Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing [version 2; peer review: 2 approved]

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5' end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach 'SMART-9N' and a version compatible rapid adapters  available from Oxford Nanopore Technologies 'Rapid SMART-9N'. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work

Markedly Divergent Tree Assemblage Responses to Tropical Forest Loss and Fragmentation across a Strong Seasonality Gradient

We examine the effects of forest fragmentation on the structure and composition of tree assemblages within three seasonal and aseasonal forest types of southern Brazil, including evergreen, Araucaria, and deciduous forests. We sampled three southernmost Atlantic Forest landscapes, including the largest continuous forest protected areas within each forest type. Tree assemblages in each forest type were sampled within 10 plots of 0.1 ha in both continuous forests and 10 adjacent forest fragments. All trees within each plot were assigned to trait categories describing their regeneration strategy, vertical stratification, seed-dispersal mode, seed size, and wood density. We detected differences among both forest types and landscape contexts in terms of overall tree species richness, and the density and species richness of different functional groups in terms of regeneration strategy, seed dispersal mode and woody density. Overall, evergreen forest fragments exhibited the largest deviations from continuous forest plots in assemblage structure. Evergreen, Araucaria and deciduous forests diverge in the functional composition of tree floras, particularly in relation to regeneration strategy and stress tolerance. By supporting a more diversified light-demanding and stress-tolerant flora with reduced richness and abundance of shade-tolerant, old-growth species, both deciduous and Araucaria forest tree assemblages are more intrinsically resilient to contemporary human-disturbances, including fragmentation-induced edge effects, in terms of species erosion and functional shifts. We suggest that these intrinsic differences in the direction and magnitude of responses to changes in landscape structure between forest types should guide a wide range of conservation strategies in restoring fragmented tropical forest landscapes worldwide

Ancient micrometeorites suggestive of an oxygen-rich Archaean upper atmosphere

It is widely accepted that Earth’s early atmosphere contained less than 0.001 per cent of the present-day atmospheric oxygen (O2) level, until the Great Oxidation Event resulted in a major rise in O2 concentration about 2.4 billion years ago1. There are multiple lines of evidence for low O2 concentrations on early Earth, but all previous observations relate to the composition of the lower atmosphere2 in the Archaean era; to date no method has been developed to sample the Archaean upper atmosphere. We have extracted fossil micrometeorites from limestone sedimentary rock that had accumulated slowly 2.7 billion years ago before being preserved in Australia’s Pilbara region. We propose that these micrometeorites formed when sand-sized particles entered Earth’s atmosphere and melted at altitudes of about 75 to 90 kilometres (given an atmospheric density similar to that of today3). Here we show that the FeNi metal in the resulting cosmic spherules was oxidized while molten, and quench-crystallized to form spheres of interlocking dendritic crystals primarily of magnetite (Fe3O4), with wüstite (FeO)+metal preserved in a few particles. Our model of atmospheric micrometeorite oxidation suggests that Archaean upper-atmosphere oxygen concentrations may have been close to those of the present-day Earth, and that the ratio of oxygen to carbon monoxide was sufficiently high to prevent noticeable inhibition of oxidation by carbon monoxide. The anomalous sulfur isotope (Δ33S) signature of pyrite (FeS2) in seafloor sediments from this period, which requires an anoxic surface environment4, implies that there may have been minimal mixing between the upper and lower atmosphere during the Archaean

First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil

<p>Abstract</p> <p>Background</p> <p>In Brazil dengue has been a major public health problem since DENV-1 introduction and spread in 1986. After a low or silent co-circulation, DENV-1 re-emerged in 2009 causing a major epidemic in the country in 2010 and 2011. In this study, the phylogeny of DENV-1 strains isolated in RJ after its first introduction in 1986 and after its emergence in 2009 and 2010 was performed in order to document possible evolutionary patterns or introductions in a re-emergent virus.</p> <p>Findings</p> <p>The analysis of the E gene sequences demonstrated that DENV-1 isolated during 2009/2010 still belong to genotype V (Americas/Africa) but grouping in a distinct clade (lineage II) of that represented by earlier DENV-1 (lineage I). However, strains isolated in 2011 grouped together forming another distinct clade (lineage III).</p> <p>Conclusions</p> <p>The monitoring of DENV is important to observe the spread of potentially virulent strains as well to evaluate its impact over the population during an outbreak. Whether explosive epidemics reported in Brazil caused mainly by DENV-1 was due to lineage replacement, or due the population susceptibility to this serotype which has not circulated for almost a decade or even due to the occurrence of secondary infections in a hyperendemic country, is not clear. This is the first report of multiple lineages of DENV-1 detected in Brazil.</p

Initiative, Personality and Leadership in Pairs of Foraging Fish

Studies of coordinated movement have found that, in many animal species, bolder individuals are more likely to initiate movement and shyer individuals to follow. Here, we show that in pairs of foraging stickleback fish, leadership is not merely a passive consequence of temperamental differences. Instead, the act of initiating a joint foraging trip out of cover itself brings about a change in the role that an individual plays throughout the subsequent trip, and success in recruiting a partner affects an individual's tendency to initiate the next trip. On each joint trip, whichever fish took the initiative in leading out of cover gains greater influence over its partner's behaviour, which persists even after several changes in position (i.e. termination attempts and re-joining). During any given trip, the initiator is less responsive to its partner's movements than during trips initiated by the partner. An individual's personality had an important effect on its response to failure to recruit a partner: while bold fish were unaffected by failures to initiate a joint trip, shy individuals were less likely to attempt another initiation after a failure. This difference provides a positive feedback mechanism that can partially stabilise social roles within the pair, but it is not strong enough to prevent occasional swaps, with individuals dynamically adjusting their responses to one another as they exchange roles

Intra-radicular dentin treatments and retention of fiber posts with self-adhesive resin cements

The aim of this study was to evaluate the effect of treating intraradicular dentin with irrigating solutions on the retention of glass-fiber posts luted with self-adhesive resin cement. Bovine incisors were endodontically treated, and 9-mm-deep postholes were prepared. Before inserting the cement, the root canals were irrigated with various solutions: 11.5% polyacrylic acid for 30 s, 17% EDTA for 60 s, or 5% NaOCl for 60 s, respectively. Irrigation with distilled water was used in the control group. After all specimens had been rinsed with distilled water, the excess moisture was removed and the posts were luted using either BisCem (Bisco) or RelyX Unicem clicker (3M ESPE). Seven days after luting, the specimens were sectioned transversally into 1-mm-thick slices, which were submitted to push-out testing on a mechanical testing machine. Bond strength data (n = 6 per group) were analyzed by two-way ANOVA and Student-Newman-Keuls' test (α = 0.05). For Unicem, EDTA showed lower bond strength than the other solutions, which had similar results. For BisCem, EDTA showed higher bond strength than the other treatments, while application of NaOCl yielded higher bond strength than polyacrylic acid whereas the control group had intermediate results. In conclusion, irrigating root canals before insertion of self-adhesive resin cements, especially EDTA, might interfere with retention of the fiber posts