The AMPK-related kinase NUAK1 controls cortical axons branching by locally modulating mitochondrial metabolic functions.

The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.

Quantification of human enteric viruses as alternative indicators of fecal pollution to evaluate wastewater treatment processes.

We investigated the potential use and quantification of human enteric viruses in municipal wastewater samples of Winnipeg (Manitoba, Canada) as alternative indicators of contamination and evaluated the processing stages of the wastewater treatment plant. During the fall 2019 and winter 2020 seasons, samples of raw sewage, activated sludge, effluents, and biosolids (sludge cake) were collected from the North End Sewage Treatment Plant (NESTP), which is the largest wastewater treatment plant in the City of Winnipeg. DNA (Adenovirus and crAssphage) and RNA enteric viruses (Pepper mild mottle virus, Norovirus genogroups GI and GII, Rotavirus Astrovirus, and Sapovirus) as well as the uidA gene found in Escherichia coli were targeted in the samples collected from the NESTP. Total nucleic acids from each wastewater treatment sample were extracted using a commercial spin-column kit. Enteric viruses were quantified in the extracted samples via quantitative PCR using TaqMan assays. Overall, the average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.1023 and 0.9921) than the average gene copies assessed in the effluents for DNA and RNA viruses and uidA in terms of both volume and biomass. A significant reduction (p-value ≤ 0.0438) of Adenovirus and Noroviruses genogroups GI and GII was observed in activated sludge samples compared with those for raw sewage per volume. Higher GCNs of enteric viruses were observed in dewatered sludge samples compared to liquid samples in terms of volume (g of sample) and biomass (ng of nucleic acids). Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA, indicating that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Viruses such as Noroviruses genogroups GI and GII, and Rotavirus were detected during colder months. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used confidently as complementary viral indicators of human fecal pollution.

Metagenomic community composition and resistome analysis in a full-scale cold climate wastewater treatment plant.

BACKGROUND: Wastewater treatment plants are an essential part of maintaining the health and safety of the general public. However, they are also an anthropogenic source of antibiotic resistance genes. In this study, we characterized the resistome, the distribution of classes 1-3 integron-integrase genes (intI1, intI2, and intI3) as mobile genetic element biomarkers, and the bacterial and phage community compositions in the North End Sewage Treatment Plant in Winnipeg, Manitoba. Samples were collected from raw sewage, returned activated sludge, final effluent, and dewatered sludge. A total of 28 bacterial and viral metagenomes were sequenced over two seasons, fall and winter. Integron-integrase genes, the 16S rRNA gene, and the coliform beta-glucuronidase gene were also quantified during this time period. RESULTS: Bacterial classes observed above 1% relative abundance in all treatments were Actinobacteria (39.24% ± 0.25%), Beta-proteobacteria (23.99% ± 0.16%), Gamma-proteobacteria (11.06% ± 0.09%), and Alpha-proteobacteria (9.18 ± 0.04%). Families within the Caudovirales order: Siphoviridae (48.69% ± 0.10%), Podoviridae (23.99% ± 0.07%), and Myoviridae (19.94% ± 0.09%) were the dominant phage observed throughout the NESTP. The most abundant bacterial genera (in terms of average percent relative abundance) in influent, returned activated sludge, final effluent, and sludge, respectively, includes Mycobacterium (37.4%, 18.3%, 46.1%, and 7.7%), Acidovorax (8.9%, 10.8%, 5.4%, and 1.3%), and Polaromonas (2.5%, 3.3%, 1.4%, and 0.4%). The most abundant class of antibiotic resistance in bacterial samples was tetracycline resistance (17.86% ± 0.03%) followed by peptide antibiotics (14.24% ± 0.03%), and macrolides (10.63% ± 0.02%). Similarly, the phage samples contained a higher prevalence of macrolide (30.12% ± 0.30%), peptide antibiotic (10.78% ± 0.13%), and tetracycline (8.69% ± 0.11%) resistance. In addition, intI1 was the most abundant integron-integrase gene throughout treatment (1.14 × 104 gene copies/mL) followed by intI3 (4.97 × 103 gene copies/mL) while intI2 abundance remained low (6.4 × 101 gene copies/mL). CONCLUSIONS: Wastewater treatment successfully reduced the abundance of bacteria, DNA phage and antibiotic resistance genes although many antibiotic resistance genes remained in effluent and biosolids. The presence of integron-integrase genes throughout treatment and in effluent suggests that antibiotic resistance genes could be actively disseminating resistance between both environmental and pathogenic bacteria.

SetRICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response.

The integrating conjugative element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA polymerase V ortholog, polVICE391, encoded by the ICE391 rumAB operon. polV and its orthologs have previously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in vivo. As a result, multiple levels of regulation are imposed on the polymerases so as to avoid aberrant mutagenesis. We report here, that the mutagenesis-promoting activity of polVICE391 is additionally regulated by a transcriptional repressor encoded by SetRICE391, since Escherichia coli expressing SetRICE391 demonstrated reduced levels of polVICE391-mediated spontaneous mutagenesis relative to cells lacking SetRICE391. SetRICE391 regulation was shown to be specific for the rumAB operon and in vitro studies with highly purified SetRICE391 revealed that under alkaline conditions, as well as in the presence of activated RecA, SetRICE391 undergoes a self-mediated cleavage reaction that inactivates repressor functions. Conversely, a non-cleavable SetRICE391 mutant capable of maintaining repressor activity, even in the presence of activated RecA, exhibited low levels of polVICE391-dependent mutagenesis. Electrophoretic mobility shift assays revealed that SetRICE391 acts as a transcriptional repressor by binding to a site overlapping the -35 region of the rumAB operon promoter. Our study therefore provides evidence indicating that SetRICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response.