Detection to trace aluminum ion of pharmaceutical wastewater using synthesis of Schiff-based chemosensor

Background: The aim of this research was to develop a fluorogenic sensor for Al3+ions, which have been identified as a possible food and drinking water pollutant by the WHO and considered to be harmful to human health. Methods: The sensing mechanism was based on excited-state intramolecular proton transfer, with the intramolecular rotation restriction occurring after binding with the analyte. The probe attaches Al3+selectively and emits strong emission in 4:1 H2 O/MeOH (v/v) solution while irradiated at 400 nm in the presence of a wide number of cations, acting as a "turn-on" fluorescence chemosensor. The range of detection for Al3+is 3.3 nM (3 method), which is more than 200 times more responsive than the WHO suggested limit of 7.4 mM (3σ method). Mass spectra, job plot, and Benesi-Hildebrand plot were used to determine the formation of the 1:1 metal-to-ligand complex. Results: Aluminum (Al) ion content in effluent obtained from the pharmaceutical sector is 0.381 mM, which is a trace amount. A separate in vitro experiment indicates that the probe can precisely perceive Al3+ions in a cell line. The sensor-based method is developed to detect 3.3 nM of Al3+ions, which is significantly less than the WHO max. Conclusion: The probe to detect Al3+ions in live cells. HL becomes a flexible sensor for recognizing intracellular Al3+in human liver cancer cell line Hep G2 and human lung fibroblast cell lines by fluorescence cell imaging procedures, and the probe’s non-toxicity has been proven by MTT tests up to 100M.

Culture of IMR90 Cells in Advanced MEM with Reduced Serum

NOTE: Methods document and worksheet versions of this method are attached as PDFs.SCOPE OF APPLICATION (LIMITATIONS)This method describes the thawing, culturing, and cryopreservation of the human lung fibroblast cell line IMR90 in Advanced MEM-based growth medium. Disclaimer: The contents of this article have been reviewed by the US Environmental Protection Agency and approved for publication and do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Different bisphenols induce non-monotonous changes in miRNA expression and LINE-1 methylation in two cell lines

4,4ʹ-Isopropylidenediphenol (bisphenol A, BPA), a chemical substance that is widely used mainly as a monomer in the production of polycarbonates, in epoxy resins, and in thermal papers, is suspected to cause epigenetic modifications with potentially toxic consequences. Due to its negative health effects, BPA is banned in several products and is replaced by other bisphenols such as bisphenol S and bisphenol F. The present study examined the effects of BPA, bisphenol S, bisphenol F, p,pʹ-oxybisphenol, and the BPA metabolite BPA β-d-glucuronide on the expression of a set of microRNAs (miRNAs) as well as long interspersed nuclear element-1 methylation in human lung fibroblast and Caco-2 cells. The results demonstrated a significant modulation of the expression of different miRNAs in both cell lines including miR-24, miR-155, miR-21, and miR-146a, known for their regulatory functions of cell cycle, metabolism, and inflammation. At concentrations between 0.001 and 10 µg/ml, especially the data of miR-155 and miR-24 displayed non-monotonous and often significant dose–response curves that were U- or bell-shaped for different substances. Additionally, BPA β-d-glucuronide also exerted significant changes in the miRNA expression. miRNA prediction analysis indicated effects on multiple molecular pathways with relevance for toxicity. Besides, long interspersed nuclear element-1 methylation, a marker for the global DNA methylation status, was significantly modulated by two concentrations of BPA and p,pʹ-oxybisphenol. This pilot study suggests that various bisphenols, including BPA β-d-glucuronide, affect epigenetic mechanisms, especially miRNAs. These results should stimulate extended toxicological studies of multiple bisphenols and a potential use of miRNAs as markers.

Hypoxia Promotes a Mixed Inflammatory-Fibrotic Macrophages Phenotype in Active Sarcoidosis

BackgroundMacrophages are pivotal cells in sarcoidosis. Monocytes-derived (MD) macrophages have recently been demonstrated to play a major role especially in pulmonary sarcoidosis. From inflammatory tissues to granulomas, they may be exposed to low oxygen tension environments. As hypoxia impact on sarcoidosis immune cells has never been addressed, we designed the present study to investigate MD-macrophages from sarcoidosis patients in this context. We hypothesized that hypoxia may induce functional changes on MD-macrophages which could have a potential impact on the course of sarcoidosis.MethodsWe studied MD-macrophages, from high active sarcoidosis (AS) (n=26), low active or inactive sarcoidosis (IS) (n=24) and healthy controls (n=34) exposed 24 hours to normoxia (21% O2) or hypoxia (1.5% O2). Different macrophage functions were explored: hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) activation, cytokines secretion, phagocytosis, CD80/CD86/HLA-DR expression, profibrotic response.ResultsWe observed that hypoxia, with a significantly more pronounced effect in AS compared with controls and IS, increased the HIF-1α trans-activity, promoted a proinflammatory response (TNFα, IL1ß) without activating NF-κB pathway and a profibrotic response (TGFß1, PDGF-BB) with PAI-1 secretion associated with human lung fibroblast migration inhibition. These results were confirmed by immunodetection of HIF-1α and PAI-1 in granulomas observed in pulmonary biopsies from patients with sarcoidosis. Hypoxia also decreased the expression of CD80/CD86 and HLA-DR on MD-macrophages in the three groups while it did not impair phagocytosis and the expression of CD36 expression on cells in AS and IS at variance with controls.ConclusionsHypoxia had a significant impact on MD-macrophages from sarcoidosis patients, with the strongest effect seen in patients with high active disease. Therefore, hypoxia could play a significant role in sarcoidosis pathogenesis by increasing the macrophage proinflammatory response, maintaining phagocytosis and reducing antigen presentation, leading to a deficient T cell response. In addition, hypoxia could favor fibrosis by promoting profibrotic cytokines response and by sequestering fibroblasts in the vicinity of granulomas.

GSK-3 Inhibition Modulates Metalloproteases in a Model of Lung Inflammation and Fibrosis

Idiopathic pulmonary fibrosis (IPF) is mainly characterized by aberrant extracellular matrix deposition, consequent to epithelial lung injury and myofibroblast activation, and inflammatory response. Glycogen synthase kinase 3 (GSK-3) is a serine–threonine kinase involved in several pathways, and its inhibition has been already suggested as a therapeutic strategy for IPF patients. There is evidence that GSK-3 is able to induce matrix metalloproteinase (MMP) expression and that its inhibition modulates MMP expression in the tissues. The aim of our study was to investigate the role of GSK-3 and its inhibition in the modulation of MMP-9 and -2 in an in vivo mouse model of lung fibrosis and in vitro using different cell lines exposed to pro-inflammatory or pro-fibrotic stimuli. We found that GSK-3 inhibition down-modulates gene expression and protein levels of MMP-9, MMP-2, and their inhibitors TIMP-1 and TIMP-2 in inflammatory cells harvested from bronchoalveolar lavage fluid (BALF) of mice treated with bleomycin as well as in interstitial alveolar macrophages and cuboidalized epithelial alveolar cells. To the same extent, GSK-3 inhibition blunted the increased MMP-9 and MMP-2 activity induced by pro-fibrotic stimuli in a human lung fibroblast cell line. Moreover, the αSMA protein level, a marker of fibroblast-to-myofibroblast transition involved in fibrosis, was decreased in primary fibroblasts treated with TGFβ following GSK-3 inhibition. Our results confirm the implication of GSK-3 in lung inflammation and fibrosis, suggesting that it might play its role by modulating MMP expression and activity but also pushing fibroblasts toward a myofibroblast phenotype and therefore enhancing extracellular matrix deposition. Thus, its inhibition could represent a possible therapeutic strategy.

A Robust Protocol for Decellularized Human Lung Bioink Generation Amenable to 2D and 3D Lung Cell Culture

Decellularization efforts must balance the preservation of the extracellular matrix (ECM) components while eliminating the nucleic acid and cellular components. Following effective removal of nucleic acid and cell components, decellularized ECM (dECM) can be solubilized in an acidic environment with the assistance of various enzymes to develop biological scaffolds in different forms, such as sheets, tubular constructs, or three-dimensional (3D) hydrogels. Each organ or tissue that undergoes decellularization requires a distinct and optimized protocol to ensure that nucleic acids are removed, and the ECM components are preserved. The objective of this study was to optimize the decellularization process for dECM isolation from human lung tissues for downstream 2D and 3D cell culture systems. Following protocol optimization and dECM isolation, we performed experiments with a wide range of dECM concentrations to form human lung dECM hydrogels that were physically stable and biologically responsive. The dECM based-hydrogels supported the growth and proliferation of primary human lung fibroblast cells in 3D cultures. The dECM is also amenable to the coating of polyester membranes in Transwell™ Inserts to improve the cell adhesion, proliferation, and barrier function of primary human bronchial epithelial cells in 2D. In conclusion, we present a robust protocol for human lung decellularization, generation of dECM substrate material, and creation of hydrogels that support primary lung cell viability in 2D and 3D culture systems

THE INSULINOTROPIC EFFECT OF THIAZOLIDINEDIONE DERIVATIVES IN TYPE II DIABETIC MODEL, RIN-5F CELLS, AND ITS ACTION ON CULTURED HUMAN LUNG FIBROBLAST SHOWS OXYGEN FREE-RADICAL SCAVENGING ACTIVITY

Objective: Diabetes mellitus is a persistent disorder caused by deficiency in insulin production. Oxidative stress plays an important role in physiopathology of diabetes. The present research determined the antidiabetic and antioxidant effects of 5-naphthalidin thiazolidinediones derivatives. Methods: Both in vitro and in vivo experiments were carried out on diabetes induced male Swiss albino rats by single intramuscular injection of alloxan (0.15 mg/kg i.m) and IDDM-rats received 4b, 4c, or 4d (36 mg/kg, p.o). The derivatives were further subjected to insulin secretion by RIN-5F cells confirmed insulinotropic effect. In vitro antioxidant activity was evaluated using enzymatic activities on cell lines In addition, all the synthesized derivatives showed non-toxic effects against a diseased human lung fibroblast (COPD), HCC7231 (TACC CCL-96). Results: The results revealed that 4b, 4c, and 4d showed promising results by substantially lowering the blood glucose levels on 21st day of post-administration. Serum insulin levels substantially rose, suggesting that derivatives exert insulinotropic effects through different pathways. 4c showed oxygen free-radical scavenging activity. Conclusion: Our study proves that oral administration of 5-naphthalidin-TZD twice a day is a valuable treatment for type II diabetes. It seems to be a good drug moiety endowed significant oxygen free-radical scavenging activity.

Liberation from fibrogenesis or tumorigenesis via cellular senescence by a noble small molecule

Uncontrolled proliferative diseases such as fibrosis or cancers are fatal human disorders. Previously, we found that a chromone-scaffold derivative called ONG41008 had a strong anti-fibrotic effect on in vitro fibrogenesis as well as in a murine lung fibrosis model. It later occurred to our attention that while ONG41008 remarkably attenuated proliferation of diseased human lung myofibroblasts (DHLF), resulting in replicative senescence (RS) typified by cell flatness, normal human lung fibroblasts were not affected. Video demonstration revealed that RS was evident within 48hr after ONG41008 treatment. No ONG41008 affected activated caspase 3 and mitochondrial membrane potential in DHLF. An interactome study suggested that metabolic shift, chromatin remodeling, or cell cycle control may be required for the RS. This observation prompted us to be engaged in the cellular senescence of tumor cells. Clearly, senescent cells were conspicuously but temporarily observed in A549, adenocarcinomic human alveolar epithelial cells, giving us confidence that dysregulated cell proliferation could be a common underlying principle conserved in both DHLF and A549. An early phase of stimulation of A549 by ONG41008 led to RS followed by multinucleation (MNC), which has been known to be oncogene-induced senescence (OIS). MNC was immediately followed by apoptosis. Concomitant with massive upregulation of p16 and translocation to the nuclei, complete cell death of the remaining A549 occurred. Induction and nuclear translocation of p21was also noted in both A549 and DHLF stimulated with ONG41008. No induction of TP53 was seen but phosphorylation of TP53 was substantially increased in A549. Both immunocytochemistry and western blots corroborated these common senescent imaging features. With comparative analyses, it is clear that ONG41008 exhibited much lesser toxicity on normal human lung fibroblast than SAHA (suberoylanilide hydroxamic acid) and Nintedanib. Taken together, all these studies strongly suggest that ONG41008 is a potent inducer of RS or OIS, presumably resulting in cessation of the cell cycle are at G1 or G2 stage and/or systemic cell death. To our best knowledge, the liberation of uncontrolled proliferative cells from fibrogenesis or tumorigenesis by a small molecule in vitro is an unprecedented case. ONG41008 could be a potential and safe drug for a broad range of fibrotic diseases or tumorigenic diseases.