The artificial sweetener neotame negatively regulates the intestinal epithelium directly through T1R3-signaling and indirectly through pathogenic changes to model gut bacteria.

Introduction: Recent studies have indicated considerable health risks associated with the consumption of artificial sweeteners. Neotame is a relatively new sweetener in the global market however there is still limited data on the impact of neotame on the intestinal epithelium or the commensal microbiota. Methods: In the present study, we use a model of the intestinal epithelium (Caco-2) and microbiota (Escherichia coli and Enterococcus faecalis) to investigate how physiologically-relevant exposure of neotame impacts intestinal epithelial cell function, gut bacterial metabolism and pathogenicity, and gut epithelium-microbiota interactions. Results: Our findings show that neotame causes intestinal epithelial cell apoptosis and death with siRNA knockdown of T1R3 expression significantly attenuating the neotame-induced loss to cell viability. Similarly, neotame exposure results in barrier disruption with enhanced monolayer leak and reduced claudin-3 cell surface expression through a T1R3-dependent pathway. Using the gut bacteria models, E. coli and E. faecalis, neotame significantly increased biofilm formation and metabolites of E. coli, but not E. faecalis, reduced Caco-2 cell viability. In co-culture studies, neotame exposure increased adhesion capacity of E. coli and E. faecalis onto Caco-2 cells and invasion capacity of E. coli. Neotame-induced biofilm formation, E.coli-specific Caco-2 cell death, adhesion and invasion was identified to be meditated through a taste-dependent pathway. Discussion: Our study identifies novel pathogenic effects of neotame on the intestinal epithelium or bacteria alone, and in co-cultures to mimic the gut microbiome. These findings demonstrate the need to better understand food additives common in the global market and the molecular mechanisms underlying potential negative health impacts.

Quercetin counteracts monosodium glutamate to mitigate immunosuppression in the thymus and spleen via redox-guided cellular signaling.

BACKGROUND: Chronic inflammation brought on by oxidative stress can result in several immunopathologies. Natural compounds with antioxidant characteristics, like quercetin, have shown effectiveness in reducing oxidative damage and regulating the immune response. PURPOSE: The commonly used food additive monosodium glutamate (M) causes immunosuppression by disrupting redox equilibrium and inducing oxidative stress. The goal of this work is to examine the therapeutic potential of quercetin against immunotoxicity brought on by M, revealing the molecular route implicated in such immunopathology by targeting the thymus and spleen, to support the development of future anti-inflammatory and antioxidant therapies. STUDY DESIGN AND METHODS: M-fed rats were employed as an immunotoxicity model and were supplemented with quercetin for four weeks. Hematological and biochemical parameters were measured; H&E staining, immunohistochemistry, flow cytometry, real-time quantitative PCR, and western blotting were performed. RESULTS: Based on the findings, TLR4 was activated by M to cause oxidative stress-mediated inflammation, which was alleviated by the supplementation of quercetin by modulating redox homeostasis to neutralize free radicals and suppress the inflammatory response. To prevent M-induced inflammation, quercetin demonstrated anti-inflammatory functions by blocking NF-kB activation, lowering the production of pro-inflammatory cytokines, and increasing the release of anti-inflammatory cytokines. By normalizing lipid profiles and lowering the potential risk of immunological deficiency caused by M, quercetin also improves lipid metabolism. Additionally, it has shown potential for modifying insulin levels, suggesting a possible function in controlling M-induced alteration in glucose metabolism. The addition of quercetin to M enhanced the immune response by improving immunoglobulin levels and CD4/CD8 expression in the thymus and spleen. Additionally, quercetin inhibited apoptosis by controlling mitochondrial caspase-mediated cellular signaling, suggesting that it may be able to halt cell death in M-fed rats. CONCLUSION: The results of this study first indicate that quercetin, via modulating redox-guided cellular signaling, has a promising role in reducing immune disturbances. This study illuminates the potential of quercetin as a safe, natural remedy for immunopathology caused by M, including thymic hypoplasia and/or splenomegaly, and paves the way for future anti-inflammatory and antioxidant supplements.

Investigating the use and awareness of artificial sweeteners among diabetic patients in Bangladesh.

BACKGROUND: As with many countries around the world, the incidence of diabetes in Bangladesh is increasing significantly. Whilst there is controversy in the field regarding the health impact of artificial sweeteners in Western communities, the link between sweetener consumption and awareness in Bangladesh has not been established. METHODS: In the present study, 260 diabetic patients completed a questionnaire survey to investigate the use and awareness of sweeteners and how this links to demographics and potential co-morbidities. RESULTS: Findings show that daily artificial sweetener consumption is significantly associated with hypertension but not other co-morbidities such as kidney disease or obesity. We further demonstrate that there is limited checking of artificial sweeteners in food or drink products by participants. the rurality of diabetic participants was found to significantly correlates with lower awareness of any health impact of artificial sweeteners. CONCLUSIONS: The findings from this study demonstrate that there is a need to increase the awareness of artificial sweetener use in diabetic patients in Bangladesh. Combined with a more robust understanding of the health impact of artificial sweeteners, these findings suggest that there is potential to improve outcomes for diabetic patients by improving this awareness.

Catharanthus roseus (L.) G. Don counteracts the ampicillin resistance in multiple antibiotic-resistant Staphylococcus aureus by downregulation of PBP2a synthesis.

It is essential to revisit the global biodiversity, search for ethnopharmacologically relevant plants, and unveil their untapped potential to overcome the complications associated while treating infections triggered by multiple antibiotic-resistant Staphylococcus aureus. Catharanthus roseus (L.) G. Don of the Apocynaceae family is a medicinal plant used for remedial purposes against infectious diseases from ancient times. In this study, we intended to evaluate the mechanism by which the ethanolic extract of C. roseus root (EECRR) causes the reversal of ampicillin resistance in S. aureus. To achieve this goal, we have stained EECRR-treated S. aureus with acridine orange, analysed DNA damage by comet assay, and studied the alteration of plasmid band pattern and expression of penicillin-binding protein 2a (PBP2a) protein. Experiments revealed better S. aureus killing efficiency of EECRR at its minimum inhibitory concentration (MIC) doses due to DNA damage and reducing plasmid band intensities along with a decline in the expression of PBP2a in EECRR-treated cells at half-MIC dose. EECRR proved to be an efficient growth inhibitor of S. aureus that reduces the expression of PBP2a. Therefore, EECRR can also render ampicillin-resistant S. aureus susceptible to the antibiotic.

In Silico Identification and Analysis of Potentially Bioactive Antiviral Phytochemicals against SARS-CoV-2: A Molecular Docking and Dynamics Simulation Approach.

SARS-CoV-2, a deadly coronavirus sparked COVID-19 pandemic around the globe. With an increased mutation rate, this infectious agent is highly transmissible inducing an escalated rate of infections and death everywhere. Hence, the discovery of a viable antiviral therapy option is urgent. Computational approaches have offered a revolutionary framework to identify novel antimicrobial treatment regimens and allow a quicker, cost-effective, and productive conversion into the health center by evaluating preliminary and safety investigations. The primary purpose of this research was to find plausible plant-derived antiviral small molecules to halt the viral entrance into individuals by clogging the adherence of Spike protein with human ACE2 receptor and to suppress their genome replication by obstructing the activity of Nsp3 (Nonstructural protein 3) and 3CLpro (main protease). An in-house library of 1163 phytochemicals were selected from the NPASS and PubChem databases for downstream analysis. Preliminary analysis with SwissADME and pkCSM revealed 149 finest small molecules from the large dataset. Virtual screening using the molecular docking scoring and the MM-GBSA data analysis revealed that three candidate ligands CHEMBL503 (Lovastatin), CHEMBL490355 (Sulfuretin), and CHEMBL4216332 (Grayanoside A) successfully formed docked complex within the active site of human ACE2 receptor, Nsp3, and 3CLpro, respectively. Dual method molecular dynamics (MD) simulation and post-MD MM-GBSA further confirmed efficient binding and stable interaction between the ligands and target proteins. Furthermore, biological activity spectra and molecular target analysis revealed that all three preselected phytochemicals were biologically active and safe for human use. Throughout the adopted methodology, all three therapeutic candidates significantly outperformed the control drugs (Molnupiravir and Paxlovid). Finally, our research implies that these SARS-CoV-2 protein antagonists might be viable therapeutic options. At the same time, enough wet lab evaluations would be needed to ensure the therapeutic potency of the recommended drug candidates for SARS-CoV-2.

Targeting Shikimate Kinase Pathway of Acinetobacter baumannii: A Structure-Based Computational Approach to Identify Antibacterial Compounds.

Acinetobacter baumannii (A. baumannii) is an opportunistic bacterium that has developed multidrug resistance (MDR) to most of today's antibiotics, posing a significant risk to human health. Considering the fact that developing novel drugs is a time-consuming and expensive procedure, this research focuses on utilizing computational resources for repurposing antibacterial agents for A. baumannii. We targeted shikimate kinase, an essential enzyme in A. baumannii, that plays a significant role in the metabolic process. The basis for generating new therapeutic compounds is to inhibit the shikimate kinase and thereby targeting the shikimate pathway. Herein, 1941 drug-like compounds were investigated in different in silico techniques for assessing drug-likeness properties, ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling, binding affinity, and conformation analysis utilizing Autodock-vina and SwissDock. CHEMBL1237, CHEMBL1237119, CHEMBL2018096, and CHEMBL39167178 were determined as potential drug candidates for suppressing shikimate kinase protein. Molecular Dynamics Simulation (MDS) results for root mean square deviation, root mean square fluctuation, hydrogen bond, and gyration radius confirm the drug candidates' molecular stability with the target protein. According to this study, CHEMBL1237 (Lisinopril) could be the most suitable candidate for A. baumannii. Our investigation suggests that the inhibitors of shikimate kinase could represent promising treatment options for A. baumannii. However, further in vitro and in vivo studies are necessary to validate the therapeutic potential of the suggested drug candidates.

Correction: Oxa-376 and Oxa-530 variants of ß-lactamase: computational study uncovers potential therapeutic targets of Acinetobacter baumannii.

[This corrects the article DOI: 10.1039/D2RA02939A.].

Oxa-376 and Oxa-530 variants of ß-lactamase: computational study uncovers potential therapeutic targets of Acinetobacter baumannii.

Antimicrobial resistance is a major global health crisis, resulting in thousands of deaths each year. Antibiotics' effectiveness against microorganisms deteriorates over time as multidrug resistance (MDR) develops, which is exacerbated by irregular antibiotic use, poor disease management, and the evasive nature of bacteria. The World Health Organization has recognized multidrug resistance as a critical public health concern, and Acinetobacter baumannii has been at the center of attention due to its ability to develop multidrug resistance (MDR). It generally produces carbapenem-hydrolyzing oxacillinase, which has been identified as the primary source of beta-lactam resistance in MDR bacteria. Recently, point mutations in A. baumannii have been identified as a key factor of multidrug resistance, making them a prime concern for researchers. The goal of the current work was to establish a unique way of finding multidrug-resistant variants and identify the most damaging mutations in the existing databases. We characterized the deleterious variants of oxacillinases using several computational tools. Following a thorough analysis, Oxa-376 and Oxa-530 were found to be more damaging when compared with the wild-type Oxa-51. The mutants' 3D structures were then prepared and refined with RaptorX, GalaxyRefine, and SAVES servers. Our research incorporates seven antimicrobial agents to illustrate the resistance capability of the variants of oxacillinase by evaluating binding affinity in Autodock-vina and Schrodinger software. RMSD, RMSF, Radius of gyration analysis, the solvent-accessible surface area (SASA), hydrogen bonding analysis and MM-GBSA from Molecular Dynamics Simulation revealed the dynamic nature and stability of wild-type and Oxa-376 and Oxa-530 variants. Our findings will benefit researchers looking for the deleterious mutations of Acinetobacter baumannii and new therapeutics to combat those variants. However, further studies are necessary to evaluate the mechanism of hydrolyzing activity and antibiotic resistance of these variants.

Investigating the Opposing Effect of Two Different Green Tea Supplements on Oxidative Stress, Mitochondrial Function and Cell Viability in HepG2 Cells.

Green tea extract (GTE) improves exercise outcomes and reduces obesity. However, case studies indicate contradictory physiology regarding liver function and toxicity. We studied the effect of two different decaffeinated GTE (dGTE) products, from a non-commercial (dGTE1) and commercial (dGTE2) supplier, on hepatocyte function using the human cell model, HepG2. dGTE1 was protective against hydrogen peroxide (H2O2)-induced apoptosis and cell death by attenuating oxidative stress pathways. Conversely, dGTE2 increased cellular and mitochondrial oxidative stress and apoptosis. A bioavailability study with dGTE showed the major catechin in GTE, EGCG, reached 0.263 µg·ml-1. In vitro, at this concentration, EGCG mimicked the protective effect of dGTE1. GC/MS analysis identified steric acid and higher levels of palmitic acid in dGTE2 versus dGTE1 supplements. We demonstrate the significant biological differences between two GTE supplements which may have potential implications for manufacturers and consumers to be aware of the biological effects of supplementation.

Artificial Sweeteners Negatively Regulate Pathogenic Characteristics of Two Model Gut Bacteria, E. coli and E. faecalis.

Artificial sweeteners (AS) are synthetic sugar substitutes that are commonly consumed in the diet. Recent studies have indicated considerable health risks which links the consumption of AS with metabolic derangements and gut microbiota perturbations. Despite these studies, there is still limited data on how AS impacts the commensal microbiota to cause pathogenicity. The present study sought to investigate the role of commonly consumed AS on gut bacterial pathogenicity and gut epithelium-microbiota interactions, using models of microbiota (Escherichia coli NCTC10418 and Enterococcus faecalis ATCC19433) and the intestinal epithelium (Caco-2 cells). Model gut bacteria were exposed to different concentrations of the AS saccharin, sucralose, and aspartame, and their pathogenicity and changes in interactions with Caco-2 cells were measured using in vitro studies. Findings show that sweeteners differentially increase the ability of bacteria to form a biofilm. Co-culture with human intestinal epithelial cells shows an increase in the ability of model gut bacteria to adhere to, invade and kill the host epithelium. The pan-sweet taste inhibitor, zinc sulphate, effectively blocked these negative impacts. Since AS consumption in the diet continues to increase, understanding how this food additive affects gut microbiota and how these damaging effects can be ameliorated is vital.

Extracellular vesicles released from p18 overexpressing pulmonary endothelial cells are barrier protective - potential implications for acute respiratory distress syndrome.

The novel endosome protein, p18, and the early endosome GTPase, Rab4, play a significant role in protecting the pulmonary vasculature against permeability associated with acute respiratory distress syndrome. Recently, endothelial-derived extracellular vesicles have been identified to play a key role in the endothelial permeability associated with acute respiratory distress syndrome. Therefore, we investigated the effect of these microparticles, released from endothelial cells overexpressing p18 and Rab4, on pulmonary endothelial barrier function. Endothelial-derived extracellular vesicles isolated from lung microvascular endothelial cells which overexpressed cDNA for wild-type p18 protected a naïve monolayer against lipopolysaccharide-induced permeability. In contrast, endothelial-derived extracellular vesicles from cells overexpressing the non-endosomal binding p18 mutant (p18N39) exerted no protective effect on the endothelial monolayer. Cells overexpressing either dominant active or inactive Rab4 released endothelial-derived extracellular vesicles which had no effect on lipopolysaccharide-induced permeability. miRNA analysis and permeability studies of endothelial-derived extracellular vesicle isolated from wild-type p18-overexpressing cells demonstrates that let-7i-5p, miR-96-5p, and miR-137-3p are endothelial-derived extracellular vesicle cargo which exert protective effects on the pulmonary endothelium. Finally, we observed down-regulation of p18 protein expression in both the lung and endothelium in an in vivo and in vitro model of acute respiratory distress syndrome. These results demonstrate that endothelial-derived extracellular vesicle released from cells overexpressing p18, but not Rab4, contain miRNA cargo which likely promote a barrier-protective effect on the pulmonary endothelium in settings of acute respiratory distress syndrome. Findings indicate the importance of p18 in the pulmonary vasculature and demonstrate that targeting this protein may provide a novel therapeutic strategy to reduce endothelial permeability associated with acute respiratory distress syndrome.

Artificial Sweeteners Disrupt Tight Junctions and Barrier Function in the Intestinal Epithelium through Activation of the Sweet Taste Receptor, T1R3.

The breakdown of the intestinal epithelial barrier and subsequent increase in intestinal permeability can lead to systemic inflammatory diseases and multiple-organ failure. Nutrition impacts the intestinal barrier, with dietary components such as gluten increasing permeability. Artificial sweeteners are increasingly consumed by the general public in a range of foods and drinks. The sweet taste receptor (T1R3) is activated by artificial sweeteners and has been identified in the intestine to play a role in incretin release and glucose transport; however, T1R3 has not been previously linked to intestinal permeability. Here, the intestinal epithelial cell line, Caco-2, was used to study the effect of commonly-consumed artificial sweeteners, sucralose, aspartame and saccharin, on permeability. At high concentrations, aspartame and saccharin were found to induce apoptosis and cell death in intestinal epithelial cells, while at low concentrations, sucralose and aspartame increased epithelial barrier permeability and down-regulated claudin 3 at the cell surface. T1R3 knockdown was found to attenuate these effects of artificial sweeteners. Aspartame induced reactive oxygen species (ROS) production to cause permeability and claudin 3 internalization, while sweetener-induced permeability and oxidative stress was rescued by the overexpression of claudin 3. Taken together, our findings demonstrate that the artificial sweeteners sucralose, aspartame, and saccharin exert a range of negative effects on the intestinal epithelium through the sweet taste receptor T1R3.

Activation of the sweet taste receptor, T1R3, by the artificial sweetener sucralose regulates the pulmonary endothelium.

A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin remodeling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor T1R3 is a G protein-coupled receptor, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified; however, no studies have focused on T1R3 within the vasculature. We hypothesize that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier-disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule sucralose attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signaling, through zinc sulfate, T1R3, or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of the key signaling molecules Src, p21-activated kinase (PAK), myosin light chain-2 (MLC2), heat shock protein 27 (HSP27), and p110α phosphatidylinositol 3-kinase (p110αPI3K). Activation of T1R3 by sucralose protects the pulmonary endothelium from edemagenic agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin remodeling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS.