Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and render their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to identify a previously unmapped MUC3B gene located within a cluster of four structurally related membrane mucin genes that we entitle the MUC3 cluster at q22 locus in chromosome 7. We found that MUC3B shares high sequence identity with the known MUC3A gene, and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12 and MUC17 in the human MUC3 cluster are exclusively expressed in intestinal epithelial cells. Our results complete existing genetic gaps in the MUC3 cluster that is a conserved genetic unit during primate evolution. We anticipate our results to be the starting point for detection of new polymorphisms in the MUC3 cluster associated with human diseases. Moreover, our study provides the basis for exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.

A tumor of composite cervical adenocarcinoma with MUCIN gene mutation：a case report

Background : There are reports about the coexistence of two kinds of tumors in the same patient,which is believed that this phenomenon is caused by the dedifferentiation between the two tumors.In this paper, we report an human papillomavirus (HPV) negative cervical adenocarcinoma in a patient composed of two adenocarcinoma components,which is first reported.Histologically, both minimal deviation adenocarcinoma (MDA) and poorly differentiated gastric type adenocarcinoma (GTA) components, as well as their transitional area, were observed.Methods : This case of cervical cancer was screened by gene sequencing. For detection of specific somatic mutations in MDA and GTA, we filtered out mutations in malignant cervical cancer blood sample and 7 common cervical carcinoma. Then the genes were screened and identified based on the enrichment analysis of GO and KEGG and related literature reports. Results : We found 13 specific somatic gene mutations in total. Among these genes, only Mucin gene was transformed from gene level to protein level, and was positive in both MDA and GTA components of the patient by immunohistochemistry.Both components had genes mutation of MUC4 and MUC17,the component in MDA had gene mutation of MUC3A,and we found that MUC3A and MUC17 were on the same chromosome. Moreover,MUC3A and MUC4 genes were found to be fused in FusionGDB database. Conclusion : According to the reports of MUC3A,MUC4 and MUC17 genes mutation in cervical adenocarcinoma and gene fusions in tumorigenesis, we speculate that the occurrence of the transformation of pathological type from MDA to GTA in this case of cervical cancer is related to the mechanism of MUC3A and MUC4 gene fusion.We would advice, for HPV negative or atypical cervical lesions, immunohistochemistry of MUCIN genes staining and gene sequencing should be considered, which may find unusual cancer types and change the prognosis of patients.

A Tumor of Composite Cervical Adenocarcinoma with MUCIN Gene Mutation：A Case Report

Background:There are reports about the coexistence of two kinds of tumors in the same patient,which is believed that this phenomenon is caused by the dedifferentiation between the two tumors.In this paper, we report an human papillomavirus (HPV) negative cervical adenocarcinoma in a patient composed of two adenocarcinoma components,which is first reported.Histologically, both minimal deviation adenocarcinoma (MDA) and poorly differentiated gastric type adenocarcinoma (GTA) components, as well as their transitional area, were observed.Methods:This case of cervical cancer was screened by gene sequencing. For detection of specific somatic mutations in MDA and GTA, we filtered out mutations in malignant cervical cancer blood sample and 7 common cervical carcinoma. Then the genes were screened and identified based on the enrichment analysis of GO and KEGG and related literature reports. Results:We found 13 specific somatic gene mutations in total. Among these genes, only Mucin gene was transformed from gene level to protein level, and was positive in both MDA and GTA components of the patient by immunohistochemistry.Both components had genes mutation of MUC4 and MUC17,the component in MDA had gene mutation of MUC3A,and we found that MUC3A and MUC17 were on the same chromosome. Moreover,MUC3A and MUC4 genes were found to be fused in FusionGDB database. Conclusion:According to the reports of MUC3A,MUC4 and MUC17 genes mutation in cervical adenocarcinoma and gene fusions in tumorigenesis, we speculate that the occurrence of the transformation of pathological type from MDA to GTA in this case of cervical cancer is related to the mechanism of MUC3A and MUC4 gene fusion.We would advice, for HPV negative or atypical cervical lesions, immunohistochemistry of MUCIN genes staining and gene sequencing should be considered, which may find unusual cancer types and change the prognosis of patients.

A50 MODULATION OF GOBLET CELL ACTIVITY DURING GIARDIA DUODENALIS INFECTION: A ROLE FOR PAR2

Background Giardia duodenalis has been shown to alter the structure of the intestinal mucus layers during infection via obscure mechanisms. We hypothesize that goblet cell activity may be disrupted in part due to proteolytic activation of protease-activated receptor 2 (PAR2) by Giardia proteases, resulting in disruption of mucus production and secretion by intestinal goblet cells. Aims Characterize alterations in goblet cell activity during Giardia infection, focusing on the roles of Giardia protease activity and PAR2. Methods Chinese hamster ovary cells transfected with nano-luciferase tagged PAR2 were incubated with Giardia NF or GSM trophozoites. Cleavage within the activation domain results in release of enzymes into the supernatant. Luminescence in the supernatant was measured as an indication of PAR cleavage by Giardia. LS174T, a human colonic mucus-producing cell line, was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a PAR2 antagonist, a calcium chelator, or an ERK1/2 inhibitor. Quantitative PCR (qPCR) was performed for the MUC2 mucin gene. Wild-type (WT) and PAR2 knockout (KO) mice were infected with Giardia. Colonic mucus was stained using fluorescein-coupled wheat-germ agglutinin (WGA), and qPCR was performed for Muc2 and Muc5ac. Results Giardia trophozoites cleaved PAR2 within the N-terminal activation domain in a cysteine protease-dependent manner. Cleavage was isolate dependent, with isolates that show higher protease activity cleaving at a higher rate. High protease activity Giardia isolates increased MUC2 gene expression in LS714T. This increase was attenuated by inhibition of Giardia cysteine protease activity, and by antagonism of PAR2, inhibition of calcium release, or inhibition of ERK1/2 activity in LS174T cells. Both Muc2 and Muc5ac expression were upregulated in the colons of WT mice in response to Giardia infection, while in the jejunum Muc2 expression decreased and Muc5ac expression increased. In KO, no changes in gene expression were seen in the colon in response to Giardia infection, while in the jejunum, Muc2 expression was unchanged and Muc5ac expression decreased. Both WT infected and KO noninfected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in KO infected mice. Conclusions PAR2 plays a significant role in the regulation of mucin gene expression in mice and in a human colonic cell line. Results suggest that Giardia cysteine proteases cleave and activate PAR2, leading to calcium release and activation of the MAPK pathway in goblet cells, ultimately leading to altered mucin gene expression. Findings identify a novel regulatory pathway for mucus production by intestinal goblet cells. Funding Agencies CAG, CCC

Amelioration of Cigarette Smoke-Induced Mucus Hypersecretion and Viscosity by Dendrobium officinale Polysaccharides In Vitro and In Vivo

Chronic obstructive pulmonary disease (COPD), characterized by oxidative stress and inflammation, is one of the leading causes of death worldwide, in which cigarette smoke (CS) is the major risk factor. Dendrobium officinale polysaccharides (DOPs) are the main active ingredients extracted from Dendrobium officinale, which have been reported to have antioxidant and anti-inflammatory activity as well as inhibition of mucin gene expression. This study is aimed at investigating the effect of DOPs on CS-induced mucus hypersecretion and viscosity in vitro and in vivo. For in vitro study, primary normal human bronchial epithelial cells (HBECs) differentiated at the air-liquid interface (ALI) culture for 28 days were stimulated with cigarette smoke medium (CSM) in the absence or presence of various concentrations of DOPs or N-acetylcysteine (NAC) for 24 hours. For in vivo study, male Sprague-Dawley rats were randomized to sham air (SA) as control group or CS group for 56 days. At day 29, rats were subdivided and given water as control, DOPs, or NAC as positive control as a mucolytic drug via oral gavage for the remaining duration. Samples collected from apical washing, cell lysates, bronchoalveolar lavage (BAL), and lung tissues were evaluated for mucin gene expression, mucus secretion, and viscosity. DOPs ameliorated the CS-induced mucus hypersecretion and viscosity as shown by the downregulation of MUC5AC mRNA, MUC5AC secretary protein, and mucus viscosity via inhibition of mucus secretory granules in both in vitro and in vivo models. DOPs produced its effective effects on the CS-induced mucus hypersecretion and viscosity via the inhibition of the mucus secretory granules. These findings could be a starting point for considering the potential role of DOPs in the management of the smoking-mediated COPD. However, further research is needed.

Concentration of Digestible Threonine in Diet on Production Performance and Intestinal Morphometry of WL Layers

A study was conducted to evaluate the effect of digestible threonine in graded concentrations on production performance and mucosal barrier function in layers. A total of 180 layers at 25 weeks of age were randomly distributed into six treatment groups each with five replicates of 6 birds each. The experimental diets were isocaloric with three crude protein(CP) levels i.e. low 13.46% CP(LCP), medium 15.56% CP (MCP) and high 17.05% CP(HCP) and three lysine levels (13.46/0.65%; 15.56/0.60%; 17.00/0.70%) each with 63 and 66% threonine concentrations and were offered to treatment groups for 20 weeks. Increase (p less than 0.05) in egg production was observed with an increase in protein/lysine level and threonine concentration. Mucin gene (MUC2) expression was increased (p less than 0.05) in LCP group and was evident at 66% threonine concentrations in LCP and MCP and 63% threonine concentration in HCP. The results of the study concluded that the diet with 13.46% CP and 0.65% lysine at 66% threonine were optimum for layers at 25-44 weeks of age.