A colorimetric sensing strategy based on enzyme@metal-organic framework and oxidase-like IrO2/MnO2 nanocomposite for α-glucosidase inhibitor screening.

A highly sensitive colorimetric sensing strategy based on enzyme@metal-organic framework (GAA@Cu-MOF) and IrO2/MnO2 nanocomposite was exploited innovatively for screening of α-glucosidase (GAA) inhibitors. IrO2/MnO2 nanocomposite exhibits excellent oxidase-mimicking activity which can directly catalyze the oxidation of 3,3,5,5,-tetramethylbenzidine (TMB) into a blue product with an absorption maximum at 652 nm. And GAA@Cu-MOF can decompose L-ascorbic acid-2-O-α-D-glucopyranosyl (AAG) to ascorbic acid (AA). The produced AA can destroy the IrO2/MnO2 nanocomposite and reduce its oxidase-like activity. However, the generation of AA is restricted when GAA inhibitors are added to the system, which allows the oxidase-like activity of the IrO2/MnO2 nanocomposite to be maintained. In view of this, a method for screening of GAA inhibitors was developed. In addition to enhancing the stability of GAA, the method can also effectively avoid the potential interference of H2O2 in the screening process of GAA inhibitors, which helps to improve the sensitivity of the method. Therefore, highly sensitive determination for acarbose and ascorbic acid are achieved with detection limits of 6.27 nM and 1.23 µM, respectively. The proposed method was successfully applied to screen potential GAA inhibitors from oleanolic acid derivatives. Graphical abstract.

Ecdysone Elicits Chronic Renal Impairment via Mineralocorticoid-Like Pathogenic Activities.

BACKGROUND/AIMS: Ecdysteroids are steroidal insect molting hormones that also exist in herbs. Ecdysteroid-containing adaptogens have been popularly used to improve well-being and by bodybuilders for muscle growth. However, the use of ecdysone in mammals is also associated with kidney growth and enlargement, indications of disturbed kidney homeostasis. The underlying pathogenic mechanism remains to be clarified. METHODS: Virtual screening tools were employed to identify compounds that are homologous to ecdysone and to predict putative ecdysone-interacting proteins. The kidney effect of ecdysone was examined in vitro and in vivo and compared with that of aldosterone. Cellular apoptosis was estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Cell motility was assessed by scratch-wound cell migration assay. Blood urea nitrogen was measured to evaluate renal function. Western immunblot analysis was employed to determine the expression profile of interested proteins. RESULTS: Computational molecular structure analysis revealed that ecdysone is highly homologous to aldosterone. Moreover, virtual screening based on compound-protein interaction profiles identified the Mineralocorticoid Receptor (MR) to potentially interact with ecdysone. Accordingly, to assess potential biological functions of ecdysone in mammals, ecdysone was applied to mineralocorticoid-sensitive inner medullar collecting duct cells. Ecdysone induced mesenchymal accumulation of extracellular matrix and epithelial dedifferentiation characterized by de novo expression of α-smooth muscle actin. In addition, ecdysone elicited cellular apoptosis and retarded cell motility, akin to the effect of aldosterone. In vivo, daily treatment of mice with ecdysone increased cell apoptosis in the kidney, impaired renal function and elicited early signs of renal fibrogenesis, marked by deposition of collagen and fibronectin in tubulointerstitium, reminiscent of the action of aldosterone. The MR signaling pathway is likely responsible for the cellular and pathobiological effects of ecdysone, as evidenced by strong ecdysone-induced MR nuclear translocation in renal tubular cells both in vitro and in vivo, while blockade of MR by concomitant spironolactone treatment largely abolished the detrimental effects of ecdysone. CONCLUSION: Our findings suggest that ecdysone induces mineralocorticoid-dependent activities that impair renal function and elicit renal injury.

An ultrasensitive colorimetric assay based on a multi-amplification strategy employing Pt/IrO2@SA@HRP nanoflowers for the detection of progesterone in saliva samples.

Progesterone (P4) belongs to a factor that affects stress response and is a potential carcinogen, and saliva levels are expected to be a standard measurement for clinical diagnosis. In this study, a new type of nanoflower with both recognition functionality and catalytic substrate ability was prepared by copper phosphate, Pt/IrO2 nanocomposites (Pt/IrO2 NPs), streptavidin (SA) and horseradish peroxidase (HRP) via a one-pot co-precipitation strategy. Due to the enhanced catalytic activity and stability of Pt/IrO2@SA@HRP nanoflowers, we developed a powerful and sensitive multiple-catalysis ELISA to monitor progesterone in saliva. Multiple-catalysis ELISA based on a specific antibody and Pt/IrO2@SA@HRP nanoflowers exhibited a linear interval range from 0.217 ng mL-1 to 7.934 ng mL-1. The median inhibitory concentration (IC50) for progesterone is 1.311 ng mL-1 and the limit of detection (LOD = IC10) is 0.076 ng mL-1 in the proposed method. Satisfactory recoveries were in a range of 79.6-107% with an acceptable coefficient of variation (below 10.6%). Results of the multiple-catalysis ELISA and LC-MS/MS had a good coincidence. Our result unraveled that multiple-catalysis ELISA is a potentially serviceable tool for the detection of progesterone in saliva.

Au-Au/IrO2@Cu(PABA) Reactor with Tandem Enzyme-Mimicking Catalytic Activity for Organic Dye Degradation and Antibacterial Application.

Herein, a Au-Au/IrO2 nanocomposite with tandem enzyme-mimicking activity was innovatively synthesized, which can show outstanding glucose oxidase (GOx)-like activity and peroxidase-like activity simultaneously under neutral conditions. Moreover, a Au-Au/IrO2@Cu(PABA) reactor was prepared via encapsulation of the Au-Au/IrO2 nanocomposite in a Cu(PABA) metal organic framework. The reactor not only exhibits excellent organic solvent stability, acid resistance, and reusability but also displays better cascade reaction catalytic efficiency (kcat/Km = 148.86 min-1 mM-1) than the natural free enzyme system (GOx/HRP) (kcat/Km = 98.20 min-1 mM-1) and Au-Au/IrO2 nanocomposite (kcat/Km = 135.24 min-1 mM-1). In addition, it is found that the reactor can catalyze glucose or dissolved oxygen to produce active oxygen species (ROS) including HO, 1O2, and O2-· through its enzyme-mimicking activity. Finally, the novel reactor was successfully used in organic dye degradation and antibacterial application. The results show that it can effectively degrade methyl orange, methylene blue, and rhodamine B, which all can reach a degradation rate of nearly 100% after interacting with Au-Au/IrO2@Cu (PABA) for 3.5 h. Furthermore, the reactor also exhibits excellent antibacterial activity, so as to achieve a complete bactericidal effect to Staphylococcus aureus and Escherichia coli at a concentration of 12.5 µg mL-1.

Production and characteristics of a novel chicken egg yolk antibody (IgY) against periodontitis-associated pathogens.

Periodontitis is a bacterial biofilm-induced oral disease, mostly caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas gingivalis (P. gingivalis). Oral administration of chicken egg yolk antibody (IgY) is a promising nutritional strategy to control pathogen infections. The objective of this study was to produce an A. actinomycetemcomitans- and P. gingivalis-specific IgY and evaluate its effects on bacterial agglutination and biofilm formation. Thirty laying hens were immunized with a complex of lysate containing typical molecular weights of membrane proteins of A. actinomycetemcomitans and P. gingivalis. IgY was isolated by polyethylene glycol 6000 and ammonium sulfate and purified by dialysis. The results of enzyme-linked immunosorbent assay showed that the obtained IgY were specific to both A. actinomycetemcomitans and P. gingivalis. In addition, immunoelectron microscopy scanning and crystal violet staining showed that the IgY could bind to cell wall of the pathogens and efficiently accelerate agglutination and inhibit biofilm formation. Furthermore, the activity of the IgY remained stable at different temperature, pH, and storage period. This is the first report that a novel two-in-one IgY was produced to modulate the agglutination and biofilm formation of A. actinomycetemcomitans and P. gingivalis, suggesting the potential of IgY to control periodontitis caused by oral pathogens.

GSK3ß-mediated Keap1-independent regulation of Nrf2 antioxidant response: A molecular rheostat of acute kidney injury to chronic kidney disease transition.

Transition of acute kidney injury (AKI) to chronic kidney disease (CKD) represents an important cause of kidney failure. However, how AKI is transformed into CKD remains elusive. Following folic acid injury, mice developed AKI with ensuing CKD transition, featured by variable degrees of interstitial fibrosis and tubular cell atrophy and growth arrest. This lingering injury of renal tubules was associated with sustained oxidative stress that was concomitant with an impaired Nrf2 antioxidant defense, marked by mitigated Nrf2 nuclear accumulation and blunted induction of its target antioxidant enzymes, like heme oxygenase (HO)-1. Activation of the canonical Keap1/Nrf2 signaling, nevertheless, seems intact during CKD transition because Nrf2 in injured tubules remained activated and elevated in cytoplasm. Moreover, oxidative thiol modification and activation of Keap1, the cytoplasmic repressor of Nrf2, was barely associated with CKD transition. In contrast, glycogen synthase kinase (GSK)3ß, a key modulator of the Keap1-independent Nrf2 regulation, was persistently overexpressed and hyperactive in injured tubules. Likewise, in patients who developed CKD following AKI due to diverse etiologies, like volume depletion and exposure to radiocontrast agents or aristolochic acid, sustained GSK3ß overexpression was evident in renal tubules and coincided with oxidative damages, impaired Nrf2 nuclear accumulation and mitigated induction of antioxidant gene expression. Mechanistically, the Nrf2 response against oxidative insult was sabotaged in renal tubular cells expressing a constitutively active mutant of GSK3ß, but reinforced by ectopic expression of dominant negative GSK3ß in a Keap1-independent manner. In vivo in folic acid-injured mice, targeting GSK3ß in renal tubules via conditional knockout or by weekly microdose lithium treatment reinstated Nrf2 antioxidant response in the kidney and hindered AKI to CKD transition. Ergo, our findings suggest that GSK3ß-mediated Keap1-independent regulation of Nrf2 may serve as an actionable therapeutic target for modifying the long-term sequelae of AKI.

Activation of mineralocorticoid receptor by ecdysone, an adaptogenic and anabolic ecdysteroid, promotes glomerular injury and proteinuria involving overactive GSK3ß pathway signaling.

Ecdysone is an arthropod molting hormone and has been marketed as a non-androgenic natural anabolic and adaptogen. However, the safety profile of ecdysone is largely undetermined. After ecdysone treatment for 2 weeks, mice developed albuminuria with histologic signs of glomerular injury, including hypertrophy, mesangial expansion, mild glomerulosclerosis and podocyte injury. A direct glomerulopathic activity of ecdysone seems to contribute, since addition of ecdysone to cultured glomerular cells induced cytopathic changes, including apoptosis, activation of mesangial cells, podocyte shape changes and a decreased expression of podocyte markers. To explore the molecular target responsible for the pathogenic actions, we employed an in silico modeling system of compound-protein interaction and identified mineralocorticoid receptor (MR) as one of the top-ranking proteins with putative interactions with ecdysone. The molecular structure of ecdysone was highly homologous to mineralocorticoids, like aldosterone. Moreover, ecdysone was capable of both inducing and activating MR, as evidenced by MR nuclear accumulation in glomerular cells both in vitro and in vivo following ecdysone treatment. Mechanistically, glycogen synthase kinase (GSK) 3ß, which has been recently implicated in pathogenesis of glomerular injury and proteinuria, was hyperactivated in glomeruli in ecdysone-treated mice, concomitant with diverse glomerulopathic changes. In contrast, spironolactone, a selective blockade of MR, largely abolished the cytopathic effect of ecdysone in vitro and attenuated albuminuria and glomerular lesions in ecdysone treated mice, associated with a mitigated GSK3ß overactivity in glomeruli. Altogether, ecdysone seems able to activate MR and thereby promote glomerular injury and proteinuria involving overactive GSK3ß pathway signaling.

Valproate hampers podocyte acquisition of immune phenotypes via intercepting the GSK3ß facilitated NFkB activation.

Glomerular podocytes are able to transdifferentiate under disease conditions, acquire de novo immune phenotypes and behave as immunocompetent cells, like phagocytes or antigen-presenting cells. Upon stimulation with lipopolysaccharide (LPS), a prototypical pathogen-associated molecular pattern, podocytes demonstrated de novo expression of a variety of NFkB-dependent immune molecules that are pivotal for immune response, including major histocompatibility complex (MHC) class II, costimulatory molecule CD80, lysosomal protease cathepsin L as well as CC chemokine ligand 2 and 5, ultimately resulting in podocyte dysfunction, characterized by cellular shrinkage, a spindle-like or asterlike cell shape and impairment of actin cytoskeleton integrity. The LPS-elicited podocyte phenotypic changes were concurrent with nuclear factor (NF) kB phosphorylation, which was associated with glycogen synthase kinase (GSK) 3ß overactivity, marked by a diminished inhibitory phosphorylation of GSK3ß. In contrast, valproate, an anticonvulsant and mood stabilizer, offset GSK3ß overactivity in LPS-injured podocytes and mitigated NFkB activation and podocyte acquisition of immune phenotypes as well as the ensuing cytopathic changes, podocyte cytoskeleton disorganization and dysfunction. The protective effect of valproate was strikingly blunted in podocytes expressing the constitutively active GSK3ß, suggesting an essential role of inhibitory phosphorylation of GSK3ß. In vivo in LPS-injured mice, valproate therapy abolished GSK3ß overactivity in glomeruli and attenuated podocyte injury and albuminuria, concomitant with a lessened NFkB activation and diminished induction of diverse podocytopathic immune molecules in podocytes and glomeruli. Taken together, valproate directly protects against podocyte injury and hampers podocyte acquisition of de novo immune phenotypes via intercepting the GSK3ß facilitated NFkB activation.