Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility.

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

Bioactivity responses to changes in mucus-associated bacterial composition between healthy and bleached Porites lobata corals.

This study aims to investigate how bioactivities of the coral surface mucus layer (SML) respond to changes in mucus-associated bacterial communities between bleached and healthy Porites lobata corals in Nha Trang Bay, Vietnam. The findings suggested that significant shifts in the mucus-associated bacterial communities were related to changes in coral health states from bleached to healthy P. lobata colonies (p < 0.05), while bacterial compositions were not significantly different across seasons and locations (p > 0.05). Of which 8 genera, Shewanella, Fusibacter, Halodesulfovibrio, Marinifilum, Endozoicomonas, Litoribacillus, Algicola, and Vibrio were present only in the SML of bleached coral while absent in the SML of the healthy one. As compared with the bleached SML, the healthy SML demonstrated stronger antibacterial activity against a coral bleaching pathogen, V. coralliilyticus, higher antitumor activity against HCT116 cell accompanied with increased induction of cleaved PARP and accelerated cell nucleic apoptosis and cycle arrest at S and G2/M phases exhibiting several typical characteristics, cell shrinkage, lost cell contact, and apoptotic body formation. Moreover, putative compounds detected at 280 nm in the healthy SML were obviously higher than those in the bleached one, probably they could be bioactive molecules responsible for competitively exclusion of pathogens, Algicola and Vibrio, from the healthy SML.

Mass Spectrometry Analysis of Shark Skin Proteins.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage in which proteins play a key role. While proteins in the skin mucus layer of various common bony fish species have been explored, the proteins of shark skin mucus remain unexplored. In this pilot study, we examine the protein composition of the skin mucus in spiny dogfish sharks and chain catsharks through mass spectrometry (NanoLC-MS/MS). Overall, we identified 206 and 72 proteins in spiny dogfish (Squalus acanthias) and chain catsharks (Scyliorhinus retifer), respectively. Categorization showed that the proteins belonged to diverse biological processes and that most proteins were cellular albeit a significant minority were secreted, indicative of mucosal immune roles. The secreted proteins are reviewed in detail with emphasis on their immune potentials. Moreover, STRING protein-protein association network analysis showed that proteins of closely related shark species were more similar as compared to a more distantly related shark and a bony fish, although there were also significant overlaps. This study contributes to the growing field of molecular shark studies and provides a foundation for further research into the functional roles and potential human biomedical implications of shark skin mucus proteins.

Identification of Novel Glycans in the Mucus Layer of Shark and Skate Skin.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage. While the mucus layers of various bony fish species have been investigated, the composition and glycan profiles of shark skin mucus remain relatively unexplored. In this pilot study, we aimed to explore the structure and composition of shark skin mucus through histological analysis and glycan profiling. Histological examination of skin samples from Atlantic spiny dogfish (Squalus acanthias) sharks and chain catsharks (Scyliorhinus retifer) revealed distinct mucin-producing cells and a mucus layer, indicating the presence of a functional mucus layer similar to bony fish mucus albeit thinner. Glycan profiling using liquid chromatography-electrospray ionization tandem mass spectrometry unveiled a diverse repertoire of mostly O-glycans in the mucus of the two sharks as well as little skate (Leucoraja erinacea). Elasmobranch glycans differ significantly from bony fish, especially in being more sulfated, and some bear resemblance to human glycans, such as gastric mucin O-glycans and H blood group-type glycans. This study contributes to the concept of shark skin having unique properties and provides a foundation for further research into the functional roles and potential biomedical implications of shark skin mucus glycans.

Metagenomes of rectal swabs in larger, advanced stage cervical cancers have enhanced mucus degrading functionalities and distinct taxonomic structure.

BACKGROUND: Gut microbiome community composition differs between cervical cancer (CC) patients and healthy controls, and increased gut diversity is associated with improved outcomes after treatment. We proposed that functions of specific microbial species adjoining the mucus layer may directly impact the biology of CC. METHOD: Metagenomes of rectal swabs in 41 CC patients were examined by whole-genome shotgun sequencing to link taxonomic structures, molecular functions, and metabolic pathway to patient's clinical characteristics. RESULTS: Significant association of molecular functions encoded by the metagenomes was found with initial tumor size and stage. Profiling of the molecular function abundances and their distributions identified 2 microbial communities co-existing in each metagenome but having distinct metabolism and taxonomic structures. Community A (Clostridia and Proteobacteria predominant) was characterized by high activity of pathways involved in stress response, mucus glycan degradation and utilization of degradation byproducts. This community was prevalent in patients with larger, advanced stage tumors. Conversely, community B (Bacteroidia predominant) was characterized by fast growth, active oxidative phosphorylation, and production of vitamins. This community was prevalent in patients with smaller, early-stage tumors. CONCLUSIONS: In this study, enrichment of mucus degrading microbial communities in rectal metagenomes of CC patients was associated with larger, more advanced stage tumors.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Self-Emulsifying Drug Delivery System Enhances Tissue Distribution of Cinnamaldehyde by Altering the Properties of the Mucus Layer.

Oral delivery is considered the preferred route of administration due to its convenience and favorable compliance. However, this delivery often faces difficulties, such as poor solubility, limited absorption, and undesirable stability, especially for some volatile oils. The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) containing cinnamaldehyde (CA) to overcome these shortcomings. The CA-SEDDS were spherical and smooth with an average size of 14.96 ± 0.18 nm. Differential scanning calorimetry (DSC) and attenuated total reflection by Fourier transform infrared (ATR-FTIR) showed that CA has been successfully loaded into SEDDS. The accumulative release of CA-SEDDS (73.39%) was approximately 2.14-fold that of free CA when using simulated intestinal fluid as the release medium. A scanning electron microscope was used to observe the mucus network structure. Rheological tests found that CA-SEDDS can appropriately enhance the viscosity of the mucus system. We found from tissue distribution studies that CA was more widely distributed in various tissues in the CA-SEDDS group compared to the free CA group. The cinnamaldehyde and cinnamon acid also accumulated more in various tissues in the CA-SEDDS group than in the free CA group, especially in the kidney. These findings hinted that SEDDS exhibited lower irritation, good release, and penetration, which demonstrated great potential for utilizing CA. Our research supports the rational implications of SEDDS in delivering similar volatile substances by improving the solubility, mucus penetration, and stability, resulting in excellent clinical efficacy.

λ-carrageenan exacerbates Citrobacter rodentium-induced infectious colitis in mice by targeting gut microbiota and intestinal barrier integrity.

For nearly half a century, the scientific community has been unable to agree upon the safety profile of carrageenan (CGN), a ubiquitous food additive. Little is known about the mechanisms by which consumption of CGN aggravates the etiopathogenesis of murine colitis. However, analyses of gut microbiota and intestinal barrier integrity have provided a breakthrough in explaining the synergistic effect of CGN upon colitis. In Citrobacter rodentium-induced infectious murine colitis, inflammation and the clinical severity of gut tissue were aggravated in the presence of λ-CGN. Using fecal transplantation and germ-free mice experiments, we evaluated the role of intestinal microbiota on the pro-inflammatory effect of λ-CGN. Mice with high dietary λ-CGN consumption showed altered colonic microbiota composition that resulted in degradation of the colonic mucus layer, a raised fecal LPS level, and a decrease in the presence of bacterially derived short-chain fatty acids (SCFAs). Mucus layer defects and altered fecal LPS and SCFA levels could be reproduced in germ-free mice by fecal transplantation from CGN-H-fed mice, but not from germ-free CGN-H-fed mice. Our results confirm that λ-CGN may create an environment that favors inflammation by altering gut microbiota composition and gut bacterial metabolism. The present study provides evidence that the "gut microbiota-barrier axis" could be an alternative target for ameliorating the colitis promoting effect of λ-CGN.

Increased mucin-degrading bacteria by high protein diet leads to thinner mucus layer and aggravates experimental colitis.

BACKGROUND AND AIM: Westernized high-fat diet increases the risk for inflammatory bowel diseases (IBDs), yet with insufficient understanding of the role of high-protein diet. We aimed to identify the effect of high-protein diets from different dietary proteins (casein, whey protein, soy protein) on experimental colitis and its impact on microbiota, structure and function of colonic mucus layer. METHODS: Female BALB/c mice were fed by standard diet, high-casein diet (HCD), high whey protein diet or high soy protein diet for 4 weeks. The susceptibility of dextran sulfate sodium (DSS)-induced colitis in mice and thickness of colonic mucus layer were compared after different dietary interventions, associated with the identification of the reversal effect of broad-spectrum antibiotic intervention (0.5 g/L of vancomycin and 1 g/L of neomycin sulfate, metronidazole and ampicillin in drinking water). Further analysis was performed on the synthesis of mucin, microbiota and sialidase involved in degradation of mucus layer. RESULTS: High-protein diets aggravated acute DSS-induced colitis independent of protein composition, while broad-spectrum antibiotics reversed this effect. HCD significantly altered the composition of bacteria in the colonic mucus layer, especially Bacteroides thetaiotaomicron and total mucin-degrading bacteria; besides, it increased sialidase concentration and reduced the thickness of mucus layer. However, it exhibited no significant effect on the synthesis of Muc2. Broad-spectrum antibiotics decreased the abundance of mucin-degrading bacteria and sialidase concentration while increased the thickness of mucus layer. CONCLUSION: High-protein diet shifts microbial composition and thickness of colonic mucus layer, leading to the aggravation of acute DSS-induced colitis.

Research on the fate of polymeric nanoparticles in the process of the intestinal absorption based on model nanoparticles with various characteristics: size, surface charge and pro-hydrophobics.

BACKGROUND: The use of drug nanocarriers to encapsulate drugs for oral administration may become an important strategy in addressing the challenging oral absorption of some drugs. In this study-with the premise of controlling single variables-we prepared model nanoparticles with different particle sizes, surface charges, and surface hydrophobicity/hydrophilicity. The two key stages of intestinal nanoparticles (NPs) absorption-the intestinal mucus layer penetration stage and the trans-intestinal epithelial cell stage-were decoupled and analyzed. The intestinal absorption of each group of model NPs was then investigated. RESULTS: Differences in the behavioral trends of NPs in each stage of intestinal absorption were found to result from differences in particle properties. Small size, low-magnitude negative charge, and moderate hydrophilicity helped NPs pass through the small intestinal mucus layer more easily. Once through the mucus layer, an appropriate size, positive surface charge, and hydrophobic properties helped NPs complete the process of transintestinal epithelial cell transport. CONCLUSIONS: To achieve high drug bioavailability, the basic properties of the delivery system must be suitable for overcoming the physiological barrier of the gastrointestinal tract.

Zearalenone induces immuno-compromised status via TOR/NF/κB pathway and aggravates the spread of Aeromonas hydrophila to grass carp gut (Ctenopharyngodon idella).

The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0-2507 µg kg-1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of ß-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease.

Gut Microbiota and Dietary Factors as Modulators of the Mucus Layer in Inflammatory Bowel Disease.

The gastrointestinal tract is optimized to efficiently absorb nutrients and provide a competent barrier against a variety of lumen environmental compounds. Different regulatory mechanisms jointly collaborate to maintain intestinal homeostasis, but alterations in these mechanisms lead to a dysfunctional gastrointestinal barrier and are associated to several inflammatory conditions usually found in chronic pathologies such as inflammatory bowel disease (IBD). The gastrointestinal mucus, mostly composed of mucin glycoproteins, covers the epithelium and plays an essential role in digestive and barrier functions. However, its regulation is very dynamic and is still poorly understood. This review presents some aspects concerning the role of mucus in gut health and its alterations in IBD. In addition, the impact of gut microbiota and dietary compounds as environmental factors modulating the mucus layer is addressed. To date, studies have evidenced the impact of the three-way interplay between the microbiome, diet and the mucus layer on the gut barrier, host immune system and IBD. This review emphasizes the need to address current limitations on this topic, especially regarding the design of robust human trials and highlights the potential interest of improving our understanding of the regulation of the intestinal mucus barrier in IBD.

Oxyresveratrol Ameliorates Dextran Sulfate Sodium-Induced Colitis in Rats by Suppressing Inflammation.

Colitis causes destruction of the intestinal mucus layer and increases intestinal inflammation. The use of antioxidants and anti-inflammatory agents derived from natural sources has been recently highlighted as a new approach for the treatment of colitis. Oxyresveratrol (OXY) is an antioxidant known to have various beneficial effects on human health, such as anti-inflammatory, antibacterial activity, and antiviral activity. The aim of this study was to investigate the therapeutic effect of OXY in rats with dextran sulfate sodium (DSS)-induced acute colitis. OXY ameliorated DSS-induced colitis and repaired damaged intestinal mucosa. OXY downregulated the expression of pro-inflammatory cytokine genes (TNF-α, IL-6, and IL-1ß) and chemokine gene MCP-1, while promoting the production of anti-inflammatory cytokine IL-10. OXY treatment also suppressed inflammation via inhibiting cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in the colon, as well as the activity of myeloperoxidase (MPO). OXY exhibited anti-apoptotic effects, shifting the Bax/Bcl-2 balance. In conclusion, OXY might improve DSS-induced colitis by restoring the intestinal mucus layer and reducing inflammation within the intestine.

Utilization of Sodium Nitroprusside as an Intestinal Permeation Enhancer for Lipophilic Drug Absorption Improvement in the Rat Proximal Intestine.

As advanced synthetic technology has enabled drug candidate development with complex structure, resulting in low solubility and membrane permeability, the strategies to improve poorly absorbed drug bioavailability have attracted the attention of pharmaceutical companies. It has been demonstrated that nitric oxide (NO), a vital signaling molecule that plays an important role in various physiological systems, affects intestinal drug absorption. However, NO and its oxidants are directly toxic to the gastrointestinal tract, thereby limiting their potential clinical application as absorption enhancers. In this study, we show that sodium nitroprusside (SNP), an FDA-approved vasodilator, enhances the intestinal absorption of lipophilic drugs in the proximal parts of the small intestine in rats. The SNP pretreatment of the rat gastrointestinal sacs significantly increased griseofulvin and flurbiprofen permeation in the duodenum and jejunum but not in the ileum and colon. These SNP-related enhancement effects were attenuated by the co-pretreatment with dithiothreitol or c-PTIO, an NO scavenger. The permeation-enhancing effects were not observed in the case of antipyrine, theophylline, and propranolol in the duodenum and jejunum. Furthermore, the SNP treatment significantly increased acidic glycoprotein release from the mucosal layers specifically in the duodenum and jejunum but not in the ileum and colon. These results suggest that SNP increases lipophilic drug membrane permeability specifically in the proximal region of the small intestine through disruption of the mucosal layer.

Influence of skin wounds on the intestinal inflammatory response and barrier function: Protective role of dietary Shewanella putrefaciens SpPdp11 administration to gilthead seabream (Sparus aurata L.).

The effects of skin wounds on the intestinal barrier function and the beneficial effects of the dietary administration of Shewanella putrefaciens (known as SpPdp11) in gilthead seabream (Sparus aurata L.) were studied. Two replicates of fish were fed a commercial diet (control, CON) or CON diet enriched with 109 cfu g-1 SpPdp11 (SP diet) for 30 days. After this time, half of the fish were sampled, while the others were injured below the lateral line (wounded fish, W) and fed the same diets for an extra week before sampling (CON + W and SP + W groups). The intestinal histology and gene expression of different genes relevant for the intestinal barrier function were studied. The results showed that injured fish had a disordered enterocyte nucleus disposition, a more intense infiltration of mixed leucocytes and a thicker lamina propria in the intestine compared to the control fish. However, the fish in the SP + W group did not present these pathological symptoms in the intestine. No significant variations in the number of goblet cells were detected among the different experimental groups. Pro-inflammatory cytokines (colony-stimulating factor receptor 1, CSF1R, myeloperoxidase, MPO and interleukin-1ß, IL-1ß), mucins (intestinal mucin, IMUC and mucin 2, MUC2), and immunoglobulin T heavy chain (IGHT) were up-regulated, while tight junction protein occludin was down-regulated in the intestine from fish of the CON + W group. Similarly, the dietary administration of SpPdp11 markedly depressed the gene expression of pro-inflammatory cytokines, MUC2 and IGHT, but increased the gene expression of anti-inflammatory cytokine transforming growth factor-ß1 (TGF-ß1) and the tight junction proteins tricellulin and occluding after wounding. In brief, the skin wounds provoked an intestinal inflammatory response that included changes in the mucus layer and tight junction disruptions. Besides this, preventive administration of SpPdp11 alleviated the intestinal dysfunctions caused by skin wounds in gilthead seabream.

Porcine Gastric Mucin Triggers Toxin Production of Enteropathogenic Bacillus cereus.

Enteropathogenic Bacillus cereus causes foodborne infections due to the production of pore-forming enterotoxins in the intestine. Before that, spores have to be ingested, survive the stomach passage, and germinate. Thus, before reaching epithelial cells, B. cereus comes in contact with the intestinal mucus layer. In the present study, different aspects of this interaction were analyzed. Total RNA sequencing revealed major transcriptional changes of B. cereus strain F837/76 upon incubation with porcine gastric mucin (PGM), comprising genes encoding enterotoxins and further putative virulence factors, as well as proteins involved in adhesion to and degradation of mucin. Indeed, PGM was partially degraded by B. cereus via secreted, EDTA-sensitive proteases. The amount of enterotoxins detectable in culture media supplemented with PGM was also clearly increased. Tests of further strains revealed that enhancement of enterotoxin production upon contact with PGM is broadly distributed among B. cereus strains. Interestingly, evidence was found that PGM can also strain-specifically trigger germination of B. cereus spores and that vegetative cells actively move toward mucin. Overall, our data suggest that B. cereus is well adapted to the host environment due to massive transcriptome changes upon contact with PGM, attributing mucin an important and, thus far, neglected role in pathogenesis.

Optimizing microbiome sequencing for small intestinal aspirates: validation of novel techniques through the REIMAGINE study.

BACKGROUND: The human small intestine plays a central role in the processes of digestion and nutrient absorption. However, characterizations of the human gut microbiome have largely relied on stool samples, and the associated methodologies are ill-suited for the viscosity and low microbial biomass of small intestine samples. As part of the REIMAGINE study to examine the specific roles of the small bowel microbiome in human health and disease, this study aimed to develop and validate methodologies to optimize microbial analysis of the small intestine. RESULTS: Subjects undergoing esophagogastroduodenoscopy without colon preparation for standard of care were prospectively recruited, and ~ 2 ml samples of luminal fluid were obtained from the duodenum using a custom sterile aspiration catheter. Samples of duodenal aspirates were either untreated (DA-U, N = 127) or pretreated with dithiothreitol (DA-DTT, N = 101), then cultured on MacConkey agar for quantitation of aerobic gram-negative bacteria, typically from the class Gammaproteobacteria, and on blood agar for quantitation of anaerobic microorganisms. DA-DTT exhibited 2.86-fold greater anaerobic bacterial counts compared to DA-U (P = 0.0101), but were not statistically different on MacConkey agar. DNA isolation from DA-U (N = 112) and DA-DTT (N = 43) samples and library preparation for 16S rRNA gene sequencing were also performed using modified protocols. DA-DTT samples exhibited 3.81-fold higher DNA concentrations (P = 0.0014) and 4.18-fold higher 16S library concentrations (P < 0.0001) then DA-U samples. 16S rRNA gene sequencing revealed increases in the detected relative abundances of obligate and facultative anaerobes in DA-DTT samples, including increases in the genera Clostridium (false discovery rate (FDR) P = 4.38E-6), Enterococcus (FDR P = 2.57E-8), Fusobacterium (FDR P = 0.02) and Bacteroides (FDR P = 5.43E-9). Detected levels of Gram-negative enteropathogens from the phylum Proteobacteria, such as Klebsiella (FDR P = 2.73E-6) and Providencia (FDR P < 0.0001) (family Enterobacteriaceae) and Pseudomonas (family Pseudomonadaceae) (FDR P = 0.04), were also increased in DA-DTT samples. CONCLUSIONS: This study validates novel DTT-based methodology which optimizes microbial culture and 16S rRNA gene sequencing for the study of the small bowel microbiome. The microbial analyses indicate increased isolation of facultative and obligate anaerobes from the mucus layer using these novel techniques.

Mucins are Involved in the Intestinal Permeation of Lipophilic Drugs in the Proximal Region of Rat Small Intestine.

PURPOSE: Mucins are the principal glycoproteins in mucus and have been implicated in the limitation of intestinal drug absorption; however, the contribution of these molecules to intestinal drug absorption remains unclear. In this study, the relationship between the effect of the mucus layer on intestinal drug permeation and mucin distribution in different parts of the rat gastrointestinal tract was evaluated. METHODS: The intestinal permeability of various lipophilic drugs in rat small intestine was evaluated using the in vitro sac method. The expression profiles of mucin mRNA and proteins were evaluated by quantitative real-time RT-PCR and western blotting, respectively. RESULTS: The intestinal permeability of griseofulvin and antipyrine was enhanced by dithiothreitol (DTT) treatment in the proximal small intestine, such as duodenum and jejunum, but not in the distal regions. The mRNA expression analysis of rat mucin genes revealed that the intestinal expression of Muc5ac was considerably higher in the duodenum, whereas that of Muc1, Muc2, and Muc3A was gradually increased toward the lower intestine. In addition, Muc5ac protein was detected only in the luminal fluids from the proximal small intestine after DTT treatment. CONCLUSIONS: Mucus limits the intestinal permeation of lipophilic drugs in the rat proximal small intestine, in which Muc5ac may be involved.

Modeling the Effect of Mucin Binding in the Gut on Drug Delivery.

An important part the absorption, distribution, metabolism and excretion of an oral therapeutic is the flux rate of drug compound crossing the mucus lining of the gut. To understand this part of the absorption process, we develop a mathematical model of advection, diffusion and binding of drug compounds within the mucus layer of the intestines. Analysis of this model yields simple, measurable criteria for the successful mucin layer traversal of drug compound.