Characterization and safety assessment of bamboo shoot shell cellulose nanofiber: Prepared by acidolysis combined with dynamic high-pressure microfluidization.

The preparation of cellulose nanofiber (CNF) using traditional methods is currently facing challenges due to concerns regarding environmental pollution and safety. Herein, a novel CNF was obtained from bamboo shoot shell (BSS) by low-concentration acid and dynamic high-pressure microfluidization (DHPM) treatment. The resulting CNF was then characterized, followed by in vitro and in vivo safety assessments. Compared to insoluble dietary fiber (IDF), the diameters of HIDF (IDF after low-concentration acid hydrolysis) and CNF were significantly decreased to 167.13 nm and 70.97 nm, respectively. Meanwhile, HIDF and CNF showed a higher crystallinity index (71.32 % and 74.35 %). Structural analysis results indicated the successful removal of lignin and hemicellulose of HIDF and CNF, with CNF demonstrating improved thermostability. In vitro, a high dose of CNF (1500 µg/mL) did not show any signs of cytotoxicity on Caco-2 cells. In vivo, no death was observed in the experimental mice, and there was no significant difference between CNF (1000 mg/kg·bw) and control group in hematological index and histopathological analysis. Overall, this study presents an environmentally friendly method for preparing CNF from BSS while providing evidence regarding its safety through in vitro and in vivo assessments, laying the foundation for its potential application in food.

Anti-Inflammatory Effects of a Novel Acetonitrile-Water Extract of Lens Culinaris against LPS-Induced Damage in Caco-2 Cells.

Natural compounds like flavonoids preserve intestinal mucosal integrity through their antioxidant, anti-inflammatory, and antimicrobial properties. Additionally, some flavonoids show prebiotic abilities, promoting the growth and activity of beneficial gut bacteria. This study investigates the protective impact of Lens culinaris extract (LE), which is abundant in flavonoids, on intestinal mucosal integrity during LPS-induced inflammation. Using Caco-2 cells as a model for the intestinal barrier, the study found that LE did not affect cell viability but played a cytoprotective role in the presence of LPS. LE improved transepithelial electrical resistance (TEER) and tight junction (TJ) protein levels, which are crucial for barrier integrity. It also countered the upregulation of pro-inflammatory genes TRPA1 and TRPV1 induced by LPS and reduced pro-inflammatory markers like TNF-α, NF-κB, IL-1ß, and IL-8. Moreover, LE reversed the LPS-induced upregulation of AQP8 and TLR-4 expression. These findings emphasize the potential of natural compounds like LE to regulate the intestinal barrier and reduce inflammation's harmful effects on intestinal cells. More research is required to understand their mechanisms and explore therapeutic applications, especially for gastrointestinal inflammatory conditions.

Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

Preventative Effects of Milk Fat Globule Membrane Ingredients on DSS-Induced Mucosal Injury in Intestinal Epithelial Cells.

The goblet cells of the gastrointestinal tract (GIT) produce glycoproteins called mucins that form a protective barrier from digestive contents and external stimuli. Recent evidence suggests that the milk fat globule membrane (MFGM) and its milk phospholipid component (MPL) can benefit the GIT through improving barrier function. Our objective was to compare the effects of two digested MFGM ingredients with or without dextran sodium sulfate (DSS)-induced barrier stress on mucin proteins. Co-cultured Caco-2/HT29-MTX intestinal cells were treated with in vitro digests of 2%, 5%, and 10% (w/v) MFGM or MPL alone for 6 h or followed by challenge with 2.5% DSS (6 h). Transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran (FD4) permeability measurements were used to measure changes in barrier integrity. Mucin characterization was performed using a combination of slot blotting techniques for secreted (MUC5AC, MUC2) and transmembrane (MUC3A, MUC1) mucins, scanning electron microscopy (SEM), and periodic acid Schiff (PAS)/Alcian blue staining. Digested MFGM and MPL prevented a DSS-induced reduction in secreted mucins, which corresponded to the prevention of DSS-induced increases in FD4 permeability. SEM and PAS/Alcian blue staining showed similar visual trends for secreted mucin production. A predictive bioinformatic approach was also used to identify potential KEGG pathways involved in MFGM-mediated mucosal maintenance under colitis conditions. This preliminary in silico evidence, combined with our in vitro findings, suggests the role of MFGM in inducing repair and maintenance of the mucosal barrier.

Characterization and Mechanism of a Novel Rice Protein Peptide (AHVGMSGEEPE) Calcium Chelate in Enhancing Calcium Absorption in Caco-2 Cells.

Rice protein peptides (RPP) are a potentially valuable source of high-quality calcium chelating properties. However, there is a lack of information regarding the calcium-absorption-promoting effect of RPP and its underlying mechanism. The present study adopted molecular docking methodologies to analyze the 10 most potent peptide segments from RPP. Results revealed that the peptide AHVGMSGEEPE (AHV) displayed optimal calcium binding properties (calcium-chelating capacity 55.69 ± 0.66 mg/g). Quantum chemistry analysis revealed that the AHV peptide effectively binds and forms stable complexes with calcium via the carbonyl oxygen atoms in valine at position 3 and the carbonyl of the C-terminal carboxyl group of glutamate at position 11. The spectral analysis results indicated that AHV may bind to calcium through carboxyl oxygen atoms, resulting in a transition from a smooth surface block-like structure to a dense granular structure. Furthermore, this study demonstrated that the 4 mmol/L AHV-Ca chelate (61.75 ± 13.23 µg/well) significantly increases calcium absorption compared to 1 mM CaCl2 (28.57 ± 8.59 µg/well) in the Caco-2 cell monolayer. In terms of mechanisms, the novel peptide-calcium chelate AHV-Ca derived from RPP exerts a cell-level effect by upregulating the expression of TRPV6 calcium-ion-channel-related genes and proteins (TRPV6 and Calbindin-D9k). This study provides a theoretical basis for developing functional foods with the AHV peptide as ingredients to improve calcium absorption.

Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit­8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans­epithelial electrical resistance and fluorescein isothiocyanate­dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence­quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3­II/LC3­I conversion and autophagic flow. The results indicated that SSTR2­dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy­related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2­dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

Mathematical model explains differences in Omicron and Delta SARS-CoV-2 dynamics in Caco-2 and Calu-3 cells.

Within-host infection dynamics of Omicron dramatically differs from previous variants of SARS-CoV-2. However, little is still known about which parameters of virus-cell interplay contribute to the observed attenuated replication and pathogenicity of Omicron. Mathematical models, often expressed as systems of differential equations, are frequently employed to study the infection dynamics of various viruses. Adopting such models for results of in vitro experiments can be beneficial in a number of aspects, such as model simplification (e.g., the absence of adaptive immune response and innate immunity cells), better measurement accuracy, and the possibility to measure additional data types in comparison with in vivo case. In this study, we consider a refinement of our previously developed and validated model based on a system of integro-differential equations. We fit the model to the experimental data of Omicron and Delta infections in Caco-2 (human intestinal epithelium model) and Calu-3 (lung epithelium model) cell lines. The data include known information on initial conditions, infectious virus titers, and intracellular viral RNA measurements at several time points post-infection. The model accurately explains the experimental data for both variants in both cell lines using only three variant- and cell-line-specific parameters. Namely, the cell entry rate is significantly lower for Omicron, and Omicron triggers a stronger cytokine production rate (i.e., innate immune response) in infected cells, ultimately making uninfected cells resistant to the virus. Notably, differences in only a single parameter (e.g., cell entry rate) are insufficient to obtain a reliable model fit for the experimental data.

Vitamin C enhances co-localization of novel TET1 nuclear bodies with both Cajal and PML bodies in colorectal cancer cells.

Deregulation of ten-eleven Translocation protein 1 (TET1) is commonly reported to induce imbalances in gene expression and subsequently to colorectal cancer development (CRC). On the other hand, vitamin C (VitC) improves the prognosis of colorectal cancer by reprogramming the cancer epigenome and limiting chemotherapeutic drug resistance events. In this study, we aimed to characterize TET1-specific subcellular compartments and evaluate the effect of VitC on TET1 compartmentalization in colonic tumour cells. We demonstrated that TET1 is concentrated in coarse nuclear bodies (NB) and 5-hydroxymethylcytosine (5hmC) in foci in colorectal cancer cells (HCT116, Caco-2, and HT-29). To our knowledge, this is the first report of a novel intracellular localization profile of TET1 and its demethylation marker, 5hmC, in CRC cells. Interestingly, we found that TET1-NBs frequently interacted with Cajal bodies, but not with promyelocytic leukaemia (PML) bodies. In addition, we report that VitC treatment of HCT116 cells induces 5hmC foci biogenesis and triggers 5hmC marks to form active complexes with nuclear body components, including both Cajal and PML proteins. Our data highlight novel NB-concentrating TET1 in CRC cells and demonstrate that VitC modulates TET1-NBs' interactions with other nuclear structures. These findings reveal novel TET1-dependent cellular functions and potentially provide new insights for CRC management.

Impact of Micro- and Nano-Plastics on Human Intestinal Organoid-Derived Epithelium.

The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.

Short communication: a novel multispecies bacteria-based direct-fed microbial supports in vitro gut barrier integrity challenged with a pathogen or pro-inflammatory cytokines.

We conducted two experiments to evaluate the effects of a novel bacterial-based direct-fed microbial (DFM) on intestinal barrier integrity using the in vitro transepithelial electrical resistance (TEER) assay. In experiment 1, human-derived Caco-2 cells received or not (CON) a DFM containing Ligilactobacillus (formerly Lactobacillus) animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and B. subtilis 597 (BDP; BOVAMINE DEFEND® Plus) at a rate of 1 × 108 CFU/transwell. Concurrently with treatment application (CON or BDP), a pathogenic challenge of Clostridium perfringens type A was added alone (PAT) or with BDP (PAT + BDP) at a rate of 2.8 × 107 CFU/transwell in a 2 × 2 factorial arrangement. In experiment 2, Caco-2 cells were also assigned in a 2 × 2 factorial design to CON or BDP and then, 2 h post-treatment administration (CON and BDP), a mixture of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) was added alone (CYT) or with BDP (CYT + BDP) at a 10:1 ratio, respectively. In both experiments, TEER was measured for 18 h. In experiment 1, a DFM × pathogen × hour interaction was observed for TEER (P < 0.0001). Adding the PAT alone initially tended to increase TEER vs. CON from 1.1 to 2.2 h (P ≤ 0.09), increased TEER at 3.2 h (P < 0.01), but reduced TEER from 5.4 to the end of the experimental period at 18.4 h (P ≤ 0.01). On the other hand, adding DFM, with or without the pathogenic challenge, yielded greater TEER vs. CON-CON and CON-PAT for most of the experimental period (P ≤ 0.04). A similar interaction was detected and reported in experiment 2 (P < 0.0001). The CYT challenge reduced mean TEER compared with all other treatments from 3.2 h to the remainder of the study (P ≤ 0.03). On the other hand, BDP-CYT was able to maintain the integrity of the epithelial cells when compared with CON-CON throughout the experimental period (P ≤ 0.03), the exception being at 3.2 h (P = 0.20). Moreover, BDP-CON increased (P ≤ 0.04) TEER when compared with CON-CON from 3.2 to 18.4 h, but also in comparison with BDP-CYT from 4.3 to 18.4 h post-DFM and challenge administration into the cells. In summary, C. perfringens type A and a pro-inflammatory cytokine cocktail compromised the integrity of intestinal epithelial cell monolayers in vitro, whereas adding a multispecies bacteria-based DFM counteracted these damaging effects.

Two experiments were designed to evaluate the effects of adding a bacterial-based direct-fed microbial (DFM) containing Lactobacillus animalis 506, Propionibacterium freudenreichii 507, Bacillus paralicheniformis 809, and Bacillus subtilis 597 on the integrity of intestinal epithelial cells challenged with Clostridium perfringens type A or a pro-inflammatory cytokine cocktail. Regardless of the challenge, the addition of the DFM maintained the integrity of the intestinal epithelial cells in vitro. These results help to elucidate the potential beneficial effects that the bacterial-based DFM containing L. animalis 506, P. freudenreichii 507, B. paralicheniformis 809, and B. subtilis 597 may bring to livestock species.

Dissolution and antioxidant potential of apigenin self nanoemulsifying drug delivery system (SNEDDS) for oral delivery.

Self-nanoemulsifying drug delivery systems (SNEDDS) have been used to improve the oral bioavailability of various drugs. In the current study, apigenin was developed as SNEDDS to solve its dissolution problem and enhance oral bioavailability and antioxidant potential. SNEDDS were prepared by mixing Gelucire 44/14, Tween 80, and PEG 400 under controlled conditions. The droplet of diluted SNEDDS demonstrated a spherical shape with a size of less than 100 nm and a neutral charge. The very fast self-emulsification was obtained within 32 s, and the transmittance values exceeded 99%. The highest drug loading was 90.10 ± 0.24% of the initial load with the highest %encapsulation efficiency of 84.20 ± 0.03%. FT-IR and DSC spectra showed no interaction between components. The dissolution in buffer pH 1.2, 4.5, and 6.8 showed significantly higher dissolved apigenin than the apigenin coarse powder. The dissolution profiles were fitted to the Korsmeyer-Peppas kinetics. The cellular antioxidant activities in Caco-2 cells were approximately 52.25-54.64% compared to no treatment and were higher than the apigenin coarse powder (12.70%). Our work highlights the potential of SNEDDS to enhance the dissolution and permeability of apigenin and promote antioxidant efficacy, which has a strong chance of being developed as a bioactive compound for nutraceuticals.

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliCEcN have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 has more missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Introduction: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 µg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

Biofilm-producing Escherichia coli O104:H4 overcomes bile salts toxicity by expressing virulence and resistance proteins.

We investigated bile salts' ability to induce phenotypic changes in biofilm production and protein expression of pathogenic Escherichia coli strains. For this purpose, 82 pathogenic E. coli strains isolated from humans (n = 70), and animals (n = 12), were examined for their ability to form biofilms in the presence or absence of bile salts. We also identified bacterial proteins expressed in response to bile salts using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-electrophoresis) and liquid chromatography-mass spectrometry (LC-MS/MS). Lastly, we evaluated the ability of these strains to adhere to Caco-2 epithelial cells in the presence of bile salts. Regarding biofilm formation, two strains isolated from an outbreak in Republic of Georgia in 2009 were the only ones that showed a high and moderate capacity to form biofilm in the presence of bile salts. Further, we observed that those isolates, when in the presence of bile salts, expressed different proteins identified as outer membrane proteins (i.e. OmpC), and resistance to adverse growth conditions (i.e. F0F1, HN-S, and L7/L12). We also found that these isolates exhibited high adhesion to epithelial cells in the presence of bile salts. Together, these results contribute to the phenotypic characterization of E. coli O104: H4 strains.

Uncontrolled Post-Industrial Landfill-Source of Metals, Potential Toxic Compounds, Dust, and Pathogens in Environment-A Case Study.

The aim of this case study was the evaluation of the selected metals' concentration, potential toxic compound identification, cytotoxicity analysis, estimation of the airborne dust concentration, biodiversity, and number of microorganisms in the environment (leachate, soil, air) of the biggest uncontrolled post-industrial landfills in Poland. Based on the results obtained, preliminary solutions for the future management of post-industrial objects that have become an uncontrolled landfill were indicated. In the air, the PM1 fraction dominated, constituting 78.1-98.2% of the particulate matter. Bacterial counts were in the ranges of 9.33 × 101-3.21 × 103 CFU m-3 (air), 1.87 × 105-2.30 × 106 CFU mL-1 (leachates), and 8.33 × 104-2.69 × 106 CFU g-1 (soil). In the air, the predominant bacteria were Cellulosimicrobium and Stenotrophomonas. The predominant fungi were Mycosphaerella, Cladosporium, and Chalastospora. The main bacteria in the leachates and soils were Acinetobacter, Mortierella, Proteiniclasticum, Caloramator, and Shewanella. The main fungi in the leachates and soils were Lindtneria. Elevated concentrations of Pb, Zn, and Hg were detected. The soil showed the most pronounced cytotoxic potential, with rates of 36.55%, 63.08%, and 100% for the A-549, Caco-2, and A-549 cell lines. Nine compounds were identified which may be responsible for this cytotoxic effect, including 2,4,8-trimethylquinoline, benzo(f)quinoline, and 1-(m-tolyl)isoquinoline. The microbiome included bacteria and fungi potentially metabolizing toxic compounds and pathogenic species.

Identification of Corn Peptides with Alcohol Dehydrogenase Activating Activity Absorbed by Caco-2 Cell Monolayers.

Alcohol dehydrogenase (ADH) plays a pivotal role in constraining alcohol metabolism. Assessing the ADH-activating activity in vitro can provide insight into the capacity to accelerate ethanol metabolism in vivo. In this study, ADH-activating peptides were prepared from corn protein meal (CGM) using enzymatic hydrolysis, and these peptides were subsequently identified following simulated gastrointestinal digestion and their absorption through the Caco-2 cell monolayer membrane. The current investigation revealed that corn protein hydrolysate hydrolyzed using alcalase exhibited the highest ADH activation capability, maintaining an ADH activation rate of 52.93 ± 2.07% following simulated gastrointestinal digestion in vitro. After absorption through the Caco-2 cell monolayer membrane, ADH-activating peptides were identified. Among them, SSNCQPF, TGCPVLQ, and QPQQPW were validated to possess strong ADH activation activity, with EC50 values of 1.35 ± 0.22 mM, 2.26 ± 0.16 mM, and 2.73 ± 0.13 mM, respectively. Molecular Docking revealed that the activation of ADH occurred via the formation of a stable complex between the peptide and the active center of ADH by hydrogen bonds and hydrophobic interactions. The results of this study also suggest that corn protein hydrolysate could be a novel functional dietary element that helps protects the liver from damage caused by alcohol and aids in alcohol metabolism.

Evaluation of the Lipophilicity of Angularly Condensed Diquino- and Quinonaphthothiazines as Potential Candidates for New Drugs.

Lipophilicity is one of the most important properties of compounds required to estimate the absorption, distribution, and transport in biological systems, in addition to solubility, stability, and acid-base nature. It is crucial in predicting the ADME profile of bioactive compounds. The study assessed the usefulness of computational and chromatographic methods (thin-layer chromatography in a reversed-phase system, RP-TLC) for estimating the lipophilicity of 21 newly synthesized compounds belonging to diquinothiazines and quinonaphthiazines. In order to obtain reliable values of the relative lipophilicities of diquinothiazines and quinonaphthiazines, the partition coefficients obtained using different algorithms such as AlogPs, AClogP, AlogP, MLOGP, XLOGP2, XLOGP3, logP, and ClogP were compared with the chromatographic RM0 values of all the tested compounds measured by the experimental RP-TLC method (logPTLC). Additionally, logPTLC values were also correlated with other descriptors, as well as the predicted ADME and drug safety profiling parameters. The linear correlations of logPTLC values of the tested compounds with other calculated molecular descriptors such as molar refractivity, as well as ADME parameters (Caco-2 substrates, P-gp inhibitors, CYP2C19, and CYP3A4) generally show poor predictive power. Therefore, in silico ADME profiling can only be helpful at the initial step of designing these new candidates for drugs. The compliance of all discussed diquinothiazines and naphthoquinothiazines with the rules of Lipinski, Veber, and Egan suggests that the tested pentacyclic phenothiazine analogs have a chance to become therapeutic drugs, especially orally active drugs.

Intestinal Piezo1 aggravates intestinal barrier dysfunction during sepsis by mediating Ca2+ influx.

INTRODUCTION: Intestinal barrier dysfunction is a pivotal factor in sepsis progression. The mechanosensitive ion channel Piezo1 is associated with barrier function; however, its role in sepsis-induced intestinal barrier dysfunction remains poorly understood. METHODS: The application of cecal ligation and puncture (CLP) modeling was performed on both mice of the wild-type (WT) variety and those with Villin-Piezo1flox/flox genetic makeup to assess the barrier function using in vivo FITC-dextran permeability measurements and immunofluorescence microscopy analysis of tight junctions (TJs) and apoptosis levels. In vitro, Caco-2 monolayers were subjected to TNF-α incubation. Moreover, to modulate Piezo1 activation, GsMTx4 was applied to inhibit Piezo1 activation. The barrier function, intracellular calcium levels, and mitochondrial function were monitored using calcium imaging and immunofluorescence techniques. RESULTS: In the intestinal tissues of CLP-induced septic mice, Piezo1 protein levels were notably elevated compared with those in normal mice. Piezo1 has been implicated in the sepsis-mediated disruption of TJs, apoptosis of intestinal epithelial cells, elevated intestinal mucosal permeability, and systemic inflammation in WT mice, whereas these effects were absent in Villin-Piezo1flox/flox CLP mice. In Caco-2 cells, TNF-α prompted calcium influx, an effect reversed by GsMTx4 treatment. Elevated calcium concentrations are correlated with increased accumulation of reactive oxygen species, diminished mitochondrial membrane potential, and TJ disruption. CONCLUSIONS: Thus, Piezo1 is a potential contributor to sepsis-induced intestinal barrier dysfunction, influencing apoptosis and TJ modification through calcium influx-mediated mitochondrial dysfunction.

Biogenic Zinc Oxide Nanoparticles synthesized from Tinospora Cordifolia induce oxidative stress, mitochondrial damage and apoptosis in Colorectal Cancer.

Cancer chemotherapy remains a serious challenge, and new approaches to therapy are urgently needed to build novel treatment regimens. The methanol extract of the stem of Tinospora Cordifolia was used to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) that display anticancer activities against colorectal cancer. Biogenic ZnO-NPs synthesized from methanol extract of Tinospora Cordifolia stem (ZnO-NPs TM) were tested against HCT-116 cell lines to assess anticancer activity. UV-Vis, FTIR, XRD, SEM, and TEM analysis characterized the biogenic ZnO-NPs. To see how well biogenic ZnO-NPs fight cancer, cytotoxicity, AO/EtBr staining, Annexin V/PI staining, mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) analysis, and caspase cascade activity analysis were performed to assess the anticancer efficacy of biogenic ZnO-NPs. The IC50 values of biogenic ZnO-NPs treated cells (HCT-116 and Caco-2) were 31.419 ± 0.682µg/ml and 36.675 ± 0.916µg/ml, respectively. qRT-PCR analysis showed that cells treated with biogenic ZnO-NPs Bax and P53 mRNA levels increased significantly (p ≤ 0.001). It showed to have impaired MMP and increased ROS generation. In a corollary, our in vivo study showed that biogenic ZnO-NPs have an anti-tumour effect. Biogenic ZnO-NPs TM showed both in vitro and in vivo anticancer effects that could be employed as anticancer drugs.

Synthesis of salcaprozate sodium and its significance in enhancing pancreatic kininogenase absorption performance.

We conducted pharmacokinetic research wherein salcaprozate sodium (SNAC) was utilized as a penetration enhancer by incorporating it into pancreatic kininogenase (PK) to improve the bioavailability of pancreatic kininogenase enteric-coated tablets. We conducted in vitro studies on PK using the Caco-2 cell model and quantified PK levels using the enzyme-linked immunosorbent assay (ELISA) method. We conducted methodological verification by blending SNAC and PK powders into enteric-coated capsules, and studied the pharmacokinetic characteristics. Based on the PK transport assay, the cumulative permeation rates of the test group that employed a SNAC to PK ratio of 32:1, 16:1, 8:1, 4:1, and 2:1 were 13.574%, 7.597%, 10.653%, 3.755%, and 2.523%, respectively. We conducted a uniformity test on the powder that contained a blend of SNAC and PK. The relative standard deviations (RSDs) for both the power containing SNAC and the power not containing SNAC were less than 10%. Based on the methodological verification, in vivo pharmacokinetic study of PK met the experimental requirements. As indicated by the results of in vivo pharmacokinetic research on rats, the test group (This group used SNAC) had a PK AUC0-12 h of 5679.747 ng/L*h and t1/2 of 4.569 h, while the control group (This group did not use SNAC) had a PK AUC0-12 h of 4639.665 ng/L*h and t1/2 of 3.13 h. This study has established a low-cost, environmentally friendly, and safe SNAC synthesis route with high process yield suitable for industrial production. SNAC demonstrates an absorption-enhancing effect on PK, and the optimal ratio of SNAC to PK is determined to be 32:1.