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Standard DNA-based analyses of microbial communities cannot distinguish between active microbes and dead or dormant cells. Here, Couradeau et al. use BONCAT (bioorthogonal non-canonical amino acid tagging), flow cytometry, and 16S rRNA gene amplicon sequencing to identify active microbial cells in soils.

This paper reports observations and reflections from a pedagogic intervention intended to enhance mathematics students' learning of course content in a course in multivariable calculus. The intervention design required students to structure, practice, and deliver an oral presentation that was a synthesis of the teacher's weekly lectures, for the benefit of themselves and their peers. The intervention appears to have incentivized students to engage with and learn mathematics contents in an effective way, and it also helped the mathematics teacher to reflect on, adapt, and develop his teaching strategies. To conclude, it would seem that both students and teachers in mathematics have much to gain from giving students opportunities to develop their own voice in learning contexts otherwise relying on books and lectures.

We performed whole genome analyses of DNA methylation in Shewanella17 oneidensis MR-1 to examine its possible role in regulating gene expression and18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing19 revealed extensive methylation of adenine (N6mA) throughout the20 genome. These methylated bases were located in five sequence motifs,21 including three novel targets for Type I restriction/modification enzymes. The22 sequence motifs targeted by putative methyltranferases were determined via23 SMRT sequencing of gene knockout mutants. In addition, we found S.24 oneidensis MR-1 cultures grown under various culture conditions displayed25 different DNA methylation patterns. However, the small number of differentially26 methylated sites could not be directly linked to the much larger number of27 differentially expressed genes in these conditions, suggesting DNA methylation is28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment29 of methylated GATC motifs in the origin of replication indicate DNA methylation30 may regulate genome replication in a manner similar to that seen in Escherichia31 coli. Furthermore, comparative analyses suggest that many32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.

Bladder cancer, one of the most prevalent malignancies worldwide, remains hard to classify due to a staggering molecular complexity. Despite a plethora of diagnostic tools and therapies, it is hard to outline the key steps leading up to the transition from high-risk non-muscle-invasive bladder cancer (NMIBC) to muscle-invasive bladder cancer (MIBC). Carcinogen-induced murine models can recapitulate urothelial carcinogenesis and natural anti-tumor immunity. Herein, we have developed and profiled a novel model of progressive NMIBC based on 10 weeks of OH-BBN exposure in hepatocyte growth factor/cyclin dependent kinase 4 (R24C) (Hgf-Cdk4R24C) mice. The profiling of the model was performed by histology grading, single cell transcriptomic and proteomic analysis, while the derivation of a tumorigenic cell line was validated and used to assess in vivo anti-tumor effects in response to immunotherapy. Established NMIBC was present in females at 10 weeks post OH-BBN exposure while neoplasia was not as advanced in male mice, however all mice progressed to MIBC. Single cell RNA sequencing analysis revealed an intratumoral heterogeneity also described in the human disease trajectory. Moreover, although immune activation biomarkers were elevated in urine during carcinogen exposure, anti-programmed cell death protein 1 (anti-PD1) monotherapy did not prevent tumor progression. Furthermore, anti-PD1 immunotherapy did not control the growth of subcutaneous tumors formed by the newly derived urothelial cancer cell line. However, treatment with CpG-oligodeoxynucleotides (ODN) significantly decreased tumor volume, but only in females. In conclusion, the molecular map of this novel preclinical model of bladder cancer provides an opportunity to further investigate pharmacological therapies ahead with regards to both targeted drugs and immunotherapies to improve the strategies of how we should tackle the heterogeneous tumor microenvironment in urothelial bladder cancer to improve responses rates in the clinic.

The ecology of plant viruses began to be explored at the end of the 19th century. Since then, major advances have revealed mechanisms of virus-host-vector interactions in various environments. These advances have been accelerated by new technlogies for virus detection and characterization, most recently including high throughput sequencing (HTS). HTS allows investigators, for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present. This powerful approach has spurred new investigation of the viral metagenome (virome). The rich virome datasets accumulated illuminate important ecological phenomena such as virus spread among host reservoirs (wild and domestic), effects of ecosystem simplification caused by human activities (and agriculture) on the biodiversity and the emergence of new viruses in crops. To be effective, however, HTS-based virome studies must successfully navigate challenges and pitfalls at each procedural step, from plant sampling to library preparation and bioinformatic analyses. This review summarizes major advances in plant virus ecology associated with technological developments, and then presents important considerations and best practices for HTS use in virome studies.

ABSTRACT Quantitative stable isotope probing (qSIP) estimates isotope tracer incorporation into DNA of individual microbes and can link microbial biodiversity and biogeochemistry in complex communities. As with any quantitative estimation technique, qSIP involves measurement error, and a fuller understanding of error, precision, and statistical power benefits qSIP experimental design and data interpretation. We used several qSIP data sets—from soil and seawater microbiomes—to evaluate how variance in isotope incorporation estimates depends on organism abundance and resolution of the density fractionation scheme. We assessed statistical power for replicated qSIP studies, plus sensitivity and specificity for unreplicated designs. As a taxon’s abundance increases, the variance of its weighted mean density declines. Nine fractions appear to be a reasonable trade-off between cost and precision for most qSIP applications. Increasing the number of density fractions beyond that reduces variance, although the magnitude of this benefit declines with additional fractions. Our analysis suggests that, if a taxon has an isotope enrichment of 10 atom% excess, there is a 60% chance that this will be detected as significantly different from zero (with alpha 0.1). With five replicates, isotope enrichment of 5 atom% could be detected with power (0.6) and alpha (0.1). Finally, we illustrate the importance of internal standards, which can help to calibrate per sample conversions of %GC to mean weighted density. These results should benefit researchers designing future SIP experiments and provide a useful reference for metagenomic SIP applications where both financial and computational limitations constrain experimental scope. IMPORTANCE One of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about their genomic content and potential functions but may not reflect an organism’s activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions but often fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and functional activity of specific microbes within a naturally occurring community. Here, we explore how the resolution of density gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional experimental replication, and discuss cost-benefit balanced scenarios for SIP experimental design.

ABSTRACT Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

Current knowledge of giant virus diversity is largely based on samples from aquatic environments. Here the authors employ cultivation-independent metagenomics and mini-metagenomics on forest soil and identify 16 novel giant virus genomes, some of which representing novel lineages, including the so far largest genome in Mimiviridae.

Abstract Background Nanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity. Results From 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs. Conclusions This population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

This article explores the redefinition and reframing of the American landscape by contemporary artists and thinkers. It acknowledges the historical dominance of White European-American men in landscape art and the need for diverse perspectives. The article focuses on an exhibition at the Thomas Cole National Historic Site that showcases the work of women artists, both historical and contemporary, aiming to expand our understanding of land and landscape. It also discusses the work of several contemporary artists who challenge traditional depictions of the American landscape, exploring themes of gender, race, history, and the environment. These artists invite viewers to question their understanding of the natural world and its representation in art. The article emphasizes the importance of sustainable and generative relationships with the land. [Extracted from the article]