To define microRNA (miRNA) involvement during arbovirus infection of Aedes aegypti, we mined deep sequencing libraries of Dengue type 2 (DENV2) -exposed mosquitoes. Three biological replicates for each timepoint (2, 4, and 9 days post-exposure (dpe)) and treatment group allowed us to remove outliers associated with sample-to-sample variability. Using edgeR (R Bioconductor), designed for use with replicate deep sequencing data, we determined the log fold-change (logFC) of miRNA levels (18–23 nts). The number of significantly modulated miRNAs increased from 5 or fewer at 2 and 4 dpe to 23 unique miRNAs by 9 dpe. Putative miRNA targets were predicted by aligning miRNAs to the transcriptome, and the list was reduced to include the intersection of hits found using the Miranda, PITA, and TargetScan algorithms. To further reduce false positives, putative targets were validated by cross-checking them to mRNAs reported in recent DENV2 host response transcriptome reports; 4076 targets were identified. Of these, 464 gene targets have predicted miRNA binding sites in 3′UTRs. Context-specific target functional groups include proteins involved in transport, transcriptional regulation, mitochondrial function, chromatin modification and signal transduction processes known to be required for viral replication and dissemination. The miRNA response is placed in context with other vector host response studies by comparing the predicted targets to those of transcriptome studies. Together, these data are consistent with the hypothesis that profound and persistent changes to gene expression occur in DENV2-exposed mosquitoes.

Mitochondria play an important role in maintaining metabolic and energy homeostasis in the cell. In plants, impairment in mitochondrial functions usually has detrimental effects on growth and development. To study genes that are important for plant growth, we have isolated a collection of slow growth (slo) mutants in Arabidopsis (Arabidopsis thaliana). One of the slo mutants, slo3, has a significant reduction in mitochondrial complex I activity. The slo3 mutant has a four-nucleotide deletion in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. The SLO3 protein contains nine classic PPR domains belonging to the P subfamily. The small deletion in the slo3 mutant changes the reading frame and creates a premature stop codon in the first PPR domain. We demonstrated that the SLO3-GFP is localized to the mitochondrion. Further analysis of mitochondrial RNA metabolism revealed that the slo3 mutant was defective in splicing of NADH dehydrogenase subunit7 (nad7) intron 2. This specific splicing defect led to a dramatic reduction in complex I activity in the mutant as revealed by blue native gel analysis. Complementation of slo3 by 35S:SLO3 or 35S:SLO3-GFP restored the splicing of nad7 intron 2, the complex I activity, and the growth defects of the mutant. Together, these results indicate that the SLO3 PPR protein is a splicing factor of nad7 intron 2 in Arabidopsis mitochondria.

This is a repository copy of Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

bTo test the hypothesis that RNA interference (RNAi) imposes diversifying selection on RNA virus genomes, we quantified West Nile virus (WNV) quasispecies diversity after passage in Drosophila cells in which RNAi was left intact, depleted, or stimulated against WNV. As predicted, WNV diversity was significantly lower in RNAi-depleted cells and significantly greater in RNAistimulated cells relative to that in controls. These findings reveal that an innate immune defense can shape viral population structure.

Traditional diagnostic assays often lack sensitivity and can be difficult to multiplex across many pathogens. Next-generation sequencing (NGS) can overcome some of these problems but has limited application in the detection of low-copy-number pathogens in complex samples. Targeted genome capture (TGC) utilizes oligonucleotide probes to enrich specific nucleic acids in heterogeneous extracts and can therefore increase the proportion of NGS reads for low-abundance targets. While earlier studies have demonstrated the utility of this technology for detection of novel pathogens in human clinical samples, the capacity and practicality of TGC-NGS in a veterinary diagnostic setting have not yet been evaluated. Here we report the use of TGC-NGS assays for the detection and characterization of diverse feline pathogen taxa. We detected 31 pathogens comprising nine pathogen taxa in 28 felid samples analyzed. This included 20 pathogens detected via traditional PCR and 11 additional pathogens that had not been previously detected in the same samples. Most of the pathogens detected were sequenced at sufficient breadth and depth to confidently classify them at the species or subspecies level. Target nucleic acids were enriched from a low of 58-fold to 56 million-fold relative to host nucleic acids. Despite the promising performance of these assays, a number of pathogens detected by conventional PCR or serology were not isolated by TGC-NGS, suggesting that further validation is required before this technology can be used in lieu of quality-controlled standard assays. We conclude that TGC-NGS offers great potential as a broad multiplex pathogen characterization assay in veterinary diagnostic and research settings.