PM10 dysregulates epithelial barrier function in human corneal epithelial cells that is restored by antioxidant SKQ1.

Exposure to airborne particulate <10 µm (PM10) adversely affects the ocular surface. This study tested PM10 on epithelial barrier integrity in immortalized human corneal epithelial cells (HCE-2) and mouse cornea, and whether antioxidant SKQ1 is restorative. HCE-2 were exposed to 100 µg/ml PM10 ± SKQ1 for 24 h. An Electric Cell-Substrate Impedance Sensing (ECIS) system monitored the impact of PM10. RT-PCR, western blotting and immunofluorescence measured levels of barrier and associated proteins, stanniocalcin 2 (STC2), and a kit measured total calcium. In vivo, female C57BL/6 mice were exposed to either control air or PM10 (±SKQ1) in a whole-body exposure chamber, and barrier associated proteins tested. Tight junction and mucins proteins in the cornea were tested. In HCE-2, PM0 vs control significantly reduced mRNA and protein levels of tight junction and adherence proteins, and mucins. ECIS data demonstrated that PM10 vs control cells exhibited a significant decrease in epithelial barrier strength at 4000 Hz indicated by reduced impedance and resistance. PM10 also upregulated STC2 protein and total calcium levels. In vivo, PM10 vs control reduced zonula occludens 1 and mucins. SKQ1 pre-treatment reversed PM10 effects both in vitro and in vivo. In conclusion, PM10 exposure reduced tight junction and mucin proteins, and compromised the seal between cells in the corneal epithelium leading to decreased epithelial barrier strength. This effect was reversed by SKQ1. Since the corneal epithelium forms the first line of defense against air pollutants, including PM10, preserving its integrity using antioxidants such as SKQ1 is crucial in reducing the occurrence of ocular surface disorders.

The role of sialoglycans in modulating dendritic cell function and tumour immunity.

Dendritic cells (DCs) are crucial for initiating immune responses against tumours by presenting antigens to T cells. Glycosylation, particularly sialylation, plays a significant role in regulating cell functions, by modulating protein folding and signalling. This review aimed to provide a comprehensive overview of how sialic acids influence key aspects of DC biology, including maturation, migration, antigen presentation, and T cell interactions. Sialic acids influence DC endocytosis, affecting their ability to uptake and present antigens, while guiding their migration to lymph nodes and inflamed tissues. Removing sialic acids enhances DC-mediated antigen presentation to T cells, potentially boosting immune responses. Additionally, sialylated glycans on DCs modulate immune checkpoints, which can impact tumour immunity. Hypersialylation of tumour mucins further promotes immune evasion by interacting with DCs. Understanding the interplay between sialylation and DC functions offers promising avenues for enhancing cancer immunotherapy.

Milk Fat Globules: 2024 Updates.

Milk fat globules (MFGs) are a remarkable example of nature's ingenuity. Human milk (HM) carries contains 3-5% fat, 0.8-0.9% protein, 6.9-7.2% carbohydrate calculated as lactose, and 0.2% mineral constituents. Most of these nutrients are carried in these MFGs, which are composed of an energy-rich triacylglycerol (TAG) core surrounded by a triple membrane structure. The membrane contains polar lipids, specialized proteins, glycoproteins, and cholesterol. Each of these bioactive components serves important nutritional, immunological, neurological, and digestive functions. These MFGs are designed to release energy rapidly in the upper gastrointestinal tract and then persist for some time in the gut lumen so that the protective bioactive molecules are conveyed to the colon. These properties may shape the microbial colonization and innate immune properties of the developing gastrointestinal tract. Milk fat globules in milk from humans and ruminants may resemble in structure but there are considerable differences in size, profile, composition, and specific constituents. There are possibilities to not only enhance the nutritional composition in a goal-oriented fashion to correct specific deficiencies in the infant but also to use these fat globules as a nutraceutical in infants who require specific treatments. To mention a few, there might be possibilities in enhancing neurodevelopment, in defense against gastrointestinal and respiratory tract infections, improving insulin sensitivity, treating chronic inflammation, and altering plasma lipids. This review provides an overview of the composition, structure, and biological activities of the various components of the MFGs. We have assimilated research findings from our own laboratory with an extensive review of the literature utilizing key terms in multiple databases including PubMed, EMBASE, and Science Direct. To avoid bias in the identification of studies, keywords were short-listed a priori from anecdotal experience and PubMed's Medical Subject Heading (MeSH) thesaurus.

Spatiotemporal characteristics of eustachian tube development in C57BL/6 mice: Correlation between morphological and functional maturation.

BACKGROUND: The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due to its complexity and the challenges of in vitro manipulation. Historically, these challenges have hindered research into the morphology and function development of the ET. This study elucidates the spatiotemporal relationship of ET morpho-functional maturation in mice, identifying key periods and factors that lay the theoretical foundation for exploring the molecular mechanisms of ET-related diseases. RESULTS: We comprehensively characterized the ET development in C57BL/6 mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the development of cilia, secretory cells, surrounding glands, and macrophages. Immunostaining identified the localization and secretion patterns of the mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET function assessment tools, we evaluated the developmental features of ET mucociliary clearance and ventilation functions. CONCLUSIONS: In C57BL/6 mice, E16.5 marks a critical period for middle ear cavity and ET formation. Muc5b plays a foundational role during early stages, while Muc5ac enhances function in later stages. During P7-11, despite morphological maturity, ET function remains underdeveloped but continues to improve with growth.

Age-related decline in goblet cell numbers and mucin content of the human colon: Implications for lower bowel functions in the elderly.

BACKGROUND & AIMS: Older people experience a greater incidence of lower bowel disorders, including constipation. Causes can include factors associated with growing older, such as use of medications or disease, but compounded by degenerative changes within the bowel wall. It has been suggested that the latter is exacerbated by loss of an effective mucosal barrier to luminal contents. In human colon, little is known about the impact of ageing on key components of this barrier, namely the goblet cells and mucin content. METHODS: Changes in the number of goblet cells and density of mucin content were investigated in macroscopically normal human ascending (AC; n = 13) and descending (DC; n = 14) colon from elderly (≥ 67 years) and younger adults (60 years and below). Samples were serially sectioned and stained for haematoxylin and eosin to assess tissue morphology, and alcian blue periodic acid Schiff (ABPAS) and MUC-2 antibody to identify goblet cells producing mucins. New procedures in visualization and identification of goblet cells and mucin contents were employed to ensure unbiased counting and densitometric analysis. RESULTS: Compared with the younger adults, the numbers of goblet cells per crypt were significantly lower in the elderly AC (72 ± 1.2 vs 51 ± 0.5) and DC (75 ± 2.6 vs. 54 ± 1.9), although this reduction did not reach statistical significance when assessed per mucosal area (AC: P = 0.068; DC: P = 0.096). In both regions from the elderly, numerous empty vesicles (normally containing mucins) were observed, and some areas of epithelium were devoid of goblet cells. Thus, the density of mucin content per unit mucosal area were significantly reduced with age. CONCLUSIONS: Ageing could result in a reduced number of goblet cells and development of degenerative changes in mucin production. Together, these have implications for the mucus barrier function in the colon of elderly individuals.

Emerging role of mucins in antibody drug conjugates for ovarian cancer therapy.

Ovarian cancer stands as the deadliest gynecologic malignancy, responsible for nearly 65% of all gynecologic cancer-related deaths. The challenges in early detection and diagnosis, coupled with the widespread intraperitoneal spread of cancer cells and resistance to chemotherapy, contribute significantly to the high mortality rate of this disease. Due to the absence of specific symptoms and the lack of effective screening methods, most ovarian cancer cases are diagnosed at advanced stages. While chemotherapy is a common treatment, it often leads to tumor recurrence, necessitating further interventions. In recent years, antibody-drug conjugates (ADCs) have emerged as a valuable tool in targeted cancer therapy. These complex biotherapeutics combine an antibody that specifically targets tumor specific/associated antigen(s) with a high potency anti-cancer drug through a linker, offering a promising approach for ovarian cancer treatment. The identification of molecular targets in various human tumors has paved the way for the development of targeted therapies, with ADCs being at the forefront of this innovation. By delivering cytotoxic agents directly to tumors and metastatic lesions, ADCs show potential in managing chemo-resistant ovarian cancers. Mucins such as MUC16, MUC13, and MUC1 have shown significantly higher expression in ovarian tumors as compared to normal and/or benign samples, thus have become promising targets for ADC generation. While traditional markers are limited by their elevated levels in non-cancerous conditions, mucins offer a new possibility for targeted treatment in ovarian cancer. This review comprehensively described the potential of mucins for the generation of ADC therapy, highlighting their importance in the quest to improve the outcome of ovarian cancer patients.

Impact of saliva incorporation on the rheological properties of in vitro gastric contents formulated from sour cream.

Rheological properties of gastric contents depend on the food ingested, and on the volume and composition of secretions from the host, which may vary. This study investigates the impact of saliva regular incorporation in the stomach after a meal on the rheological properties of gastric contents, considering two levels of salivary flow (low = 0.5 and high = 1.5 mL/min). In vitro chymes were obtained by mixing sour cream, simulated gastric fluid, two different volumes of oral fluid (at-rest human saliva, SSF for Simulated Salivary Fluid or water) and adjusting pH at 3. Chymes samples were characterized at 37°C for their particle size and rheological properties. Overall, particle size distribution was not different between samples: incorporating a larger volume of saliva resulted in more heterogeneity, but the surface area moment D[3,2] and volume moment D[4,3] did not differ significantly with the oral fluid type. Shear viscosity of chyme samples was higher when saliva was incorporated, in comparison with water or SSF. In addition, as shown from data extracted at γ ̇ $$ \dot{\gamma} $$ = 20 s-1 the higher the fluid volume the lower the shear viscosity, which is attributed to a dilution effect. However, this dilution effect was attenuated in the case of saliva, most likely due to its composition in organic compounds (e.g., mucins) contributing to the rheological properties of this biological fluid. In these in vitro conditions, both saliva and the salivation rate had a significant but slight impact on the rheological properties of gastric contents (of the order of 1-5 mPa s at γ ̇ $$ \dot{\gamma} $$ = 20 s-1).

Analysis of Mucin-Domain Glycoproteins Using Mass Spectrometry.

Mucin-domain glycoproteins are characterized by their high density of glycosylated serine and threonine residues, which complicates their analysis by mass spectrometry. The dense glycosylation renders the protein backbone inaccessible to workhorse proteases like trypsin, the vast heterogeneity of glycosylation often results in ion suppression from unmodified peptides, and search algorithms struggle to confidently analyze and site-localize O-glycosites. We have made a number of advances to address these challenges, rendering mucinomics possible for the first time. Here, we summarize these contributions and provide a detailed protocol for mass spectrometric analysis of mucin-domain glycoproteins. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Enrichment of mucin-domain glycoproteins Basic Protocol 2: Enzymatic digestion of mucin-domain glycoprotein(s) Basic Protocol 3: Mass spectrometry data collection for O-glycopeptides Basic Protocol 4: Mass spectrometry data analysis of O-glycopeptides.

Potential New Inflammatory Markers in Bronchiectasis: A Literature Review.

Specific molecular and inflammatory endotypes have been identified for chronic respiratory disorders, including asthma and COPD (chronic obstructive pulmonary disease). These endotypes correspond with clinical aspects of disease, enabling targeted medicines to address certain pathophysiologic pathways, often referred to as "precision medicine". With respect to bronchiectasis, many comorbidities and underlying causes have been identified. Inflammatory endotypes have also been widely studied and reported. Additionally, several genes have been shown to affect disease progression. However, the lack of a clear classification has also hampered our understanding of the disease's natural course. The aim of this review is, thus, to summarize the current knowledge on biomarkers and actionable targets of this complex pathologic condition and to point out unmet needs, which are required in the design of effective diagnostic and therapeutic trials.

Infection and the Glycome─New Insights into Host Response.

Glycans play critical roles in the host-pathogen interactions leading to infection. However, we still understand very little about the dynamic nature of glycosylation in response to infection and its function in modulating host immunity. Many of the host proteins involved in immune defense are glycoproteins. Furthermore, the innate immune system recognizes glycans. The glycoform of a protein can impact proteolytic stability, receptor interactions, serum half-life, and other aspects. New, cutting-edge chemical biology tools are shedding light on the interplay between infection and the host glycome. In this review, we highlight new work on the importance of dynamic glycosylation of host proteins in the innate and adaptive immune pathways in response to infection. These include recent findings on altered glycoprofiles of mucins, complement components, and antibodies.

Quantification and site-specific analysis of co-occupied N- and O-glycopeptides.

Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.

The MUC2 Gene Product: Polymerisation and Post-Secretory Organisation-Current Models.

MUC2 mucin, the primary gel-forming component of intestinal mucus, is well researched and a model of polymerisation and post-secretory organisation has been published previously. Recently, several significant developments have been made which either introduce new ideas or challenge previous theories. New ideas include an overhaul of the MUC2 C-terminal globular structure which is proposed to harbour several previously unobserved domains, and include a site for an extra intermolecular disulphide bridge dimer between the cysteine 4379 of adjacent MUC2 C-termini. MUC2 polymers are also now thought to be secreted attached to the epithelial surface of goblet cells in the small intestine and removed following secretion via a metalloprotease meprin ß-mediated cleavage of the von Willebrand D2 domain of the N-terminus. It remains unclear whether MUC2 forms intermolecular dimers, trimers, or both, at the N-termini during polymerisation, with several articles supporting either trimer or dimer formation. The presence of a firm inner mucus layer in the small intestine is similarly unclear. Considering this recent research, this review proposes an update to the previous model of MUC2 polymerisation and secretion, considers conflicting theories and data, and highlights the importance of this research to the understanding of MUC2 mucus layers in health and disease.

Prognostic Impact of Mucin Expression in Curatively Resected Ampulla of Vater Cancer.

Introduction: Mucins play a pivotal role in epithelial carcinogenesis; however, their role remains elusive in ampulla of Vater (AoV) cancer, regardless of histological subtype. Therefore, we investigated the clinical significance of MUC1, MUC2, MUC5AC, and MUC6 expression in AoV cancer. Methods: Using samples from 68 patients with AoV cancer, we performed immunohistochemical staining for MUC1, MUC2, MUC5AC, and MUC6 using a tissue microarray. Subsequently, we analyzed their expression patterns in relation to clinicopathological parameters and patient outcomes. Results: Of the patients, 98.5% exhibited positive expression for MUC1, while MUC2, MUC5AC, and MUC6 were expressed in 44.1%, 47.1%, and 41.2% of the patients, respectively. Correlation analyses between mucin expression and clinicopathological factors revealed no significant associations, except between MUC5AC expression and N stage. Univariate analysis demonstrated significant associations between MUC5AC expression and overall survival (OS). Multivariate analysis further confirmed that MUC5AC expression was a significant predictor of OS, along with the N stage. However, MUC5AC expression was not meaningfully associated with recurrence-free survival (RFS). The patients positive for MUC5AC expression had a considerably shorter OS than those with negative expression. Conclusions: Our study provides insights into the clinical impact of mucins on AoV cancer, regardless of the histological subtype. Although MUC1 expression is universal, MUC5AC expression is a significant prognostic indicator that correlates with lymph node metastasis and poor OS. These results emphasize the possible utility of MUC5AC as a biomarker for extensive lymph node dissection and the prognostic evaluation of patients with AoV cancer.

The Acute Inflammatory Potential of Particles From the Echinococcus granulosus Laminated Layer Is Moderated by Its Calcium Inositol Hexakisphosphate Component.

Cystic echinococcosis is caused by the tissue-dwelling larva (hydatid) of Echinococcus granulosus sensu lato. A salient feature is that this larva is protected by the acellular laminated layer (LL). As the parasite grows, the LL sheds abundant particles that can accumulate in the parasite's vicinity. The potential of LL particles to induce inflammation in vivo has not been specifically analysed. It is not known how each of its two major components, namely highly glycosylated mucins and calcium inositol hexakisphosphate (InsP6) deposits, impacts inflammation induced by the LL as a whole. In this work, we show that LL particles injected intraperitoneally cause infiltration of eosinophils, neutrophils and monocytes/macrophages as well as the disappearance of resident (large peritoneal) macrophages. Strikingly, the absence of calcium InsP6 enhanced the recruitment of all the inflammatory cell types analysed. In contrast, oxidation of the mucin carbohydrates caused decreased recruitment of neutrophils. The carbohydrate-oxidised particles caused cell influx nonetheless, which may be explained by possible receptor-independent effects of LL particles on innate immune cells, as suggested by previous works from our group. In summary, LL particles can induce acute inflammatory cell recruitment partly dependent on its mucin glycans, and this recruitment is attenuated by the calcium InsP6 component.

Prognostic role of MUCIN family and its relationship with immune characteristics and tumor biology in diffuse-type gastric cancer.

The main component of O-glycoproteins, mucin, is known to play important roles in physiological conditions and oncogenic processes, particularly correlated with poor prognosis in different carcinomas. Diffuse-type gastric cancer (DGC) has long been associated with genomic stability and unfavorable clinical outcomes. To investigate further, we obtained clinical information and the RNA-seq data of the TCGA-STAD cohort. Through the use of unsupervised clustering methods and GSEA, we identified two distinct clusters, characterized by higher and lower expression of MUC2 and MUC20, denoted as cluster 1 and cluster 2, respectively. Subsequently, employing CIBERSORT, it was determined that cluster 2 exhibited a higher tumor mutation burden (TMB) and a greater abundance of CD8+ T cells and activated CD4+ memory T cells, in addition to immune checkpoints (ICPs). On the other hand, cluster 1 showed a lower TIDE score estimation, indicating a higher probability of tumor immune escape. Furthermore, overexpression of MUC15 and MUC20 was confirmed through qPCR and Western blotting, and their specific roles in mediating the epithelial-mesenchymal transition (EMT) process of GC cells (SNU484 and Hs746t) were validated via CCK-8 assay and wound healing assay in vitro. These findings highlight the potential prognostic value of MUC20 and offer insights into the prospects of immunotherapy for DGC by targeting MUC20.

Environmental and Metabolic Risk Factors Linked to Gallbladder Dysplasia.

Gallbladder disorders encompass a spectrum from congenital anomalies to inflammatory and neoplastic conditions, frequently requiring surgical intervention. Epithelial abnormalities like adenoma and metaplasia have the potential to progress to carcinoma, emphasizing the importance of histopathological assessment for early detection of malignancy. Gallbladder cancer (GBC) may be incidentally discovered during cholecystectomy for presumed benign conditions, underscoring the need for a thorough examination. However, the lack of clarity regarding the molecular mechanisms of GBC has impeded diagnostic and therapeutic advancements. Timely detection is crucial due to GBC's aggressive nature and poor prognosis. Chronic inflammation plays a central role in carcinogenesis, causing DNA damage and oncogenic alterations due to persistent insults. Inflammatory cytokines and microRNAs are among the various mediators contributing to this process. Gallbladder calcifications, particularly stippled ones, may signal malignancy and warrant preemptive removal. Molecular pathways involving mutations in oncogenes and tumor suppressor genes drive GBC pathogenesis, with proposed sequences such as gallstone-induced inflammation leading to carcinoma formation. Understanding these mechanisms, alongside evaluating mucin characteristics and gene mutations, can deepen comprehension of GBC's pathophysiology. This, in turn, facilitates the identification of high-risk individuals and the development of improved treatment strategies, ultimately enhancing patient outcomes. Thus, in this review, our aim has been to underscore the primary mechanisms underlying the development of gallbladder dysplasia and neoplasia.

Conserved cysteines prevent C-mannosylation of mucin Cys domains.

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucin CysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.

Tumor-associated carbohydrate antigens of MUC1 - Implication in cancer development.

Glycosylation of cancerous epithelial MUC1 protein is specifically altered in comparison to that which is presented by healthy cells. One of such changes is appearing tumor-associated carbohydrate antigens (TACAs) which are rare in normal tissues and are highly correlated with poor clinical outcomes and cancer progression. This review summarizes and describes the role of Tn, T antigens, their sialylated forms as well as fucosylated Lewis epitopes in different aspects of tumor development, progression, and metastasis. Finally, applications of MUC1 glycan epitopes as potential targets for therapeutic strategy of cancers are notified. One of the novelties of this review is presentation of TACAs as inherently connected with MUC1 mucin.

Stress-induced mucin 13 reductions drive intestinal microbiome shifts and despair behaviors.

Depression is a prevalent psychological condition with limited treatment options. While its etiology is multifactorial, both chronic stress and changes in microbiome composition are associated with disease pathology. Stress is known to induce microbiome dysbiosis, defined here as a change in microbial composition associated with a pathological condition. This state of dysbiosis is known to feedback on depressive symptoms. While studies have demonstrated that targeted restoration of the microbiome can alleviate depressive-like symptoms in mice, translating these findings to human patients has proven challenging due to the complexity of the human microbiome. As such, there is an urgent need to identify factors upstream of microbial dysbiosis. Here we investigate the role of mucin 13 as an upstream mediator of microbiome composition changes in the context of stress. Using a model of chronic stress, we show that the glycocalyx protein, mucin 13, is selectively reduced after psychological stress exposure. We further demonstrate that the reduction of Muc13 is mediated by the Hnf4 transcription factor family. Finally, we determine that deleting Muc13 is sufficient to drive microbiome shifts and despair behaviors. These findings shed light on the mechanisms behind stress-induced microbial changes and reveal a novel regulator of mucin 13 expression.

A mathematical model of competition between fiber and mucin degraders in the gut provides a possible explanation for mucus thinning.

The human gut microbiota relies on complex carbohydrates (glycans) for energy and growth, primarily dietary fiber and host-derived mucins. We introduce a mathematical model of a glycan generalist and a mucin specialist in a two-compartment chemostat model of the human colon. Our objective is to characterize the influence of dietary fiber and mucin supply on the abundance of mucin-degrading species within the gut ecosystem. Current mathematical gut reactor models that include the enzymatic degradation of glycans do not differentiate between glycan types and their degraders. The model we present distinguishes between a generalist that can degrade both dietary fiber and mucin, and a specialist species that can only degrade mucin. The integrity of the colonic mucus barrier is essential for overall human health and well-being, with the mucin specialist Akkermanisa muciniphila being associated with a healthy mucus layer. Competition, particularly between the specialist and generalists like Bacteroides thetaiotaomicron, may lead to mucus layer erosion, especially during periods of dietary fiber deprivation. Our model treats the colon as a gut reactor system, dividing it into two compartments that represent the lumen and the mucus of the gut, resulting in a complex system of ordinary differential equations with a large and uncertain parameter space. To understand the influence of model parameters on long-term behavior, we employ a random forest classifier, a supervised machine learning method. Additionally, a variance-based sensitivity analysis is utilized to determine the sensitivity of steady-state values to changes in model parameter inputs. By constructing this model, we can investigate the underlying mechanisms that control gut microbiota composition and function, free from confounding factors.