Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium.

The genus Pythium (nom. cons.) sensu lato (s.l.) is composed of many important species of plant pathogens. Early molecular phylogenetic studies suggested paraphyly of Pythium, which led to a formal proposal by Uzuhashi and colleagues in 2010 to split the genus into Pythium sensu stricto (s.s.), Elongisporangium, Globisporangium, Ovatisporangium (= Phytopythium), and Pilasporangium using morphological characters and phylogenies of the mt cytochrome c oxidase subunit 2 (cox2) and D1-D2 domains of nuc 28S rDNA. Although the split was fairly justified by the delineating morphological characters, there were weaknesses in the molecular analyses, which created reluctance in the scientific community to adopt these new genera for the description of new species. In this study, this issue was addressed using phylogenomics. Whole genomes of 109 strains of Pythium and close relatives were sequenced, assembled, and annotated. These data were combined with 10 genomes sequenced in previous studies. Phylogenomic analyses were performed with 148 single-copy genes represented in at least 90% of the taxa in the data set. The results showed support for the division of Pythium s.l. The status of alternative generic names that have been used for species of Pythium in the past (e.g., Artotrogus, Cystosiphon, Eupythium, Nematosporangium, Rheosporangium, Sphaerosporangium) was investigated. Based on our molecular analyses and review of the Pythium generic concepts, we urge the scientific community to adopt the generic names Pythium, Elongisporangium, Globisporangium, and their concepts as proposed by Uzuhashi and colleagues in 2010 in their work going forward. In order to consolidate the taxonomy of these genera, some of the recently described Pythium spp. are transferred to Elongisporangium and Globisporangium.

Elevated cortisol lowers thermal tolerance but results in limited cardiac remodelling in rainbow trout (Oncorhynchus mykiss) experiencing chronic social stress.

Juvenile rainbow trout (Oncorhynchus mykiss) held in pairs form dominance hierarchies in which subordinate individuals experience chronic social stress accompanied by lowered thermal tolerance (assessed as the critical thermal maximum, CTmax). Here, we tested the hypothesis that chronic elevation of circulating cortisol levels reduces thermal tolerance in subordinate trout. In support of this hypothesis, subordinate trout that recovered from social stress for 48 h, a period sufficient to return cortisol to normal baseline levels, no longer showed reduced CTmax. Further, thermal tolerance was not restored in subordinates treated with cortisol during recovery from social stress. To explore possible mechanisms underlying the effect of chronic stress on CTmax, we also tested the hypothesis that chronic cortisol elevation induces cardiac remodelling in subordinate trout, as previously reported for cortisol-treated rainbow trout. Ventricle mass and cardiac hypertrophy markers were unaffected by social stress. Picrosirius Red staining revealed a trend for lower collagen levels in the ventricles of subordinate relative to dominant trout. However, collagen type I transcript and protein levels, and markers of collagen turnover were unaffected. Indicators of cardiac function, including ventricle passive stiffness and intrinsic heart rate (fH), similarly were unaffected. In vivo fH was also similar between subordinate and dominant fish. Nevertheless, in keeping with their lower CTmax, subordinate fish exhibited cardiac arrhythmia at significantly lower temperatures than dominant fish during CTmax trials. Thus, high baseline cortisol levels in subordinate trout result in lowered thermal tolerance, but 5 days of social stress did not greatly affect cardiac structure or function.

Soil exposed to silver nanoparticles reveals significant changes in community structure and altered microbial transcriptional profiles.

Anthropogenic activities can disrupt soil ecosystems, normally resulting in reduced soil microbial health. Regulatory agencies need to determine the effects of uncharacterized substances on soil microbial health to establish the safety of these chemicals if they end up in the environment. Previous work has focused on measuring traditional ecotoxicologial endpoints within the categories of microbial biomass, activity, and community structure/diversity. Because these tests can be labor intensive, lengthy to conduct, and cannot measure changes in individual gene functions, we wanted to establish whether metatranscriptomics could be used as a more sensitive endpoint and provide a perspective on community function that is more informative than taxonomic identification of microbes alone. We spiked a freshly collected sandy loam soil (Vulcan, Alberta, Canada) with 0, 60, 145, 347, 833, and 2000 mg kg-1 of silver nanoparticles (AgNPs), a known antagonist of microorganisms due to its propensity for dissolution of toxic silver ions. Assessments performed in our previous work using traditional tests demonstrated the toxicity of AgNPs on soil microbial processes. We expanded this analysis with genomics-based tests by measuring changes in community taxonomic structure and function using 16S rDNA profiling and metatranscriptomics. In addition to identifying bacterial taxa affected by AgNPs, we found that genes involved in heavy metal resistance (e.g., the CzcA efflux pump) and other toxicity response pathways were highly upregulated in the presence of silver. Dose-response analysis using BMDExpress2 software successfully modeled many physiologically relevant genes responding to low concentrations of AgNPs. We found that the transcriptomic point of departure (BMD50) was lower than the IC50s calculated using the traditional tests in our previous work. These results suggest that dose-response modeling of metatranscriptomic gene expression is a useful tool in soil microbial health assessment. SUMMARY: Genomics-based endpoints for the assessment of soil microbial health can be used to perform quantitative dose-response modeling, and soil-based RNAseq adds functional insights.

Draft Genome Sequence of the Industrially Significant Bacterium Bacillus amyloliquefaciens NRRL 942.

Bacillus amyloliquefaciens NRRL 942 is a Gram-positive bacterium with several potential industrial uses. We have sequenced the whole genome of this organism to assist in understanding the biological mechanisms that might modulate human health or environmental risk in the event of its release into the environment.