within A549 cells. Taken together, all our results suggest that GSP could be a potent therapeutic agent to suppress excessive mucus production and viral replication in RSV-induced airway inflammatory disorders.

Nutrients central to maintaining intestinal absorptive efficiency and barrier integrity with fowl

The small intestinal mucosa acts to recover nutrients from the lumen while providing a barrier against potential hazards. Its unstirred water layer (USWL) at the lumen interface involves membrane associated mucin linearly protruding from underlying microvilli that entangles secretory mucin released from local goblet cells. Both mucin sources are dominated by repetitive O-glycosylated areas dependant on threonine, serine, glycine, and proline. Secretory mucin differs from membrane attached mucin by further employing multiple cystines that interconnect these areas into a net-like molecular sieve. All of the glycosylated areas have ionizable acidic groups credited with reducing pH from that in the lumen to create a micro environment favoring enzymes finalizing digestion while optimizing nutrient terms for absorption. Erosion of the USWL and/or abuse of the membrane due to lumen threats require continuous repair. The aforementioned amino acids are necessary in substantial amounts while vitamin B6 collaborates with vitamin A as meaningful cofactors for mucin synthesis. Marginal inadequacies of these nutrients during inordinate demand are expected to impair mucin replacement. In turn, marginal increases in feed conversion likely occur while fostering the probability of necrotic enteritis together with gizzard erosions. Abuse of the absorptive membrane is of particular concern from fatty acid hydroperoxides because of their continual presence in feed and inability of the USWL to provide protection. These hydroperoxides threaten membrane integrity by their inclusion in micelles during digestive events with fat thereby permitting transit through the USWL. Once coalesced with membrane phospholipids, structural aberrations are visualized as interfering with nutrient recovery while enabling leakage of cell contents to potentiate wet excreta. Inclusion of dietary vitamin E along with vitamin A into micelles with fatty acid hydroperoxides provides relief by quenching further peroxidation. Assuring cystine, threonine, glycine, and serine that are directly available as such together with vitamins A, E, and B6 represents one approach toward optimizing maintenance of the intestinal mucosa.

Exercise and Dietary Factors Affecting the Microbiota: Current Knowledge and Future Perspectives

Gut micro biota degrades complex polysaccharides and synthesizes short chain fatty acids that regulate the intestinal barrier by stimulating mucin synthesis. Mucin maintains the integrity of the gut by forming a mucus layer that covers and protects the intestinal epithelium. Lactate- and butyrate-producing bacteria maintain gut integrity in healthy individuals, while non-butyrate-producing, lactate-utilizing bacteria prevent optimal mucin synthesis. The effect of the micro biota on the host physiology was first observed with animal experiments involving fecal transplantation, in which impressive metabolic effects were passed from the donor to the host. These experiments showed that the micro biota from obese donors has an increased capacity to harvest energy from the diet, thus affecting body composition in the long term. One of the potential mechanisms through which the micro biota could affect body composition is by regulating the delivery of orexigenic and anorexigenic hormones, as it has been shown that the concentration of these hormones is correlated with several groups of the gut micro biota. Gut micro biota has not only been linked to body composition outcomes but also to the development and severity of certain metabolic abnormalities such as autoimmune diseases (type I diabetes and lupus), necrotizing enterocolitis, irritable bowel syndrome, ulcerative colitis, acute diarrheal infections, and frailty. It is well established that lifestyle factors such as exercise, diet, and supplementation with pre- and/or pro biotics affects the gut micro biota. The purpose of this review is to evaluate the effect of exercise, diet, and supplementation with pre- and/or pro biotics on the gut micro biota of humans and animals models. Clinical perspectives and future research areas will also be discussed.