Human Colonic Cell Line Research Articles

315. Modeling Strain-Dependent Variability in Carbapenem-Resistant Klebsiella pneumoniae Colonization in the Malnourished Host

Abstract Background Particular strains of Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) have achieved global dominance extending to developing countries where malnutrition is a potential risk factor for infection. Bacteria and host properties that determine colonization dynamics in this context are not understood. We hypothesized that strain variability in host cell adhesion and nutrient-utilization in vitro predicts in vivo intestinal colonization by CR-Kp in malnourished hosts.Figure 1:Strain-dependent cell adhesion by carbapenem-resistant Klebsiella pneumoniae(A) Adhesion to Colo-201 cells and/or (B) Caco-2 cells as percentage of bacteria recovered after co-culturing then washing off non-adherent cells. Bacterial quantification performed by culturing on MacConkey agar + Meropenem. Strains of interest indicated with arrows and colored-bars. All experiments showing results of 3 replicates. Methods Patient-derived isolates of CR-Kp were subject to in vitro assays testing adhesion to human colon cell lines. Select isolates were further tested for in vitro growth in minimal media with limiting protein availability. These isolates were additionally inoculated via orogastric lavage into C57BL/6 mice (n=6 per group) on a nutrient replete control diet (CD) or isocaloric protein-deficient diet (PD) and intestinal colonization was quantified with selective culture of feces and intestinal specimens on MacConkey agar containing meropenem.Figure 2:Nutrient limited growth of carbapenem-resistant Klebsiella pneumoniaeGrowth in vitro determined by serial absorption measurement of bacteria culture under different nutrient conditions. M9 minimal medium (M9mm; M9 solution +0.4% glucose) was used with or without casein peptone (0.16% or 0.016%) as a protein/amino acid source. Results Across >100 screened human isolates of CR-Kp, adhesion to human colon cell lines ranged from < 1% to 30% (Figure 1). Three high-adhesion isolates (Kp175, Kp4493 and Kp4605) and one low-adhesion isolate (Kp4404) were selected for further investigation (Table 1). All three high-adhesion isolates grew to varying degrees in protein-limited conditions, while Kp4404 required a protein source for growth (Figure 2). Mice inoculated with CR-Kp were stably colonized only if they were fed a PD rather than CD diet. Kp175 and Kp4605 demonstrated the highest fecal shedding and intestinal burden (102-104 CFU per mg), which was significantly greater on a PD versus CD diet (p< 0.05 by Mann-Whitney test). In contrast, Kp4493 and Kp4404 inconsistently colonized mice regardless of diet (Figure 3).Figure 3:Diet-dependent intestinal colonization by carbapenem-resistant Klebsiella pneumoniae(A) 3-4 wk old C57BL/6 male mice initiated on nutrient-replete control diet (CD) or isocaloric protein-deficient diet (PD) and inoculated via orogastric lavage with 10^6 CFU of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). (B) Weight measured over time along with bacterial burden in (C) fecal pellets and (D) intestinal tissue by culturing on MacConkey agar + Meropenem. 6 animals used per condition. * = p<0.05 by Mann Whitney Test. Conclusion Our findings suggest a wide variability in intestinal cell adhesion and nutrient utilization by clinical isolates of CR-Kp. These in vitro properties incompletely predict in vivo colonization in a mouse model. Notably, host dietary deficiency promotes intestinal colonization by certain nutritionally resilient CR-Kp isolates, with potential implications for the global spread of high-risk strains in malnourished populations.Table 1:Genotypic, clinical and phenotypic characteristics of carbapenem-resistant Klebsiella pneumoniae Adhesion percentage express as mean (standard deviation). Disclosures David van Duin, MD, PhD, Merck: Advisor/Consultant|Merck: Grant/Research Support|Pfizer: Advisor/Consultant|Qpex: Advisor/Consultant|Roche: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Grant/Research Support

P0135 Effect of human recombinant IL-33 on fibrosis markers in colonic fibroblasts

Abstract Background IBD is frequently complicated by intestinal fibrosis and strictures formation, occurring in more than 50% of Crohn’s disease (CD) and 5% of ulcerative colitis. Intestinal fibrosis leads to surgery in CD and strictures frequently recur leading to repeated surgeries1. There is currently no specific therapy to prevent or inhibit intestinal fibrosis and this therefore constitutes a major treatment challenge. Intestinal fibrosis is characterized by excessive deposition of extracellular matrix (ECM) synthesized by intestinal myofibroblasts1. Interleukin-33 (IL-33) is a pleiotropic nuclear cytokine involved in immunoregulation and tissue repair2. Following cellular injury, IL-33 is released by epithelial, endothelial or fibroblast cells. Binding to the ST2 (suppressor of tumorigenicity 2) receptor, it induces translocation of the transcription factor NF-□□□□B, which regulates the expression of pro-inflammatory genes2. IBD patients have increased colonic expression of IL-33 and ST2 in epithelial cells compared to control patients3. Neutralization of IL-33 enables the repair of lung epithelial cells by limiting the release of pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α in a mouse model of pulmonary epithelial lesions4. We hypothesized that IL-33 may be involved in IBD-associated intestinal fibrosis. We therefore aimed to understand the role of IL-33 in a human colonic fibroblast cell line (CCD-18Co). Methods First, we measured IL-33 and ST2 mRNA expression in CCD-18Co in response to TGF-ß1 (10 ng/mL) and/or TNF-ɑ (100 ng/mL) for 24h. Then, CCD-18Co were induced by increasing doses of IL-33 treatment (10, 50, 100 ng/mL) for 24h and inflammatory and fibrosis markers were measured. Results TGF-ß1 significantly induced mRNA levels encoding ECM-associated proteins such as α-SMA (***p<0.001) or COL1A1 (*p<0.05, Figure 1).Treatment with TGF-ß1 and/or TNF-α had no effect on the expression of mRNAs encoding IL-33 and ST2 in CCD-18Co in our experimental conditions. IL-33 treatment significantly increased mRNA levels for IL-1ß and ECM-associated proteins (COL3A1, CTGF and VIM) (*p<0.05, Figure 1) in CCD-18Co but these effects are abolished in presence of TGF-ß1 and/or TNF-α treatment. Conclusion Treatment with TGF-ß1 and/or TNF-α had no significative impact on IL-33 mRNA levels in the CCD-18Co in our experimental conditions. IL-33 treatment increased ECM-associated proteins in CCD-18Co line under basal conditions but not in TGF-ß1 and/or TNF-α conditions.

P0097 STING pathway drives cellular tryptophan degradation and pro-inflammatory responses in intestinal fibroblasts

Abstract Background Tryptophan metabolism is severely disrupted in IBD. Pro-inflammatory cytokines activate the first rate-limiting enzyme IDO1 leading to tryptophan degradation through the kynurenine pathway. Although Trp degradation typically support de novo NAD synthesis via KP activation, evidence suggests a metabolic block at the downstream enzyme QPRT in IBD. STING has been described as inducing the secretion of type I interferons and other inflammatory cytokines, suggesting its important role in regulating intestinal homeostasis. Several studies have reported an association between STING-mediated IDO1 activity and its role in promoting tumour immune evasion. However, the relationship between STING pathway and tryptophan metabolism in intestine has not be understood. This study aims to investigate the effect of STING signaling on tryptophan degradation and explore how the metabolic blockage in tryptophan-NAD axis may influence STING-mediated immune response. Methods To investigate the STING pathway's role in tryptophan metabolism in intestine, we utilized human colonic fibroblast cell line CCD-18co. Quantitative RT-PCR and Western blot were employed to measure mRNA and protein expression. Tryptophan metabolism was quantified via LC-MS. siRNA was performed to mediate QPRT knockdown. Intracellular NAD level were assessed by NAD-glo assay. Seahorse assay was conducted to evaluate mitochondrial function. Results We screened a range of established IDO1-inducing cytokines (IFNγ, TNFα, etc.) and compared their effects on IDO1 expression with STING agonists diABZI, dsDNA and 2’3’cGAMP. STING agonists induced significantly higher IDO1 expression than any of the cytokines tested. While cultivation of in presence of Anifrolumab (type I IFN receptor antagonist) and Tofacitinib (pan-JAK inhibitor) strongly inhibited STING-induced IDO1 upregulation. LC-MS showed upregulation of IDO1 drive tryptophan into kynurenine pathway, which was again inhibited by Anifrolumab and Tofacitinib. To mimic the reduced QPRT expression in IBD, we performed QPRT knockdown. QPRT deficiency led to STING pathway overactivation upon diABZI treatment, resulting in elevated expression of pro-inflammatory cytokines and phosphorylation of STING and TBK1. QPRT-deficient cells exhibited reduced intracellular NAD level and impaired mitochondrial function. Supplementation with the nicotinamide riboside restored NAD levels, which effectively reduced the elevated cytokine expression caused by QPRT knockdown. Conclusion Our data provide a novel link of how STING activation in intestinal fibroblasts drives tryptophan degradation via KP pathway. Additionally, QPRT-related metabolic blockage enhance STING-mediated pro-inflammatory response, which can be mitigated by NAD precursor supplementation

Exploration of the cytotoxic and microtubule disruption potential of novel imidazo[1,5-a]pyridine-based chalcones.

In continuation of our efforts to develop new anticancer compounds, a new series of imidazo[1,5-a]pyridine-chalcone derivatives was designed, synthesized, characterized, and evaluated for its cytotoxicity against five human cancer cell lines, i.e., breast (MDA-MB-231), colon (RKO), bone (Mg-63), prostate (PC-3), and liver (HepG2) cell lines, as well as a normal cell line (HEK). Among the synthesized compounds, two exhibited promising cytotoxicity against the MDA-MB-231 cell line with IC50 values of 4.23 ± 0.25 μM and 3.26 ± 0.56 μM. We also studied apoptotic induction of the compounds using annexin V-FITC/PI staining, and ROS-mediated mitochondrial damage was elucidated using DCFDA, followed by JC-1 staining. The potential activity of the compounds was further confirmed by immuno-fluorescence and molecular docking studies, which revealed the anticancer activity of active compounds through binding and microtubule disruption.

Genomic characteristics and virulence of common but overlooked Yersinia intermedia, Y. frederiksenii, and Y. kristensenii in food.

Yersinia intermedia, Y. frederiksenii, and Y. kristensenii are a group of pathogens that are commonly found in food and are often overlooked in terms of their pathogenic potential. This study conducted a systematic and comprehensive genomic analysis of 114 Y. intermedia genomes, 20 Y. frederiksenii genomes, and 65 Y. kristensenii genomes from public database and our previous study. The results showed that these species were most frequently detected in Europe (56.28%, 112/199), followed by in Asia (20.6%, 41/199). Additionally, 33.17% (66/199) genomes were isolated from food. Y. intermedia were grouped into Bayesian analysis of population structure (Baps) groups 3 and 4, demonstrating significant genomic diversity. This species has a high proportion of accessory genes (79.43%), approximately 50% of which have unknown functions, indicating a high degree of genomic plasticity. The three species carried a large number of mobile genetic elements (MGEs), including plasmids such as ColRNAI_1, ColE10_1, Col440II_1, Col440I_1, and Col (Ye4449) _1; insertion sequences (ISs) like MITEYpe1, MITEEc1, and IS1635; genomic islands (GIs); and prophages. In Y. intermedia, the following antibiotics resistance genes (ARGs) were detected: qnrD1 in 3.51% (4/114), aph(3')-Ia in 2.63% (3/114), blaA in 1.75% (2/114), and catA1, vat(F), and tet(C) each in 0.88% (1/114). In Y. kristensenii, vat(F) was present in 98.46% (64/65), blaTEM-116 in 7.69% (5/65), and aph(3')-Ia in 1.54% (1/65). However, only one Y. frederiksenii genome carried vat(F). There were differences in the virulence gene composition of the three species, with Y. kristensenii having the highest number of virulence genes, particularly its complete cytotoxic genes (yaxA and yaxB) and flagellar motor proteins genes (motA and motB). The pathogenic mechanisms of Y. intermedia and Y. frederiksenii were more similar, especially in the carriage of O-antigen related genes. Y. frederiksenii's unique mechanisms also include the yapC gene, which encodes the autotransporter protein YapC from Y. pestis. After co-cultured with human colonic epithelial cell lines Caco-2 and HT-29, Y. intermedia and Y. kristensenii demonstrated different adhesive and invasive capabilities, particularly the Y. intermedia strain y7, which exhibited stronger adhesion and invasion in both cell lines. In strains y118 and y119 of Y. intermedia, an Arg378del mutation in the UreC protein was identified, resulting in the loss of urease activity. Therefore, this study revealed the pathogenic potential of Y. intermedia, Y. frederiksenii, and Y. kristensenii. Future research should focus on identifying their unknown virulence genes and strengthening public food safety measures to mitigate potential risks.

Bile acid–induced metabolic changes in the colon promote Enterobacteriaceae expansion and associate with dysbiosis in Crohn’s disease

Bile acids (BAs) affect the growth of potentially pathogenic commensals, including those from the Enterobacteriaceae family, which are frequently overrepresented in inflammatory bowel disease (IBD). BAs are normally reabsorbed in the ileum for recycling and are often increased in the colonic lumina of patients with IBD, including those with Crohn’s disease (CD). Here, we investigated the influence of BAs on gut colonization by Enterobacteriaceae. We found increased abundance of Enterobacteriaceae in the colonic mucosae of patients with CD with a concomitant decrease in the transporters that resorb BAs in the ileum. The increase in Enterobacteriaceae colonization was greater in the colons of patients who had undergone terminal ileum resection compared with those with intact ileum, leading us to hypothesize that BAs promote intestinal colonization by Enterobacteriaceae. Exposure of human colonic epithelial cell lines to BAs reduced mitochondrial respiration, increased oxygen availability, and enhanced the epithelial adherence of several Enterobacteriaceae members. In a publicly available human dataset, mucosal Enterobacteriaceae was negatively associated with the expression of genes related to mitochondrial function. In a murine model, increased intestinal BA availability enhanced colonization by Escherichia coli in a manner that depended on bacterial respiration. Together, our findings demonstrate that BAs reduce mitochondrial respiration in the colon, leading to an increase in oxygen availability that facilitates Enterobacteriaceae colonization. This identification of BAs as facilitators of host-commensal interactions may be relevant to multiple intestinal diseases.

Preparation and Characterization of Copper Oxide-Chitosan Nanocomposite for Colon-Targeted Drug Delivery System

This work aimed at synthesizing the chitosan-based nanocomposite for concurrent diagnosis of colon tumors and targeted drug shipping with minimum toxicity. CuO-chitosan nanocomposite was developed using CuO nanoparticles (NPs), chitosan raw materials and 1% acetic acid. FE-SEM/EDX, TEM, XRD, DLSUV-Vis, FT-IR, as well as fluorescent emission spectrum methods were used to investigate the characteristics of nanocomposite gained throughout distinct levels. Bovine serum albumin (BSA) protein release rate was estimated. We investigated the antibacterial functioning of the nanocomposite against different bacteria types (like S. aureus, S. typhi, E. coli, L. monocytogene and P. aeruginosa), which can affect in causing tense colonic diseases. Qualitative and quantitative surveys were performed. The impact of the nanocomposite on cancerous and normal human colon cell lines, specifically HCT-116 and SW-948, was examined using the MTT assay. Eventually, loading BSA onto the CuO–chitosan nanocomposite enhances its potential as a drug carrier for treating colon cancer, causing minimal harm to surrounding normal cells. Additionally, the CuO-chitosan nanocomposite demonstrated significant antibacterial activity against Escherichia coli, a key pathogen associated with Crohn’s disease and ulcerative colitis.

EGCG inhibits the oxidative damage induced by TiO2-NPs in human colon cell lines.

To assess the protective effects of (-)-Epigallocatechin-3-gallate (EGCG), a natural antioxidant, against cellular oxidative damage induced by titanium dioxide nanoparticles (TiO2-NPs), Human Colon cells NCM460 and Colon Cancer cells SW620 were selected for this study. The cells were divided into three groups: control group, TiO2-NPs (80 μg/mL) exposure group, and EGCG (20 μmol/L)+TiO2-NPs (80 μg/mL) co-exposure group. The study evaluated the precipitation rate of TiO2-NPs influenced by EGCG in a cell-free system. It also measured the levels of ROS, MDA, and total antioxidant capacity in the cells of each group. The uptake of TiO2-NPs by the cells was assessed using the SSCe/SSC0 ratio, and genome instability was evaluated. The results demonstrated that the addition of 20 μmol/L EGCG to the system resulted in greater sedimentation of TiO2-NPs compared to TiO2-NPs alone (P<0.05). The SSCe/SSC0 values in the co-exposure group were significantly lower than those in the TiO2-NPs alone group (P<0.001). TiO2-NPs induced a higher oxidative stress index in the cells (P<0.001), while the co-exposure group exhibited a lower REDOX index (P<0.001). The combination of EGCG and TiO2-NPs did not significantly affect genome instability in either cell line. Importantly, EGCG showed a certain inhibitory effect on oxidative damage to colon cells induced by TiO2-NPs, with no significant difference observed between normal and cancer cells in terms of this protective effect. Conducting a comprehensive investigation into the interaction mechanism between EGCG and TiO2-NPs is crucial for establishing a scientific foundation that can guide the optimal utilization of the antioxidant properties of EGCG to mitigate the toxicity associated with TiO2-NPs.

CGAS regulates metabolic reprogramming independently of STING pathway in colorectal cancer

BackgroundCyclic GMP-AMP synthase (cGAS) is widely acknowledged for detecting cytosolic chromatin fragments and triggering innate immune responses through the production of the second messenger cGAMP, which subsequently activates the adaptor protein STING. However, the role of cGAS in regulating metabolic reprogramming independently of STING activation has not yet been explored. MethodsGene set enrichment pathway analysis (GSEA) based on TCGA transcriptomics, combined with Seahorse metabolic analysis of CRC cell lines and human normal colonic mucosa cell line FHC, was performed to profile the metabolic features in CRC. cGAS doxycycline- (dox) inducible knockout (iKO) CRC sublines were generated to investigate the role of cGAS in CRC. Transcriptome and proteome data from COAD cohorts were utilized to evaluate the RNA and protein expression levels of cGAS in COAD tissues and normal colon tissues. Overall survival information of patients with COAD was used to evaluate the prognostic value of cGAS expression. Colony formation assays were conducted to evaluate the clonogenicity of CRC cells under different situations. Flow cytometry detecting the signal of fluorogenic reactive oxygen species (ROS) probes was performed to evaluate the total cellular and mitochondrial oxidative stress level in CRC cells. A propidium iodide (PI) staining assay was used to evaluate the cell death level in CRC cells. Quantitative PCR (qPCR) was conducted to detect the RNA level of STING pathway downstream target genes. Mass spectrometry was used for the identification of novel binding partners of cGAS in CRC cells. Co-immunoprecipitation (co-IP) was conducted to confirm the interaction between cGAS and NDUFA4L2. ResultsBy integrating metabolic pathway analysis based on TCGA transcriptomics with Seahorse metabolic analysis of a panel CRC cell lines and the human normal colonic mucosa cell line FHC, we demonstrated that CRC cells exhibit typical characteristics of metabolic reprogramming, characterized by a shift from oxidative phosphorylation (OXPHOS) to glycolysis. We found that cGAS is critical for CRC cells to maintain this metabolic switch. Specifically, the suppression of cGAS through siRNA-mediated knockdown or doxycycline-inducible knockout reversed this metabolic switch, resulting in increased OXPHOS activity, elevated production of OXPHOS byproduct reactive oxygen species (ROS), and consequently caused oxidative stress. This disruption induced oxidative stress, ultimately resulting in cell death and reduced cell viability. Moreover, significant upregulation of cGAS in CRC tissues and cell lines and its association with poor prognosis in CRC patients was observed. Subsequently, we demonstrated that the role of cGAS in regulating metabolic reprogramming does not rely on the canonical cGAS-STING pathway. Co-immunoprecipitation combined with mass spectrometry identified NDUFA4L2 as a novel interactor of cGAS. Subsequent functional experiments, including mitochondrial respiration and oxidative stress assays, demonstrated that cGAS plays a crucial role in sustaining elevated levels of NDUFA4L2 protein expression. The increased expression of NDUFA4L2 is essential for cGAS-mediated regulation of metabolic reprogramming and cell survival in CRC cells. ConclusioncGAS regulates metabolic reprogramming and promotes cell survival in CRC cells through its interaction with NDUFA4L2, independently of the canonical cGAS-STING pathway.

Enzymatic Acrolein Production System and Its Impact on Human Cells.

Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 μM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.

Effect of Milk Protein-Polyphenol Conjugate on the Regulation of GLP-1 Hormone.

Modern functional foods are designed to provide health benefits beyond basic nutrition. They are enriched with bioactive ingredients like probiotics, vitamins, minerals, and antioxidants. These foods support overall health, enhance immune function, and help prevent chronic diseases. Milk proteins and tea are known to influence satiety and regulate body weight. Studies have shown that green tea polyphenols, namely, (-)-epigallocatechin gallate (EGCG), and whey proteins, predominantly lactoferrin (LF) from milk, play a role in regulating satiety. This study aims to investigate the effect of conjugating EGCG with apo-lactoferrin (Apo-LF) and assessing these effects on satiety through monitoring glucagon-like peptide-1 (GLP-1) regulation in a human colon (NCI-H716) cell line. Apo-LF-EGCG conjugates were synthesized and characterized in terms of structural and functional properties. The effect on GLP-1 regulation was assessed by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) to monitor gene and protein expressions, respectively. The results revealed that the protein-polyphenol interaction occurs through the complex formation of hydrogen bonds at the O-H and carbonyl groups of EGCG. The conjugates also showed a significant up-regulation of gene and protein expression levels of GLP-1 while also preventing EGCG from degradation, thereby preserving its antioxidant properties. The Apo-LF-EGCG conjugates increase satiety via increasing GLP-1 secretion in human colon cells while simultaneously retaining the antioxidant properties of EGCG. Therefore, these conjugates show potential for use as dietary supplements to enhance satiety.

Establishment of a stress granule reporter system for evaluating in vitro colon toxicity

ABSTRACT Exposure to toxic molecules from food or oral medications induces toxicity in colon cells that cause various human diseases; however, in vitro monitoring systems for colon cell toxicity are not well established. Stress granules are nonmembranous foci that form in cells exposed to cellular stress. When cells sense toxic environments, they acutely and systemically promote stress granule formation, with Ras GTPase-activating protein-binding protein 1 (G3BP1) acting as a core component to protect their mRNA from abnormal degradation. Here, we knocked in green fluorescent protein (GFP)-coding sequences into the C-terminal region of the G3BP1 gene in a human colon cell line through CRISPR-Cas9-mediated homologous recombination and confirmed the formation of stress granules with the G3BP1-GFP protein in these cells under cellular stress exposure. We demonstrated the formation and dissociation of stress granules in G3BP1-GFP expressing colon cells through real-time monitoring using a fluorescence microscope. Furthermore, we validated the toxicity monitoring system in the established colon cell line by observing stress granule formation following exposure to dihydrocapsaicin, bisphenol A, and sorbitol. Taken together, we established a stress granule reporter system in a colon cell line, providing a novel assessment for the real-time monitoring of colon toxicity in response to various chemicals.

New bioactive titanium(IV) derivatives with their DFT, molecular docking, DNA/BSA interaction, antioxidant and in-vitro investigations

A set of four new metallacyclic complexes titanium(IV) associated with Schiff bases (SBs) (1a- 1d) obtained by the specific reactions of β-diketones such as acetylacetone (acacH) and benzyl acetone (bzacH) and aminophenol derivatives while treating Ti(OPri)4 and these SBs individually in 1:2 stoichiometry under 8 h reflux condition in anhydrous benzene to afford as reddish powder (2a-2d). These products have been purified in repeated washings of dry n-hexane afterwards, subjected to characterization by suitable spectroscopic techniques (NMR, IR, UV–Vis and HRMS). These hexacoordinated complexes appeared as distorted octahedral geometry from computational data studies. The synthesized titanium(IV) complexes were screened for their antioxidant abilities where 2c and 2d exhibited remarkable activity as IC50 values. The DNA & BSA binding properties of the complexes (2a-2d) have also been investigated through electronic absorption and molecular docking analysis. The obtained results indicated clearly that the complexes got bound with DNA via groove binding mode according to the intrinsic binding constant values (Kb = 1.5 × 104 M−1 to 7.5 × 105 M−1). Derivative 2c was examined in-vitro on a panel of cancerous cell line − breast cancer and human colon cell lines MCF-7 to reflect IC50 results as 53.92 μg/mL and HT-29 33.43 μg/mL, respectively.

Downregulation of O-GlcNAc transferase activity impairs basal autophagy and late endosome positioning under nutrient-rich conditions in human colon cells

Autophagy is a critical catabolic pathway that enables cells to survive and adapt to stressful conditions, especially nutrient deprivation. The fusion of autophagic vacuoles with lysosomes is the final step of autophagy, which degrades the engulfed contents into metabolic precursors for re-use by the cell. O-GlcNAc transferase (OGT) plays a crucial role in regulating autophagy flux in response to nutrient stress, particularly by targeting key proteins involved in autophagosome-lysosome fusion. However, the role of OGT in basal autophagy, which occurs at a low and constitutive levels under growth conditions, remains poorly understood. Silencing or inhibition of OGT was used to compare the effect of OGT downregulation on autophagy flux in the non-cancerous CCD841CoN and cancerous HCT116 human colon cell lines under nutrient-rich conditions. We provide evidence that the reduction of OGT activity impairs the maturation of autophagosomes, thereby blocking the completion of basal autophagy in both cell lines. Additionally, OGT inhibition results in the accumulation of lysosomes and enlarged late endosomes in the perinuclear region, as demonstrated by confocal imaging. This is associated with a defect in the localization of the small GTPase Rab7 to these organelles. The regulation of transport and fusion events between the endosomal and lysosomal compartments is crucial for maintaining the autophagic flux. These findings suggest an interplay between OGT and the homeostasis of the endolysosomal network in human cells.

TL1A Promotes Fibrogenesis in Colonic Fibroblasts via the TGF-β1/Smad3 Signaling Pathway.

Intestinal fibrosis is a refractory complication of inflammatory bowel disease (IBD). Tumor necrosis factor ligand-related molecule-1A (TL1A) is important for IBD-related intestinal fibrosis in a dextran sodium sulfate (DSS)-induced experimental colitis model. This study aimed to explore the effects of TL1A on human colonic fibroblasts. A trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model of LCK-CD2-TL1A-GFP transgenic (Tg) or wild-type (WT) mice was established to determine the effect and mechanism of TL1A on intestinal fibrosis. The human colonic fibroblast CCD-18Co cell line was treated concurrently with TL1A and human peripheral blood mononuclear cell (PBMC) supernatant. The proliferation and activation of CCD-18Co cells were detected by BrdU assays, flow cytometry, immunocytochemistry and Western blotting. Collagen metabolism was tested by Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR). The level of collagen metabolism in the TNBS+ethyl alcohol (EtOH)/Tg group was greater than that in the TNBS+EtOH/WT group. Transforming growth factor-β1 (TGF-β1) and p-Smad3 in the TNBS+EtOH/Tg group were upregulated as compared with those in the TNBS+EtOH/WT group. The proliferation of CCD-18Co cells was promoted by the addition of human PBMC supernatant supplemented with 20 ng/mL TL1A, and the addition of human PBMC supernatant and TL1A increased CCD-18Co proliferation by 24.4% at 24 h. TL1A promoted cell activation and increased the levels of COL1A2, COL3A1, and TIMP-1 in CCD-18Co cells. Treatment of CCD-18Co cells with TL1A increased the expression of TGF-β1 and p-Smad3. TL1A promotes TGF-β1-mediated intestinal fibroblast activation, proliferation, and collagen deposition and is likely related to an increase in the TGF-β1/Smad3 signaling pathway.

A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

LC-HRMS Profiling and Cytotoxic Potential of Actinomycetes Associated with the Red Sea Soft Coral Sarcophyton glaucum: In vitro and In silico Studies.

In the current study, the actinomycetes associated with the red sea-derived soft coral Sarcophyton glaucum were investigated in terms of biological and chemical diversity. Four different media, M1, ISP2, Marine Agar (MA), and Actinomycete isolation agar (AIA) were used for the isolation of three strains of actinomycetes that were identified as Streptomyces sp. UR 25, Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. LC-HRMS analysis was used to investigate the chemical diversity of the isolated actinobacteria. The LC-HRMS data were statistically processed using MetaboAnalyst 5.0 viz to differentiate the extract groups and determine the optimal growth culturing conditions. Multivariate data statistical analysis revealed that the Micromonospora sp. extract cultured on (MA) medium is the most distinctive extract in terms of chemical composition. While, the Streptomyces sp. UR 25 extracts are differ significantly from Micromonospora sp. UR32 and Saccharomonospora sp. UR 19. Biological investigation using in vitro cytotoxic assay for actinobacteria extracts revealed the prominent potentiality of the Streptomyces sp. UR 25 cultured on oligotrophic medium against human hepatoma (HepG2), human breast adenocarcinoma (MCF-7) and human colon adenocarcinoma (CACO2) cell lines (IC50 =3.3, 4.2 and 6.8 μg/mL, respectively). SwissTarget Prediction speculated that among the identified compounds, 16-deethyl, indanomycin (8) could have reasonable affinity on HDM2 active site. In this respect, molecular docking study was performed for compound (8) to reveal a substantial affinity on HDM2 active site. In addition, molecular dynamics simulations were carried out at 200 ns for the most active compound (8) compared to the co-crystallized inhibitor DIZ giving deeper information regarding their thermodynamic and dynamic properties as well.

Colonic ketogenesis, a microbiota-regulated process, contributes to blood ketones and protects against colitis in mice.

Ketogenesis is considered to occur primarily in liver to generate ketones as an alternative energy source for non-hepatic tissues when glucose availability/utilization is impaired. 3-Hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2) mediates the rate-limiting step in this mitochondrial pathway. Publicly available databases show marked down-regulation of HMGCS2 in colonic tissues in Crohn's disease and ulcerative colitis. This led us to investigate the expression and function of this pathway in colon and its relevance to colonic inflammation in mice. Hmgcs2 is expressed in cecum and colon. As global deletion of Hmgcs2 showed significant postnatal mortality, we used a conditional knockout mouse with enzyme deletion restricted to intestinal tract. These mice had no postnatal mortality. Fasting blood ketones were lower in these mice, indicating contribution of colonic ketogenesis to circulating ketones. There was also evidence of gut barrier breakdown and increased susceptibility to experimental colitis with associated elevated levels of IL-6, IL-1β, and TNF-α in circulation. Interestingly, many of these phenomena were mostly evident in male mice. Hmgcs2 expression in colon is controlled by colonic microbiota as evidenced from decreased expression in germ-free mice and antibiotic-treated conventional mice and from increased expression in a human colonic epithelial cell line upon treatment with aqueous extracts of cecal contents. Transcriptomic analysis of colonic epithelia from control mice and Hmgcs2-null mice indicated an essential role for colonic ketogenesis in the maintenance of optimal mitochondrial function, cholesterol homeostasis, and cell-cell tight-junction organization. These findings demonstrate a sex-dependent obligatory role for ketogenesis in protection against colonic inflammation in mice.

INFLAMMATION-TRIGGERED CLAUDIN-23 DOWNREGULATION IMPACTS INTESTINAL EPITHELIAL BARRIER FUNCTION

Abstract Intestinal epithelium maintains gut homeostasis by protecting the underlying tissue compartments from harmful contents. Although it is suggested that epithelial barrier function compromise contributes to the pathogenesis of inflammatory bowel disease (IBD), the underlying mechanisms are not well understood. Claudins (CLDN) are tight junctions (TJ) proteins that determine epithelial barrier properties during homeostasis and whose expression is altered in IBD, thereby contributing to a leaky barrier. We recently identified the expression of an atypical CLDN family member, CLDN23, in the regulation of homeostatic intestinal epithelial barrier function. Analysis of the human colonic crypt-luminal axis revealed increased CLDN23 expression in differentiated intestinal epithelial cells (IEC) facing the lumen compared to the proliferative crypt-base IECs. Complementary cell biologic approaches using IEC-specific Cldn23 knockout mice (Cldn23ERΔIEC) and human colon cell lines revealed that CLDN23 strengthens epithelial barrier function. Mechanistically, CLDN23 promoted redistribution of barrier-forming CLDN3 and CLDN4 from the lateral membrane to the TJ through cis- and trans-interactions, resulting in narrowed CLDN3 and CLDN4 pore diameters. Since inflammation compromises the epithelial barrier and contributes to disease pathogenesis in IBD, we first examined CLDN23 expression in colonic mucosal tissue from IBD individuals. Immunofluorescence and RNA scope labeling revealed that CLDN23 expression was significantly decreased in colonic epithelial cells of IBD individuals. Furthermore, reduced CLDN23 expression was also detected in primary colonic epithelial cells (colonoids) from mice treated with pro-inflammatory cytokines TNFα and IFNγ compared to untreated colonoids. Barrier function assays of control Cldn23f/f and Cldn23ERΔIEC mice-derived colonoids treated with TNFα and IFNγ identified decreased transepithelial electrical resistance in colonoids lacking CLDN23 and treated with these pro-inflammatory cytokines. These findings suggest a protective role for CLDN23 in regulating barrier function under homeostatic and inflammatory conditions.

Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT.

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesiumparvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P.parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11nM and 41.5 ± 0.6nM after incubations times of 3 and 24h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P.parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.