Diversity of antibiotic resistance gene variants at subsequent stages of the wastewater treatment process revealed by a metagenomic analysis of PCR amplicons.

Wastewater treatment plants have been recognised as point sources of various antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) which are considered recently emerging biological contaminants. So far, culture-based and molecular-based methods have been successfully applied to monitor antimicrobial resistance (AMR) in WWTPs. However, the methods applied do not permit the comprehensive identification of the true diversity of ARGs. In this study we applied next-generation sequencing for a metagenomic analysis of PCR amplicons of ARGs from the subsequent stages of the analysed WWTP. The presence of 14 genes conferring resistance to different antibiotic families was screened by PCR. In the next step, three genes were selected for detailed analysis of changes of the profile of ARG variants along the process. A relative abundance of 79 variants was analysed. The highest diversity was revealed in the ermF gene, with 52 variants. The relative abundance of some variants changed along the purification process, and some ARG variants might be present in novel hosts for which they were currently unassigned. Additionally, we identified a pool of novel ARG variants present in the studied WWTP. Overall, the results obtained indicated that the applied method is sufficient for analysing ARG variant diversity.

Long-term mitochondrial stress induces early steps of Tau aggregation by increasing reactive oxygen species levels and affecting cellular proteostasis.

Accumulating evidence indicates that mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases. Both of these conditions are often associated with an increase in protein aggregation. However, still unknown are the specific defects of mitochondrial biology that play a critical role in the development of Alzheimer's disease, in which Tau protein aggregates are observed in the brains of some patients. Here, we report that long-term mitochondrial stress triggered Tau dimerization, which is the first step of protein aggregation. Mitochondrial dysfunction was induced in HEK293T cells that received prolonged treatment with rotenone and in HEK293T cells with the knockout of NDUFA11 protein. To monitor changes in Tau protein aggregation, we took advantage of the bimolecular fluorescence complementation assay using HEK293T cells that were transfected with plasmids that encoded Tau. Inhibition of the ISR with ISRIB induced Tau dimerization, whereas ISR activation with salubrinal, guanabenz, and sephin1 partially reversed this process. Cells that were treated with ROS scavengers, N-acetyl-l-cysteine or MitoQ, significantly reduced the amount of ROS and Tau dimerization, indicating the involvement of oxidative stress in Tau aggregation. Our results indicate that long-term mitochondrial stress may induce early steps of Tau protein aggregation by affecting oxidative balance and cellular proteostasis.