Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx ...specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of ∼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.
Microplastics (MPs) pollution becomes an emergent threat to the ecosystem, and its joint effect with organic contaminants will cause more severe consequences. Recently, MPs has been observed in human ...feces, suggesting that we are exposed to an uncertain danger. In this study, the joint effect of polyethylene microplastics particles (PEMPs) and Tetrabromobisphenol A (TBBPA) on human gut was explored through the simulation experiment in vitro with human cell Caco-2 and gut microbiota. The toxicity of TBBPA and PEMPs on Caco-2 human cells was considered by physiological and biochemical indexes such as cell proliferation, cell cycle, reactive oxygen species, lactate dehydrogenase release, and mitochondrial membrane potential. Besides, microbial community diversity, community structure, and function changes of gut microbiota were investigated using Illumina 16S rRNA gene MiSeq sequencing to reveal the influence of TBBPA and PEMPs on human gut microbiota. The results indicated that both PEMPs and TBBPA would deteriorate the status of Caco-2 cells, and TBBPA played a major role in it; meanwhile, PEMPs affected Caco-2 cells at high concentrations. Particularly, TBBPA and PEMPs exhibited a joint effect on Caco-2 cells to a certain degree. TBBPA selectivity inhibited the growth of gram-positive bacteria such as Enterococcus and Lactobacillus, contributing to the thriving of gram-negative bacteria such as Escherichia and Bacteroides. The existence of PEMPs would enhance the proportion of Clostridium, Bacteroides, and Escherichia. Community composition changed dramatically with the interference of PEMPs and TBBPA; this was undesirable to the healthy homeostasis of the human gut. PICRUSt analysis determined both PEMPs and TBBPA interfered with the metabolism pathways of gut microbiota. Hence, the threat of MPs and TBBPA to humans should arouse vigilance.
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•The effects of PEMPs and TBBPA on the Caco-2 cells were assessed comprehensively.•PEMPs co-exposure exacerbated the TBBPA cytotoxicity while its solo effect was weak.•The change of gut microbiota caused by PEMPs and TBBPA may break the gut homeostasis.•PICRUSt showed metabolic pathways of gut microbes being disturbed by PEMPs and TBBPA.
Food-intestine interaction study has always been a hot topic in food science and nutrition due to diverse physiological functions of intestine. Compared to expensive animal models with limited ...screening capabilities, the simple, reliable and highly reproducible intestinal cell models are widely used in food-intestine interaction study. There are many functional cell models used to simulate the intestine in vitro, among which the Caco-2 cell model is one of the most widely used and classical models. Recently years, the differentiated Caco-2 cell model has been greatly developed due to the development of various technologies, which not only overcomes the limitations of the traditional model, but also further broadens its application.
This review aims to overview the current applications of the differentiated Caco-2 cell model as a specialized model of intestinal cells in vitro, as well as new approaches solving the existing challenges of utilization, which can guide its future trends in interaction between food factors and the intestine.
Key findings and conclusions: With high flexibility, high repeatability and low cost, the differentiated Caco-2 cell model has been applied to a variety of intestinal studies including intestinal absorption, intestinal transport, intestinal metabolism, intestinal barrier, intestinal immunity and intestinal adhesion. Furthermore, future study should break limitations of traditional models with the help of automation, biochemistry, molecular biology and cells co-culture, so as to make it more closer to the internal environment without sacrificing its simplicity and reliability, and more suitable for cost-effective large-scale analysis of food-intestine interaction.
•Caco-2 monolayer express some structural and functional characteristics of intestine.•Caco-2 cell models applied to nutrition absorption, transport and metabolism in gut.•Effects of food factors on intestinal functions were study using Caco-2 cell models.•Molecular biology and automation are future trends to optimize Caco-2 cell models.
Positive effects of fermented foods consumption on humans have stimulated lots of research attention. In this study, we investigated the probiotic potentials, antagonistic activities, and safety ...properties of Lactobacillus brevis gp104 isolated from Iranian traditional cheese. The results showed that the strain had high resistance to acidic conditions, simulated gastric and intestinal fluid. L. brevis gp104 was able to assimilate cholesterol from the medium; 41% in medium without bile salts and 58% in medium with bile salts. The potential of this strain was relatively low in phytate hydrolyzation and 62.02% hydrophobicity, 40.2% auto-aggregation, and 48.3% co-aggregation were observed. The adhesion value of L. brevis gp104 to adenocarcinoma Caco-2 cells was 13.4% that was also confirmed by scanning electron microscopy (SEM). Antibacterial effect of L. brevis fg104 was imposed against pathogenic strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa PTCC 1707, Salmonella typhimurium PTCC 1609, and Staphylococcus aureus ATCC 25923) and the most sensitive strain S. aureus. L. brevis gp104 was able to compete (52%), inhibit (47%) and displace (21%) the adhesion of S. aureus to Caco-2 cells. L. brevis gp104 did not show haemolytic or DNase activity, which confirms its safety aspects. Therefore, L. brevis gp104 was demonstrated promising properties for its potential health benefits for its application as novel bio-therapeutic and bio-preservation agents.
•Lactobacillus brevis gp104 showed high adhesion, hydrophobicity, and auto-aggregation.•L. brevis gp104 inhibited the adhesion of pathogens to an intestinal cell line.•L. brevis gp104 showed high cholesterol assimilation in the media with and without bile salt.
Purpose
To identify conditions allowing the use of cell-based models for studies of drug absorption during
in vitro
lipolysis of lipid-based formulations (LBFs).
Methods
Caco-2 was selected as the ...cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer.
Results
Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion.
Conclusions
Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.
The tight junctions (TJs) and barrier function of the intestinal epithelium are highly sensitive to radiation. However, polyphenols can be used to reverse the effects of radiation. Here, we ...investigated the effects of hesperidin (hesperetin-7-rhamnoglucoside) on X-ray-induced intestinal barrier dysfunction in human epithelial Caco-2 monolayers. To examine whether hesperidin mitigated the effects of X-ray exposure (2 Gy), cell survival was evaluated and intestinal barrier function was assessed by measuring the transepithelial flux, apparent permeability coefficient (Papp), and barrier integrity. Hesperidin improved the survival of Caco-2 cell monolayers and attenuated X-ray exposure-induced intestinal barrier dysfunction. For fluorescein transport experiments, transepithelial flux and Papp of fluorescein in control group were significantly elevated by X-ray, but were restored to near control by 10 μM hesperidin pretreatment. Further, X-ray exposure decreased the barrier integrity and TJ interruption by reducing TJ-related proteins occludin and claudin-4, whereas cell monolayers pretreated with hesperidin before X-ray exposure were reinstated to control level. It was concluded that hesperidin treatment before X-ray exposure alleviated X-ray-induced intestinal barrier dysfunction through regulation of TJ-related proteins. These results indicate that hesperidin prevents and mitigates X-ray-induced intestinal barrier dysfunction.
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In nonclinical studies, models that can predict the metabolism of drug candidates by cytochrome P450 (CYP), including Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) are helpful. ...CYP3A4-overexpressing human cells have been used universally to evaluate whether CYP3A4 metabolizes drug-candidate compounds. However, CYP3A4-overexpressing human cell lines are problematic because their activity levels are lower than that of in vivo human CYP3A4. Heme plays a paramount role in CYP activity. The rate-limiting step in heme biosynthesis is the generation of 5-aminolevulinic acid (5-ALA). In this study, we examined whether treatment with 5-ALA to CYP3A4-POR-UGT1A1-CES2 knockin and CES1 knockout (genome-edited) Caco-2 cells enhances CYP3A4 activity. A 7-day 5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells without cytotoxicity. Moreover, consistent with the increase in intracellular heme content, 5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells. The results of this research are expected to be applied to pharmacokinetic studies using CYP-overexpressing human cells containing CYP3A4.
•5-ALA treatment increased intracellular heme levels in genome-edited Caco-2 cells.•Long-term 5-ALA treatment in genome-edited Caco-2 cells resulted in toxicity.•5-ALA treatment increased CYP3A4 activity in genome-edited Caco-2 cells.
High levels of persistent contaminants such as microplastics (MPs) and trace organic compounds (TrOCs) in the aquatic environment have become a major threat on the ecosystem and human health. While ...MP's role as a vector of environmental TrOCs is widely discussed in the literature, the corresponding implications of the interaction between these two compounds on human health (i.e., their joint toxic effect) have not been illustrated. Using a TrOCs model (Triclosan, TCS) and primary MPs (polystyrene microbeads), this work evaluates the sorption and desorption potential of TCS and MPs in simulated environmental and cellular conditions, respectively, and estimates the single and joint toxicity of these interactions toward human cells (Caco-2). Surface functionality of the microbeads highly increased their adsorption capacity of TCS, from 2.3 mg TCS for non−functionalized microbeads to 4.6 mg and 6.1 mg TCS per gram of microbeads for amino- and carboxyl-functionalized MPs, respectively. Using non-functionalized MPs, non-specific “hydrophobic-like” interactions and π-π interactions dominated the sorption mechanism of TCS; however, the addition of hydrogen interactions between functionalized microbeads and TCS increased the microbeads' overall sorption capacity. TCS was desorbed from both functionalized and non-functionalized MPs when changing from environmental conditions to cellular conditions. Desorption was found to be dependent on the matrix complexity and protein content as well as microbead functionality. Finally, toxicity tests suggested that while low concentrations of TCS and MPs (separately) have minor toxic effect toward Caco-2 cells, TCS-sorbed MPs at similar concentrations have an order of magnitude higher toxicity than pristine MPs, potentially associated with the close interaction of both MP and TCS with the cells. Overall, this study not only elucidates the role of MPs as a TrOC vector, but also demonstrates a realistic scenario in which co-presence of these environmental contaminants poses risks to the environment and human health.
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•This study examines the role of microplastics (MPs) as a contaminant vector to the human body.•Triclosan (TCS) sorbed onto polystyrene MPs of various surface functionalities.•Desorption of TCS from polystyrene MP occurred in cellular conditions.•Augmented joint toxicity toward Caco-2 cells was observed for TCS-sorbed MPs.•MP risk assessment must consider its inevitable interaction with co-existing contaminants.
The objective of this work was to evaluate in vitro bioaccessibility, intestinal absorption, antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of peptides from rainbow trout ...viscera hydrolysate (H). Rainbow trout Viscera (V) was hydrolyzed by Alcalase® 2.4L and a degree of hydrolysis (DH) of 44.8 ± 2.5% was achieved. Viscera and its hydrolysate were subjected to simulated gastrointestinal digestion (SGID) and intestinal absorption across Caco-2/TC7 cell monolayers. After the hydrolysis with Alcalase® 2.4L and the SGID of V, the species between 60.6 kDa and 13.0 kDa were decreased, causing an increase in species less than 6.51 kDa. The SGID of H did not modify the oxygen radical absorbance capacity (ORAC) or ACE inhibitory values but caused a significant decrease in the hydroxyl radical antioxidant capacity (HORAC) (30.2%). It also produced an increase in ABTS radical cation (ABTS assay) scavenging activity and ferric reducing antioxidant power (FRAP) (9.46% and 20.2%, respectively). Bioactive peptides in H were stable after SGID and they were partially able to cross Caco-2/TC7 cell monolayer, which demonstrates their possible intestinal absorption and their potential to act inside the organism.
-Taking advantage of rainbow trout viscera (V) using Alcalase (A).-Simulated gastrointestinal digestion of V generates bioactive peptides.-The previous hydrolysis with A enhances bioactivity and bioaccessibility.-The previous hydrolysis with A improves the intestinal absorption of peptides.
Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that ...assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.
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