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

Antimicrobial activity of ethanol extract from leaves of Casearia sylvestris

Casearia sylvestris Sw., also known as 'guaatonga,' is a medicinal plant with broad use in South America. Among the popular applications attributed to this plant are anti-inflammatory, anticancer, antimicrobial, and antiulcer activities. Despite the broad popular use of this plant as a phytopharmaceutical agent, there are few studies about the antimicrobial potential of guaatonga. In this work, we have studied the antimicrobial potential of an ethanol extract obtained from C. sylvestris leaves against three yeasts, two filamentous fungus, six Gram-negative bacteria, and two Gram-positive bacteria. Through two chromatographic steps using a Sephadex LH-20 column and RP-HPLC, we isolated and characterized two derived compounds of gallic acid: isobutyl gallate-3,5-dimethyl ether (IGDE) and methyl gallate-3,5-dimethyl ether (MGDE). Both compounds showed antimicrobial activity. IGDE was much more efficient than MGDE in inhibiting yeasts (Candida albicans, Candida tropicalis, and Candida guilliermondii) and Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus). This fact is probably associated with the higher hydrophobicity degree of IDGE compared with MGDE.46534735

Genetic and morphological characterization of the endangered Austral papaya Vasconcellea chilensis (Planch. ex A. DC.) Solms

Caligari, PDS (Caligari, P. D. S.) Univ Talca, Ctr Plant Breeding, Talca, Chile; Univ Talca, Phen Ctr, Inst Ciencias Biol, Talca, Chile.-The Austral papaya (Vasconcellea chilensis) is an endangered species that has valuable characteristics for introgression into other papaya species. These characteristics include disease resistance, cold tolerance and latex with low proteolytic activity. It is a species that grows under extreme environmental conditions of drought, salinity and temperature; it is found growing naturally in Chile but today only as three remnant populations. The results presented here, using inter simple sequence repeat molecular markers, along with morphological trait analyses, suggest that these relict populations are the result of a relatively recent fragmentation. This implies that the fragmentation has not yet had its full effect on the genetic variation and so emphasises the need for clear and urgent conservation measures to preserve the remaining genetic variation, particularly for the most northern of the three populations which is presently unprotected

Effect of the Phenological Stage in the Natural Rubber Latex Properties

Natural Rubber Latex (NRL) from Hevea brasiliensis is a material studied because of their industrial applications. For its natural origin, it is possible to find rubber particles, proteins, phospholipids and ashes. These non-rubber content are responsible for the latex colloidal stability. H. brasiliensis tree goes through four stages during the year, changing its nutritional requirements and as a result the rubber yield and stability. Most studies have correlated latex characteristics and yield with tree age and clonal origin but none of them with phenological stages. The impact of the phenological stage on the material properties has not been completely identified yet. In this work, the influence of the clonal origin and the phenological stage with the material properties is studied. Thermal behavior, microstructural analysis, morphological study, colloidal stability and rheology are analyzed for FX3864, IAN710 and AIN873 clones during 1 year. NRL is an amorphous material but during the high-yield period, a melting point is observed. Flowering is the stage when phospholipids, protein and isoelectric point are higher. Phenological stages do not affect the rubber, but the main changes are in the non-rubber content. © 2018, Springer Science+Business Media, LLC, part of Springer Nature