Regulation of intracellular process by two-component systems: Exploring the mechanism of plasmid-mediated conjugative transfer.

Plasmid-mediated conjugative transfer facilitates the dissemination of antibiotic resistance, yet the comprehensive regulatory mechanisms governing this process remain elusive. Herein, we established pure bacteria and activated sludge conjugation system to investigate the regulatory mechanisms of conjugative transfer, leveraging metformin as an exogenous agent. Transcriptomic analysis unveiled that substantial upregulation of genes associated with the two-component system (e.g., AcrB/AcrA, EnvZ/Omp, and CpxA/CpxR) upon exposure to metformin. Furthermore, downstream regulators of the two-component system, including reactive oxygen species (ROS), cytoplasmic membrane permeability, and adenosine triphosphate (ATP) production, were enhanced by 1.7, 1.4 and 1.1 times, respectively, compared to the control group under 0.1 mg/L metformin exposure. Moreover, flow sorting and high-throughput sequencing revealed increased microbial community diversity among transconjugants in activated sludge systems. Notably, the antibacterial potential of human pathogenic bacteria (e.g., Bacteroides, Escherichia-Shigella, and Lactobacillus) was augmented, posing a potential threat to human health. Our findings shed light on the spread of antibiotic resistance bacteria and assess the ecological risks associated with plasmid-mediated conjugative transfer in wastewater treatment systems.

miR-124-3p and miR-194-5p regulation of the PI3K/AKT pathway via ROR2 in medulloblastoma progression.

Medulloblastoma (MB), a prevalent pediatric central nervous system tumor, is influenced by microRNAs (miRNAs) that impact tumor initiation and progression. However, the specific involvement of miRNAs in MB tumorigenesis remains unclear. Using single-cell RNA sequencing, we identified ROR2 expression in normal human fetal cerebellum. Subsequent analyses, including immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot, assessed ROR2 expression in MB tissues and cell lines. We investigated miR-124-3p and miR-194-5p and their regulatory role in ROR2 expression through the dual-luciferase reporter, qRT-PCR, and western blot assays. Mechanistic insights were gained through functional assays exploring the impact of miR-124-3p, miR-194-5p, and ROR2 on MB growth in vitro and in vivo. We observed significantly reduced miR-124-3p and miR-194-5p expression and elevated ROR2 expression in MB tissues and cell lines. High ROR2 expression inversely correlated with overall survival in WNT and SHH subgroups of MB patients. Functionally, overexpressing miR-124-3p and miR-194-5p and inhibiting ROR2 suppressed in vitro malignant transformation and in vivo tumorigenicity. Mechanistically, miR-124-3p and miR-194-5p synergistically regulated the ROR2/PI3K/Akt pathway, influencing MB progression. Our findings indicate that miR-124-3p and miR-194-5p function as tumor suppressors, inhibiting MB progression via the ROR2/PI3K/Akt axis, suggesting a key mechanism and therapeutic targets for MB patients.

Regulation of quorum sensing for the manipulation of conjugative transfer of antibiotic resistance genes in wastewater treatment system.

The emergence and transmission of antibiotic resistance genes (ARGs) through plasmid-mediated conjugation has become a significant worldwide public health threat. Biofilms are widely recognized as the primary reservoirs for ARGs, providing favorable conditions for horizontal gene transfer. Quorum sensing (QS) plays a critical role in bacterial biofilm formation, which further influences the spread of bacterial resistance. In this study, we examined the effects of vanillin, a QS inhibitor (QSI), at subinhibitory concentrations (sub-MICs) ranging from 0 - 0.1 g/L, on the transfer of ARGs between Escherichia coli and Pseudomonas aeruginosa. Our findings indicated that vanillin at sub-MICs inhibited the conjugative transfer frequency of the RP4 plasmid. This inhibition was supported by the downregulation of plasmid transfer genes. The suppression of conjugation can mainly be attributed to the inhibition of biofilm formation, the synthesis of extracellular polymeric substances (EPS), and the secretion of virulence factors, all of which are regulated by the bacterial QS system. On the other hand, the levels of ROS and cell membrane permeability were not primary explanations for this phenomenon. Furthermore, vanillin also reduced the conjugative transfer frequency of ARGs in wastewater effluent, providing a potential approach to alleviate bacterial resistance in water environments. These findings underscore the regulatory role of QSI in controlling ARGs transfer and have significant implications for manipulating the dissemination of bacterial resistance in the environment.

Preparation of Aromatic and Heterocyclic Amines by the Electrophilic Amination of Functionalized Diorganozincs with Polyfunctional O-2,6-Dichlorobenzoyl Hydroxylamines.

We report a catalyst-free electrophilic amination, which enables the synthesis of aromatic and heterocyclic amines. By subjecting diarylzinc or diheteroarylzinc compounds to readily accessible O-2,6-dichlorobenzoyl hydroxylamines in the presence of MgCl2 in dioxane at a temperature of 60 °C (8-16 h). This new electrophilic amination allowed an expedited synthesis of two pharmaceutically significant compounds: vortioxetine is a key intermediate of delamanid. This approach offers opportunities for the streamlined synthesis of amine-based molecules in the pharmaceutical industry.