Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Genetic and Anatomical Basis of the Barrier Separating Wakefulness and Anesthetic-Induced Unresponsiveness

A robust, bistable switch regulates the fluctuations between wakefulness and natural sleep as well as those between wakefulness and anesthetic-induced unresponsiveness. We previously provided experimental evidence for the existence of a behavioral barrier to transitions between these states of arousal, which we call neural inertia. Here we show that neural inertia is controlled by processes that contribute to sleep homeostasis and requires four genes involved in electrical excitability: Sh, sss, na and unc79. Although loss of function mutations in these genes can increase or decrease sensitivity to anesthesia induction, surprisingly, they all collapse neural inertia. These effects are genetically selective: neural inertia is not perturbed by loss-of-function mutations in all genes required for the sleep/wake cycle. These effects are also anatomically selective: sss acts in different neurons to influence arousal-promoting and arousal-suppressing processes underlying neural inertia. Supporting the idea that anesthesia and sleep share some, but not all, genetic and anatomical arousal-regulating pathways, we demonstrate that increasing homeostatic sleep drive widens the neural inertial barrier. We propose that processes selectively contributing to sleep homeostasis and neural inertia may be impaired in pathophysiological conditions such as coma and persistent vegetative states.     

Unifying Charge Generation,Recombination, and Extraction in Low‐Offset Non‐Fullerene Acceptor Organic Solar Cells

Even though significant breakthroughs with over 18% power conversion efficiencies (PCEs) in polymer:non‐fullerene acceptor (NFA) bulk heterojunction organic solar cells (OSCs) have been achieved, not many studies have focused on acquiring a comprehensive understanding of the underlying mechanisms governing these systems. This is because it can be challenging to delineate device photophysics in polymer:NFA blends comprehensively, and even more complicated to trace the origins of the differences in device photophysics to the subtle differences in energetics and morphology. Here, a systematic study of a series of polymer:NFA blends is conducted to unify and correlate the cumulative effects of i) voltage losses, ii) charge generation efficiencies, iii) non‐geminate recombination and extraction dynamics, and iv) nuanced morphological differences with device performances. Most importantly, a deconvolution of the major loss processes in polymer:NFA blends and their connections to the complex BHJ morphology and energetics are established. An extension to advanced morphological techniques, such as solid‐state NMR (for atomic level insights on the local ordering and donor:acceptor π? π interactions) and resonant soft X‐ray scattering (for donor and acceptor interfacial area and domain spacings), provide detailed insights on how efficient charge generation, transport, and extraction processes can outweigh increased voltage losses to yield high PCEs.     

A practical guide to measuring functional indicators and traits in biocrusts

Biocrusts are multifunctional communities that are increasingly being used to restore degraded or damaged ecosystems. Concurrently, restoration science is shifting away from the use of purely structural metrics, such as relative abundance, to more functional approaches. Although biocrust restoration technology is advancing, there is a lack of readily available information on how to monitor biocrust functioning and set appropriate restoration goals. We therefore compiled a selection of 22 functional indicators that can be used to monitor biocrust functions, such as CO₂ exchange as an indicator of productivity or soil aggregate stability as a proxy for erosion resistance. We describe the functional importance of each indicator and the available protocols with which it may be measured. The majority of indicators can be measured as a functional trait of species by using patches of biocrust or cultures that contain only one species. Practitioners wishing to track the multifunctionality of an entire biocrust community would be advised to choose one indicator from each broad functional group (erosion resistance, nutrient accumulation, productivity, energy balance, hydrology), whereas a targeted approach would be more appropriate for projects with a key function of interest. Because predisturbance data are rarely available for biocrust functions, restoration goals can be based on a closely analogous site, literature values, or an expert elicitation process. Finally, we advocate for the establishment of a global trait database for biocrusts, which would reduce the damage resulting from repeated sampling, and provide a wealth of future research opportunities.     

Balancing selection in Pattern Recognition Receptor signalling pathways is associated with gene function and pleiotropy in a wild rodent

Pathogen‐mediated balancing selection is commonly considered to play an important role in the maintenance of genetic diversity, in particular in immune genes. However, the factors that may influence which immune genes are the targets of such selection are largely unknown. To address this, here we focus on Pattern Recognition Receptor (PRR) signalling pathways, which play a key role in innate immunity. We used whole‐genome resequencing data from a population of bank voles (Myodes glareolus) to test for associations between balancing selection, pleiotropy and gene function in a set of 123 PRR signalling pathway genes. To investigate the effect of gene function, we compared genes encoding (a) receptors for microbial ligands versus downstream signalling proteins, and (b) receptors recognizing components of microbial cell walls, flagella and capsids versus receptors recognizing features of microbial nucleic acids. Analyses based on the nucleotide diversity of full coding sequences showed that balancing selection primarily targeted receptor genes with a low degree of pleiotropy. Moreover, genes encoding receptors recognizing components of microbial cell walls etc. were more important targets of balancing selection than receptors recognizing nucleic acids. Tests for localized signatures of balancing selection in coding and noncoding sequences showed that such signatures were mostly located in introns, and more evenly distributed among different functional categories of PRR pathway genes. The finding that signatures of balancing selection in full coding sequences primarily occur in receptor genes, in particular those encoding receptors for components of microbial cell walls etc., is consistent with the idea that coevolution between hosts and pathogens is an important cause of balancing selection on immune genes.