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Background: The calibrator in immunoassay plays an essential role in diagnosing Alzheimer's disease (AD). Presently, the most well-studied biomarkers for AD diagnosis are three phosphorylated Tau (p-Tau): p-Tau231, p-Tau217, and p-Tau181. Glycogen synthase-3beta (GSK3β)-phosphorated Tau-441 is the most commonly used calibrator for p-Tau immunoassays. However, the batch-tobatch inconsistency issue of the commonly used GSK3β-phosphorylated Tau-441 limits its clinical application. Methods: We have successfully generated and characterized 61 Tau monoclonal antibodies (mAbs) with distinct epitopes by using the hybridoma technique and employed them as capture or detection antibodies for p-Tau immunoassays. Through chemical synthesis, we synthesized calibrators, which are three peptides including capture and detection antibody epitopes, for application in immunoassays that detect p-Tau231, p-Tau217, and p-Tau181. The novel calibrators were applied to Enzyme-linked immunosorbent assay (ELISA) and Single-molecule array (Simoa) platforms to validate their applicability and establish a range of p-Tau immunoassays. Results: By employing the hybridoma technique, 49 mAbs recognizing Tau (1-22), nine mAbs targeting p-Tau231, one mAb targeting p-Tau217, and two mAbs targeting p-Tau181 were developed. Peptides, including recognition epitopes of capture and detection antibodies, were synthesized. These peptides were used as calibrators to develop 60 immunoassays on the ELISA platform, of which six highly sensitive immunoassays were selected and applied to the ultrasensitive Simoa platform. Remarkably, the LODs were 2.5, 2.4, 31.1, 32.9, 46.9, and 52.1 pg/ml, respectively. Conclusion: Three novel p-Tau calibrators were successfully generated and validated, which solved the batch-to-batch inconsistency issue of GSK3β-phosphorylated Tau-441. The novel calibrators exhibit the potential to promote the standardization of clinical AD diagnostic calibrators. Furthermore, we established a series of highly sensitive and specific immunoassays on the Simoa platform based on novel calibrators, which moved a steady step forward in p-Tau immunoassay application for AD diagnosis. [ABSTRACT FROM AUTHOR]

The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole. [ABSTRACT FROM AUTHOR]

The research work was aimed for isolation and identification of Aspergillus flavus from poultry feed samples using conventional and molecular techniques. Out of 23 samples, 8 (34.78%) were found positive for A. flavus. The molecular techniques comprise comparison of ITS1-5.8S-ITS2 region. In addition, the ability of the isolates to express aflatoxin synthase (CYP64A1) gene in synthetic liquid medium was also analyzed. Based on cultural characteristics, unique nature of the ITS 1 and 2 genes and expression of CYP64A1 at mRNA level, A. flavus was accurately identified from the feed samples. The isolated A. flavus would be a potential candidate for further studies and application. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) inhibition stands out as an innovative therapeutic strategy for combating infections caused by drug-resistant pathogens. In this study, we assessed the potential of 3-(2-isocyanobenzyl)-1 H -indole derivatives as novel quorum sensing inhibitors (QSIs). Initial screenings of their QS inhibitory activities were conducted against Pseudomonas aeruginosa PAO1 and Chromobacterium violaceum CV026. Notably, six 3-(2-isocyanobenzyl)-1 H -indole derivatives (4, 12, 25, 28, 32, and 33) exhibited promising QS, biofilms, and pyocyanin inhibitory activities under minimum inhibitory concentrations (MICs) against P. aeruginosa PAO1. Among them, 3-(2-isocyano-6-methylbenzyl)-1 H -indole (IMBI, 32) emerged as the most promising candidate, demonstrating superior biofilm and pyocyanin inhibition. Further comprehensive studies revealed that derivative 32 at 25 μg mL -1 inhibited biofilm formation by 70% against P. aeruginosa PAO1, as confirmed by scanning electron microscopy (SEM). Additionally, derivative 32 substantially increased the susceptibility of mature biofilms, leading to a 57% destruction of biofilm architecture. In terms of interfering with virulence factors in P. aeruginosa PAO1, derivative 32 (25 μg mL -1 ) displayed remarkable inhibitory effects on pyocyanin, protease, and extracellular polysaccharides (EPS) by 73%, 51%, and 37%, respectively, exceeding the positive control resveratrol (RSV). Derivative 32 at 25 μg mL -1 also exhibited effective inhibition of swimming and swarming motilities. Moreover, it downregulated the expressions of QS-related genes, including lasI , lasR , rhlI , rhlR , pqsR , sdhB , sucD , sodB , and PA5439 , by 1.82- to 10.87-fold. Molecular docking, molecular dynamics simulations (MD), and energy calculations further supported the stable binding of 32 to LasR, RhlI, RhlR, EsaL, and PqsR antagonizing the expression of QS-linked traits. Evaluation of the toxicity of derivative 32 on HEK293T cells via CCK-8 assay demonstrated low cytotoxicity. Overall, this study underscores the efficacy of derivative 32 in inhibiting virulence factors in P. aeruginosa . Derivative 32 emerges as a potential QSI for controlling P. aeruginosa PAO1 infections in vitro and an anti-biofilm agent for restoring or enhancing drug sensitivity in drug-resistant pathogens., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Quorum sensing (QS) inhibition is recognized as a novel antimicrobial target for infections caused by drug-resistant pathogens and is an attractive strategy for antipathogenic agent development. We designed and synthesized three parts of 3-(2-isocyanobenzyl)-1 H -indole derivatives and tested their activity as novel quorum sensing inhibitors (QSIs). 3-(2-Isocyanobenzyl)-1 H -indole derivatives demonstrated promising QS, biofilms, and prodigiosin inhibitory activities against Serratia marcescens at subminimum inhibitory concentrations (sub-MICs). In particular, 3-(2-isocyano-6-methylbenzyl)-1 H -indole (IMBI, 32 ) was identified as the best candidate based on several screening assays, including biofilm and prodigiosin inhibition. Further studies demonstrated that exposure to IMBI at 1.56 μg/mL to S. marcescens NJ01 significantly inhibited the formation of biofilms by 42%. The IMBI treatment on S. marcescens NJ01 notably enhanced the susceptibility of the formed biofilms, destroying the architecture of the biofilms by up to 40%, as evidenced by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). For interference of virulence factors in S. marcescens NJ01, IMBI at 3.12 μg/mL inhibited the activity of protease and extracellular polysaccharides (EPS) by 17% and 51%, respectively, which were higher than that of the positive control vanillic acid (VAN). Furthermore, IMBI downregulated the expression of QS- and biofilm-related genes fimA , bsmA , pigP , flhC , rssB , fimC , and rsmA by 1.02- to 2.74-fold. To confirm these findings, molecular docking was performed, which indicated that the binding of IMBI to SmaR, RhlI, RhlR, LasR, and CviR could antagonize the expression of QS-linked traits. In addition, molecular dynamic simulations (MD) and energy calculations indicated that the binding of receptors with IMBI was extremely stable. The biofilms of S. marcescens NJ01 were markedly reduced by 50% when IMBI (0.39 μg/mL) was combined with kanamycin (0.15 μg/mL). In conclusion, this study highlights the potency of IMBI in inhibiting the virulence factors of S. marcescens . IMBI has all the potential to be developed as an effective and efficient QS inhibitor and antibiofilm agent in order to restore or improve antimicrobial drug sensitivity.

Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae . Here, we identify the mutant PikC D50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikC D50N can mediate the drug-metabolizing reactions using the low-cost H 2 O 2 as a direct electron and oxygen donor.