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1.
Nature ; 598(7880): 332-337, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616040

RESUMO

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.


Assuntos
Bacteroides/enzimologia , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal , Mucinas/metabolismo , Sulfatases/metabolismo , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Animais , Colo/química , Cristalografia por Raios X , Feminino , Galactose/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Especificidade por Substrato , Sulfatases/química
2.
Adv Exp Med Biol ; 1325: 25-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495529

RESUMO

Mucin-type GalNAc O-glycosylation is one of the most abundant and unique post-translational modifications. The combination of proteome-wide mapping of GalNAc O-glycosylation sites and genetic studies with knockout animals and genome-wide analyses in humans have been instrumental in our understanding of GalNAc O-glycosylation. Combined, such studies have revealed well-defined functions of O-glycans at single sites in proteins, including the regulation of pro-protein processing and proteolytic cleavage, as well as modulation of receptor functions and ligand binding. In addition to isolated O-glycans, multiple clustered O-glycans have an important function in mammalian biology by providing structural support and stability of mucins essential for protecting our inner epithelial surfaces, especially in the airways and gastrointestinal tract. Here the many O-glycans also provide binding sites for both endogenous and pathogen-derived carbohydrate-binding proteins regulating critical developmental programs and helping maintain epithelial homeostasis with commensal organisms. Finally, O-glycan changes have been identified in several diseases, most notably in cancer and inflammation, where the disease-specific changes can be used for glycan-targeted therapies. This chapter will review the biosynthesis, the biology, and the translational perspectives of GalNAc O-glycans.


Assuntos
Estudo de Associação Genômica Ampla , Mucinas , Animais , Glicosilação , Humanos , Mucinas/genética , Mucinas/metabolismo , Polissacarídeos , Processamento de Proteína Pós-Traducional
3.
World J Microbiol Biotechnol ; 37(10): 176, 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34519941

RESUMO

All mucins are highly glycosylated and a key constituent of the mucus layer that is vigilant against pathogens in many organ systems of animals and humans. The viscous layer is organized in bilayers, i.e., an outer layer that is loosely arranged, variable in thickness, home to the commensal microbiota that grows in the complex environment, and an innermost layer that is stratified, non-aspirated, firmly adherent to the epithelial cells and devoid of any microorganisms. The O-glycosylation moiety represents the site of adhesion for pathogens and due to the increase of motility, mucolytic activity, and upregulation of virulence factors, some microorganisms can circumvent the component of the mucus layer and cause disruption in organ homeostasis. A dysbiotic microbiome, defective mucus barrier, and altered immune response often result in various diseases. In this review, paramount emphasis is given to the role played by the bacterial species directly or indirectly involved in mucin degradation, alteration in mucus secretion or its composition or mucin gene expression, which instigates many diseases in the digestive, respiratory, and other organ systems. A systematic view can help better understand the etiology of some complex disorders such as cystic fibrosis, ulcerative colitis and expand our knowledge about mucin degraders to develop new therapeutic approaches to correct ill effects caused by these mucin-dwelling pathogens.


Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Membrana Mucosa/microbiologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/metabolismo , Disbiose/metabolismo , Disbiose/microbiologia , Humanos , Microbiota , Mucinas/metabolismo
4.
Nutrients ; 13(8)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34444752

RESUMO

Threonine (Thr), an essential amino acid for animals and the limiting amino acid in swine and poultry diets, which plays a vital role in the modulation of nutritional metabolism, macromolecular biosynthesis, and gut homeostasis. Current evidence supports that the supplementation of Thr leads to benefits in terms of energy metabolism. Threonine is not only an important component of gastrointestinal mucin, but also acts as a nutritional modulator that influences the intestinal immune system via complex signaling networks, particularly mitogen-activated protein kinase (MAPK) and the target of the rapamycin (TOR) signal pathway. Threonine is also recognized as an indispensable nutrient for cell growth and proliferation. Hence, optimization of Thr requirement may exert a favorable impact on the factors linked to health and diseases in animals. This review focuses on the latest reports of Thr in metabolic pathways and nutritional regulation, as well as the relationship between Thr and relevant physiological functions.


Assuntos
Metabolismo Energético , Estado Nutricional , Treonina/metabolismo , Aminoácidos/metabolismo , Animais , Dieta , Trato Gastrointestinal/metabolismo , Redes e Vias Metabólicas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucinas/metabolismo , Sirolimo , Células-Tronco , Suínos
5.
Cells ; 10(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34440830

RESUMO

Gastrointestinal (GI) mucus plays a pivotal role in the tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro GI models with a physiologically relevant mucus layer, studies with deeper insights into structural and compositional changes upon chemical or physical manipulation of the system are rare. To obtain an improved mucus-containing cell model, we developed easy-to-use, reusable culture chambers that facilitated the application of GI shear stresses (0.002-0.08 dyn∙cm-2) to cells on solid surfaces or membranes of cell culture inserts in bioreactor systems, thus making them readily accessible for subsequent analyses, e.g., by confocal microscopy or transepithelial electrical resistance (TEER) measurement. The human mucus-producing epithelial HT29-MTX cell-line exhibited superior reorganization into 3-dimensional villi-like structures with highly proliferative tips under dynamic culture conditions when compared to static culture (up to 180 vs. 80 µm in height). Additionally, the median mucus layer thickness was significantly increased under flow (50 ± 24 vs. 29 ± 14 µm (static)), with a simultaneous accelerated maturation of the cells into a goblet-like phenotype. We demonstrated the strong impact of culture conditions on the differentiation and reorganization of HT29-MTX cells. The results comprise valuable advances towards the improvement of existing GI and mucus models or the development of novel systems using our newly designed culture chambers.


Assuntos
Diferenciação Celular , Muco/metabolismo , Resistência ao Cisalhamento , Técnicas de Cultura de Células/métodos , Proliferação de Células , Desenho Assistido por Computador , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células HT29 , Humanos , Microscopia Confocal , Mucinas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
6.
Cells ; 10(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34440808

RESUMO

Stratified mucin-producing intraepithelial lesion (SMILE) is a rare high-grade cervical precancerous lesion designated a variant of adenocarcinoma in situ (AIS) in the WHO classification. We aimed to determine HPV genotypes, immunohistochemical phenotype and mucin presence in SMILE. Between 2010 and 2018, SMILE was diagnosed in 34 out of 6958 (0.5%) cervical biopsies, in 23 patients. Twenty-six tissue samples from twenty-one patients were available for further analysis, including 13 with SMILE alone, 12 with SIL and/or AIS and one with HSIL, AIS and endocervical adenocarcinoma. HPV genotyping was performed using the Seegene Anyplex II HPV 28 assay. Of the 26 samples, a single HPV genotype was identified in the majority of cases (n = 22), including 12/13 SMILEs associated with SIL/AIS. All but one were high-risk HPV genotypes (23/24; 96.8%). We identified seven different HPV genotypes, the most common being HPV16 (n = 10; 43.5%), HPV18 (n = 8, 34.8%) and HPV 31 (n = 5, 21.7%). All SMILEs showed a strong positive reaction to p16, CK7, CK19 and high Ki67 expression comparable to adjacent HSIL and/or AIS if present. SMILE showed variable mucin presence and p40-positive squamous differentiation suggesting phenotypic diversity in cervical precancerous lesions infected by single HPV.


Assuntos
Neoplasia Intraepitelial Cervical/patologia , Papillomavirus Humano 16/isolamento & purificação , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias do Colo do Útero/patologia , Adenocarcinoma in Situ/patologia , Adenocarcinoma in Situ/virologia , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/virologia , Colo do Útero/metabolismo , Colo do Útero/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Genótipo , Papillomavirus Humano 16/genética , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Mucinas/metabolismo , Gradação de Tumores , Estudos Retrospectivos , Lesões Intraepiteliais Escamosas/patologia , Lesões Intraepiteliais Escamosas/virologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia , Adulto Jovem
7.
Nat Commun ; 12(1): 4070, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210959

RESUMO

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.


Assuntos
Mucinas/metabolismo , Membrana Mucosa/metabolismo , Polissacarídeos/genética , Polissacarídeos/metabolismo , Engenharia Genética , Glicosilação , Células HEK293 , Humanos , Microbiota , Mucina-1/genética , Mucina-1/metabolismo
8.
Front Cell Infect Microbiol ; 11: 668766, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34222040

RESUMO

C. butyricum is a common gut commensal bacterium, which has many positive functions in human intestine. In this study, we investigated the effects of monosaccharide and its derivatives on the adhesion of C. butyricum to the mucus of HT-29 cells. RNA interference was performed to assess the roles of MUC2 and glycan in the adhesion of C. butyricum to HT-29 cells. The effects of C. butyricum on the glycosylation of mucins were assayed with fluorescence microscope. The expression levels of mucins and glycotransferases were also determined. The results showed that C. butyricum could adhere to the mucins secreted by HT-29 cells. Several kinds of monosaccharides inhibited the adhesion of C. butyricum to HT-29 cells, which suggested that the mucus glycan was the attaching sites of this bacterium. Knockdown of MUC2, FUT2 or GALNT7 significantly decreased the numbers of the bacteria adhering to HT-29 cells. When colonizing on the surface of HT-29 cells, C. butyricum could increase the production of mucins, promote the expression of glycotransferase, and induce the glycosylation of mucins. These results demonstrated that the glycan of mucus played important roles in the adhesion of C. butyricum to HT-29 cells. This study indicates for the first time that C. butyricum possesses the ability to modulate the glycosylation profile of mucus secreted by HT-29 cells. These findings contribute to understanding the mechanism of interaction between colonic epithelial cells and commensal bacteria.


Assuntos
Clostridium butyricum , Mucinas , Clostridium butyricum/metabolismo , Células Epiteliais/metabolismo , Glicosilação , Células HT29 , Humanos , Mucinas/metabolismo
9.
Nat Biomed Eng ; 5(8): 897-913, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239116

RESUMO

Creating in vitro models of diseases of the pancreatic ductal compartment requires a comprehensive understanding of the developmental trajectories of pancreas-specific cell types. Here we report the single-cell characterization of the differentiation of pancreatic duct-like organoids (PDLOs) from human induced pluripotent stem cells (hiPSCs) on a microwell chip that facilitates the uniform aggregation and chemical induction of hiPSC-derived pancreatic progenitors. Using time-resolved single-cell transcriptional profiling and immunofluorescence imaging of the forming PDLOs, we identified differentiation routes from pancreatic progenitors through ductal intermediates to two types of mature duct-like cells and a few non-ductal cell types. PDLO subpopulations expressed either mucins or the cystic fibrosis transmembrane conductance regulator, and resembled human adult duct cells. We also used the chip to uncover ductal markers relevant to pancreatic carcinogenesis, and to establish PDLO co-cultures with stellate cells, which allowed for the study of epithelial-mesenchymal signalling. The PDLO microsystem could be used to establish patient-specific pancreatic duct models.


Assuntos
Diferenciação Celular , Dispositivos Lab-On-A-Chip , Organoides/citologia , Ductos Pancreáticos/citologia , Animais , Biomarcadores Tumorais/metabolismo , Reprogramação Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Filaminas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mucinas/metabolismo , Organoides/metabolismo , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Análise de Célula Única , Taxa de Sobrevida
10.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072552

RESUMO

Indirect evidence has determined the possibility that microplastics (MP) induce constipation, although direct scientific proof for constipation induction in animals remains unclear. To investigate whether oral administration of polystyrene (PS)-MP causes constipation, an alteration in the constipation parameters and mechanisms was analyzed in ICR mice, treated with 0.5 µm PS-MP for 2 weeks. Significant alterations in water consumption, stool weight, stool water contents, and stool morphology were detected in MP treated ICR mice, as compared to Vehicle treated group. Also, the gastrointestinal (GI) motility and intestinal length were decreased, while the histopathological structure and cytological structure of the mid colon were remarkably altered in treated mice. Mice exposed to MP also showed a significant decrease in the GI hormone concentration, muscarinic acetylcholine receptors (mAChRs) expression, and their downstream signaling pathway. Subsequent to MP treatment, concentrations of chloride ion and expressions of its channel (CFTR and CIC-2) were decreased, whereas expressions of aquaporin (AQP)3 and 8 for water transportation were downregulated by activation of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway. These results are the first to suggest that oral administration of PS-MP induces chronic constipation through the dysregulation of GI motility, mucin secretion, and chloride ion and water transportation in the mid colon.


Assuntos
Constipação Intestinal/diagnóstico , Constipação Intestinal/etiologia , Microplásticos/efeitos adversos , Fenótipo , Poliestirenos/efeitos adversos , Animais , Comportamento Animal , Biomarcadores , Fenômenos Químicos , Cloretos/metabolismo , Colo/patologia , Colo/ultraestrutura , Modelos Animais de Doenças , Suscetibilidade a Doenças , Hormônios Gastrointestinais/metabolismo , Motilidade Gastrointestinal , Bombas de Íon/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microplásticos/química , Mucinas/metabolismo , Poliestirenos/química , Transdução de Sinais , Água/metabolismo
11.
Nat Commun ; 12(1): 3573, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117223

RESUMO

O-GalNAc glycans (or mucin O-glycans) play pivotal roles in diverse biological and pathological processes, including tumor growth and progression. Structurally defined O-GalNAc glycans are essential for functional studies but synthetic challenges and their inherent structural diversity and complexity have limited access to these compounds. Herein, we report an efficient and robust chemoenzymatic modular assembly (CEMA) strategy to construct structurally diverse O-GalNAc glycans. The key to this strategy is the convergent assembly of O-GalNAc cores 1-4 and 6 from three chemical building blocks, followed by enzymatic diversification of the cores by 13 well-tailored enzyme modules. A total of 83 O-GalNAc glycans presenting various natural glycan epitopes are obtained and used to generate a unique synthetic mucin O-glycan microarray. Binding specificities of glycan-binding proteins (GBPs) including plant lectins and selected anti-glycan antibodies towards these O-GalNAc glycans are revealed by this microarray, promoting their applicability in functional O-glycomics. Serum samples from colorectal cancer patients and healthy controls are assayed using the array reveal higher bindings towards less common cores 3, 4, and 6 than abundant cores 1 and 2, providing insights into O-GalNAc glycan structure-activity relationships.


Assuntos
Glicômica , Mucinas/química , Mucinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Carboidratos , Proteínas de Transporte/metabolismo , Epitopos , Glicosilação , Humanos , Análise em Microsséries
13.
Carbohydr Polym ; 267: 118157, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34119131

RESUMO

The aim of the study was to investigate the effects of cross-linkers on quercetin (QUE) absorption characteristics of QUE-loaded chitosan nanoparticles (CS-NPs). CS-NPs (461.2-482.7 nm) were prepared by ionic gelation at pH 3.5 using tripolyphosphate (367.9 Da), dextran sulfate (>15 kDa), arabic gum (AG, >250 kDa), or hyaluronic acid (HA, >1000 kDa). Mucoadhesion and cell permeation of QUE were significantly increased by positive charged CS-NPs due to interactions with negatively charged mucosal layer. Moreover, CS-AG and CS-HA NPs prepared with relatively higher MW cross-linkers exhibited significantly higher adhesion and permeation than the others. These results were verified by a cellular antioxidant activity assay; CS-AG (137.5 unit) and CS-HA NPs (126.5 unit) showed significantly higher activities after internalization into Caco-2 cells. Therefore, encapsulation within CS-NPs prepared using high MW cross-linkers such as AG and HA is found to be potentially valuable techniques for improving the QUE absorption.


Assuntos
Quitosana/química , Portadores de Fármacos/química , Nanopartículas/química , Linhagem Celular Tumoral , Quitosana/metabolismo , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Goma Arábica/química , Goma Arábica/metabolismo , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Peso Molecular , Mucinas/metabolismo , Nanopartículas/metabolismo , Tamanho da Partícula , Ligação Proteica , Quercetina/química , Quercetina/farmacologia
14.
Theranostics ; 11(13): 6193-6213, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995653

RESUMO

Rationale: The pandemic caused by the novel coronavirus SARS-CoV-2 is advancing rapidly. In particular, the number of severe courses of the disease is still dramatically high. An efficient drug therapy that helps to improve significantly the fatal combination of damages in the airway epithelia, in the extensive pulmonary microvascularization and finally multiorgan failure, is missing. The physiological, inorganic polymer, polyphosphate (polyP) is a molecule which could prevent the initial phase of the virus life cycle, the attachment of the virus to the target cells, and improve the epithelial integrity as well as the mucus barrier. Results: Surprisingly, polyP matches perfectly with the cationic groove on the RBD. Subsequent binding studies disclosed that polyP, with a physiological chain length of 40 phosphate residues, abolishes the binding propensity of the RBD to the ACE2 receptor. In addition to this first mode of action of polyP, this polymer causes in epithelial cells an increased gene expression of the major mucins in the airways, of MUC5AC and MUC1, as well as a subsequent glycoprotein production. MUC5AC forms a gel-like mucus layer trapping inhaled particles which are then transported out of the airways, while MUC1 constitutes the periciliary liquid layer and supports ciliary beating. As a third mode of action, polyP undergoes enzymatic hydrolysis of the anhydride bonds in the airway system by alkaline phosphatase, releasing metabolic energy. Conclusions: This review summarizes the state of the art of the biotherapeutic potential of the polymer polyP and the findings from basic research and outlines future biomedical applications.


Assuntos
COVID-19/tratamento farmacológico , Pandemias/prevenção & controle , Polifosfatos/farmacologia , Animais , Antivirais/química , Antivirais/uso terapêutico , COVID-19/epidemiologia , COVID-19/transmissão , COVID-19/virologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Camundongos , Mucinas/metabolismo , Nanopartículas/química , Polifosfatos/química , Polifosfatos/uso terapêutico , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Ligação Viral/efeitos dos fármacos
15.
Ecotoxicol Environ Saf ; 220: 112340, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015635

RESUMO

Microplastics are plastic fragments widely distributed in the environment and accumulate in the organisms. However, the research on microplastics effects in mammals is limited. Polyethylene is the main kind of microplastics in the environment. We hypothesized that polyethylene exposure disrupts host intestine metabolism by modifying intestine microflora composition and then lipopolysaccharide (LPS) pathway. Female mice were orally exposed to 0, 0.002 and 0.2 µg/g/d polyethylene microplastics (PE MPs) for 30 days. Colon mucin density was quantized after AB-PAS staining. Mucin 2 (MUC2), inflammatory factors (IL-1ß, IL-6, IL-8 and IL-10), short-chain fatty acid receptors (GPR41 and GPR43), LPS receptors (TLR4 and MyD88) and LPS pathway downstream genes (ERK1 and NF-κB) mRNA levels in colon were measured. Feces were collected on the 15th day of exposure for gut microflora analysis. Blood biochemical analysis was performed. Results showed that 0.2 µg/g/d PE MPs exposure significantly decreased colon mucin expression (p < 0.05), decreased IL-1ß (p < 0.05) and increased IL-8 and IL-10 levels (p < 0.01 and p < 0.001 respectively). Microflora data showed that in 0.2 µg/g/d PE MPs group the number of Firmicutes decreased and the number of Bacteroides increased (both p < 0.01). Predicted KEGG metabolic pathways by piecrust method indicated that PE MPs enhanced amino acids metabolism in microflora. ERK1 and NF-κB mRNA were significantly lower in 0.2 µg/g/d PE MPs group (both p < 0.001). Blood total protein, albumin and globulin levels significantly increased after 0.2 µg/g/d PE MPs exposure (p < 0.01, p < 0.01 and p < 0.05 respectively). These results indicate that PE MPs exposure induced decreased mucin production, a slight immune response and increased the microflora amino acid metabolism in the mice colon by modifying colon microflora composition. SUMMARY: Polyethylene microplastics exposure decreased colon mucin release and increased amino acid metabolism by modifying colon microflora composition.


Assuntos
Bactérias/efeitos dos fármacos , Colo/imunologia , Microbioma Gastrointestinal/efeitos dos fármacos , Inflamação/imunologia , Microplásticos/efeitos adversos , Mucinas/metabolismo , Polietileno/efeitos adversos , Administração Oral , Animais , Bactérias/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Relação Dose-Resposta a Droga , Feminino , Inflamação/induzido quimicamente , Camundongos , Camundongos Endogâmicos ICR
16.
J Fish Dis ; 44(9): 1355-1367, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33990985

RESUMO

Amoebic gill disease (AGD) is caused by the marine amoeba Neoparamoeba perurans, a facultative parasite. Despite the significant impact this disease has on production of Atlantic salmon worldwide, the mechanisms involved in host-parasite interaction remains unknown. Excessive gill mucus secretion is reported as a host defence mechanism to prevent microbial colonization in the gill epithelium. Despite this response, N. perurans still attaches and proliferates. The present study aimed to investigate the interaction between N. perurans and mucin, the most abundant component in mucus. An in vitro adhesion assay using bovine submaxillary mucin (BSM) demonstrated that amoeba binding to mucin-coated substrate was significantly higher than to the BSA control. This binding interaction is likely glycan-mediated as pre-incubation with galactose, galactosamine, N-acetylgalactosamine and fucose reduced mucin adhesion to control levels. The ability of N. perurans to secrete proteases that target mucin was also investigated. Protease activity was detected in the amoeba culture media in the presence of BSM, but not when protease inhibitor was added. Mucin degradation was visually assessed on protein gels. This study provides preliminary evidence that N. perurans has developed mechanisms to interact with and evade mucus by binding to mucin glycan receptors and secreting proteases with mucolytic activity.


Assuntos
Amebozoários/fisiologia , Mucinas/metabolismo , Peptídeo Hidrolases/metabolismo , Amebíase , Amebozoários/enzimologia , Animais , Bovinos , Doenças dos Peixes/parasitologia , Brânquias/parasitologia , Peptídeo Hidrolases/química
17.
Sci Rep ; 11(1): 11094, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045537

RESUMO

Mucins are large glycoproteins consisting of approximately 80% of hetero-oligosaccharides. Gut mucin degraders of healthy subjects were investigated, through a culture dependent and independent approach. The faeces of five healthy adults were subjected to three steps of anaerobic enrichment in a medium with sole mucins as carbon and nitrogen sources. The bacterial community was compared before and after the enrichment by 16S rRNA gene profiling. Bacteria capable of fermenting sugars, such as Anaerotruncus, Holdemania, and Enterococcaceae likely took advantage of the carbohydrate chains. Escherichia coli and Enterobacteriaceae, Peptococcales, the Coriobacteriale Eggerthella, and a variety of Clostridia such as Oscillospiraceae, Anaerotruncus, and Lachnoclostridium, significantly increased and likely participated to the degradation of the protein backbone of mucin. The affinity of E. coli and Enterobacteriaceae for mucin may facilitate the access to the gut mucosa, promoting gut barrier damage and triggering systemic inflammatory responses. Only three species of strict anaerobes able to grow on mucin were isolated from the enrichments of five different microbiota: Clostridium disporicum, Clostridium tertium, and Paraclostridium benzoelyticum. The limited number of species isolated confirms that in the gut the degradation of these glycoproteins results from cooperation and cross-feeding among several species exhibiting different metabolic capabilities.


Assuntos
Clostridium/isolamento & purificação , Enterobacteriaceae/isolamento & purificação , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/microbiologia , Mucinas/metabolismo , Fezes/microbiologia , Humanos , Mucosa Intestinal/metabolismo
18.
PLoS One ; 16(5): e0251791, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34014990

RESUMO

A Gram-negative, strictly anaerobic mucin-degrading bacterium, which we designated strain E39T, was isolated from the rumen epithelium of Korean cattle. The cells were non-motile and had a coccus morphology. Growth of strain E39T was observed at 30-45°C (optimum, 39°C), pH 6.5-8.5 (optimum, pH 7.5), and in the presence of 0.0-1.0% (w/v) NaCl (optimum, 0.0-0.5%). Strain E39T contained C16:0, C18:0, C18:1 ω9c, iso-C15:0, and anteiso-C15:0 as the major fatty acids. The major polar lipids were phosphatidylethanolamine, unidentified aminophospholipid, and unidentified lipids. The major respiratory isoprenoid quinones were MK-8 and MK-9. The major fermented end-products of mucin were acetate and succinate. The G+C content of the genomic DNA was 46.4 mol%. Strain E39T was most closely related to Alloprevotella rava 81/4-12T with an 87.3% 16S rRNA gene sequence similarity. On the basis of phenotypic, chemotaxonomic, and molecular properties, strain E39T represents a novel genus of the family Prevotellaceae; as such, the name Pseudoprevotella muciniphila gen. nov., sp. nov. is proposed. A functional annotation of the whole genome sequences of P. muciniphila E39T revealed that this bacterium has a putative mucin-degrading pathway and biosynthetic pathways of extracellular polymeric substances and virulence factors which enable bacteria to adhere to the epithelial cells and avoid the host's immune responses.


Assuntos
Aderência Bacteriana/fisiologia , Bacteroidetes , Mucosa Gástrica/microbiologia , Genoma Bacteriano , Mucinas/metabolismo , Rúmen/microbiologia , Animais , Bacteroidetes/classificação , Bacteroidetes/genética , Bacteroidetes/metabolismo , Bovinos , Mucosa Gástrica/metabolismo
19.
Front Immunol ; 12: 670279, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054843

RESUMO

The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 days. In particular, the study focused on the effect of these phytogenic compounds on the gut condition by analyzing the transcriptomic profiling (microarray analysis) and histological structure of the intestinal mucosa, as well as the histochemical properties of mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of the intestine from fish fed the control and MPLE diets. The gut transcripteractome revealed an expression profile that favored biological mechanisms associated to the 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic pathways. The histological analysis showed that the MPLE dietary supplementation promoted an increase in the number of intestinal goblet cells and modified the composition of mucins' glycoproteins stored in goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of transcriptomic and histological results showed that the evaluated MPLE from sage and lemon verbena is responsible for the maintenance of intestinal health, supporting gut homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds.


Assuntos
Imunidade nas Mucosas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Junções Intercelulares/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Extratos Vegetais/farmacologia , Salvia officinalis , Dourada , Linfócitos T/efeitos dos fármacos , Verbenaceae , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Fatores Imunológicos/isolamento & purificação , Junções Intercelulares/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Mucinas/metabolismo , Permeabilidade/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Salvia officinalis/química , Dourada/genética , Dourada/imunologia , Dourada/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Transcriptoma , Verbenaceae/química
20.
Front Immunol ; 12: 618653, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968020

RESUMO

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.


Assuntos
Anexina A1/metabolismo , Cálcio/metabolismo , Conjuntivite/metabolismo , Células Caliciformes/metabolismo , Mucinas/metabolismo , Animais , Anexina A1/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Conjuntivite/etiologia , Conjuntivite/patologia , Espaço Intracelular/metabolismo , Masculino , Fosfolipases A2/metabolismo , Ratos , Transdução de Sinais
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