Whole-cell biosensors have attracted considerable interests because they are robust, eco-friendly, and cost-effective. However, most of the biosensors harness the naturally occurring wild-type promoter, which often suffers from high background noise and low sensitivity. In this study, we demonstrate how to design the core elements (i.e., RNA polymerase binding site and transcription factor binding site) of the promoters to obtain a significant gain in the signal-to-noise output ratio of the whole-cell biosensor circuits. As a proof of concept, we modified the arsenite-regulated promoter from Escherichia coli K-12 genome, such that it has a lower background and higher expression. This was achieved by balancing the relationship between the number of ArsR binding sites (ABS) and the activity of the promoter and adjusting the location of the auxiliary ABS. A promoter variant ParsD-ABS-8 was obtained with an induction ratio of 179 (11-fold increase over the wild-type promoter) when induced with 1 μM arsenite. Importantly, the developed biosensor exhibited good dose–response in the range of 0.1 to 4 μM (R 2 = 0.9928) of arsenite with a detection limit of ca. 10 nM. These results indicated that the engineered promoter modification approach could be used to improve the performance of whole-cell biosensors, thereby facilitating their practical application.

BackgroundFarnesol has potential antifungal activity against Candida albicans biofilms, but the molecular mechanism of this activity is still unclear. Farnesol inhibits hyphal growth by regulating the cyclic AMP (cAMP) signalling pathway in C. albicans, and CYR1 and PDE2 regulate a pair of enzymes that are directly responsible for cAMP synthesis and degradation. Here, we hypothesize that farnesol enhances the antifungal susceptibility of C. albicans biofilms by regulating CYR1 and PDE2.ResultsThe resistance of the CYR1- and PDE2-overexpressing strains to caspofungin, itraconazole and terbinafine was increased in planktonic cells, and that to amphotericin B was increased in biofilms. Meanwhile, the biofilms of the CYR1- and PDE2-overexpressing strains were thicker (all p < 0.05) and consisted of more hyphae than that of the wild strain. The intracellular cAMP levels were higher in the biofilms of the CYR1-overexpressing strain than that in the biofilms of the wild strain (all p < 0.01), while no changes were found in the PDE2-overexpressing strain. Exogenous farnesol decreased the resistance of the CYR1- and PDE2-overexpressing strains to these four antifungals, repressed the hyphal and biofilm formation of the strains, and decreased the intracellular cAMP level in the biofilms (all p < 0.05) compared to the untreated controls. In addition, farnesol decreased the expression of the gene CYR1 and the protein CYR1 in biofilms of the CYR1-overexpressing strain (all p < 0.05) but increased the expression of the gene PDE2 and the protein PDE2 in biofilms of the PDE2-overexpressing strain (all p < 0.01).ConclusionsThe results indicate that CYR1 and PDE2 regulate the resistance of C. albicans biofilms to antifungals. Farnesol suppresses the resistance of C. albicans biofilms to antifungals by regulating the expression of the gene CYR1 and PDE2, while PDE2 regulation was subordinate to CYR1 regulation.

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Purpose: Dapansutrile is an orally active β-sulfonyl nitrile compound that selectively inhibits the NLRP3 inflammasome. Clinical studies have shown that dapansutrile is active in vivo and limits the severity of endotoxin-induced inflammation and joint arthritis. However, there is currently a lack of more in-depth research on the effect of dapansutrile on protein targets such as NLRP3 in gouty arthritis. Therefore, we used molecular docking and molecular dynamics to explore the mechanism of dapansutrile on NLRP3 and other related protein targets.Methods: We use bioinformatics to screen active pharmaceutical ingredients and potential disease targets. The disease-core gene target-drug network was established and molecular docking was used for verification. Molecular dynamics simulations were utilized to verify and analyze the binding stability of small molecule drugs to target proteins. The supercomputer platform was used to measure and analyze the binding free energy, the number of hydrogen bonds, the stability of the protein target at the residue level, the radius of gyration and the solvent accessible surface area.Results: The protein interaction network screened out the core protein targets (such as: NLRP3, TNF, IL1B) of gouty arthritis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that gouty arthritis mainly played a vital role by the signaling pathways of inflammation and immune response. Molecular docking showed that dapansutrile play a role in treating gouty arthritis by acting on the related protein targets such as NLRP3, IL1B, IL6, etc. Molecular dynamics was used to prove and analyze the binding stability of active ingredients and protein targets, the simulation results found that dapansutrile forms a very stable complex with IL1B.Conclusion: We used bioinformatics analysis and computer simulation system to comprehensively explore the mechanism of dapansutrile acting on NLRP3 and other protein targets in gouty arthritis. This study found that dapansutrile may not only directly inhibit NLRP3 to reduce the inflammatory response and pyroptosis, but also hinder the chemotaxis and activation of inflammatory cells by regulating IL1B, IL6, IL17A, IL18, MMP3, CXCL8, and TNF. Therefore, dapansutrile treats gouty arthritis by attenuating inflammatory response, inflammatory cell chemotaxis and extracellular matrix degradation by acting on multiple targets.