Attenuated Codon Optimality Contributes to Neural-Specific mRNA Decay in Drosophila.

Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. We found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. We confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. We conclude that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system

Abnormal wave reflections and left ventricular hypertrophy late after coarctation of the aorta repair

Patients with repaired coarctation of the aorta are thought to have increased afterload due to abnormalities in vessel structure and function. We have developed a novel cardiovascular magnetic resonance protocol that allows assessment of central hemodynamics, including central aortic systolic blood pressure, resistance, total arterial compliance, pulse wave velocity, and wave reflections. The main study aims were to (1) characterize group differences in central aortic systolic blood pressure and peripheral systolic blood pressure, (2) comprehensively evaluate afterload (including wave reflections) in the 2 groups, and (3) identify possible biomarkers among covariates associated with elevated left ventricular mass (LVM). Fifty adult patients with repaired coarctation and 25 age- and sex-matched controls were recruited. Ascending aorta area and flow waveforms were obtained using a high temporal-resolution spiral phase-contrast cardiovascular magnetic resonance flow sequence. These data were used to derive central hemodynamics and to perform wave intensity analysis noninvasively. Covariates associated with LVM were assessed using multivariable linear regression analysis. There were no significant group differences (P≥0.1) in brachial systolic, mean, or diastolic BP. However central aortic systolic blood pressure was significantly higher in patients compared with controls (113 versus 107 mm Hg, P=0.002). Patients had reduced total arterial compliance, increased pulse wave velocity, and larger backward compression waves compared with controls. LVM index was significantly higher in patients than controls (72 versus 59 g/m(2), P<0.0005). The magnitude of the backward compression waves was independently associated with variation in LVM (P=0.01). Using a novel, noninvasive hemodynamic assessment, we have shown abnormal conduit vessel function after coarctation of the aorta repair, including abnormal wave reflections that are associated with elevated LVM

The aorta after coarctation repair : effects of calibre and curvature on arterial haemodynamics (vol 21, 22, 2019)

In the original version of this article [1], published on 11 April 2019, there is 1 error in the Conclusion' paragraph of the abstract

Phylogenetic relationships of the Wolbachia of nematodes and arthropods

Wolbachia are well known as bacterial symbionts of arthropods, where they are reproductive parasites, but have also been described from nematode hosts, where the symbiotic interaction has features of mutualism. The majority of arthropod Wolbachia belong to clades A and B, while nematode Wolbachia mostly belong to clades C and D, but these relationships have been based on analysis of a small number of genes. To investigate the evolution and relationships of Wolbachia symbionts we have sequenced over 70 kb of the genome of wOvo, a Wolbachia from the human-parasitic nematode Onchocerca volvulus, and compared the genes identified to orthologues in other sequenced Wolbachia genomes. In comparisons of conserved local synteny, we find that wBm, from the nematode Brugia malayi, and wMel, from Drosophila melanogaster, are more similar to each other than either is to wOvo. Phylogenetic analysis of the protein-coding and ribosomal RNA genes on the sequenced fragments supports reciprocal monophyly of nematode and arthropod Wolbachia. The nematode Wolbachia did not arise from within the A clade of arthropod Wolbachia, and the root of the Wolbachia clade lies between the nematode and arthropod symbionts. Using the wOvo sequence, we identified a lateral transfer event whereby segments of the Wolbachia genome were inserted into the Onchocerca nuclear genome. This event predated the separation of the human parasite O. volvulus from its cattle-parasitic sister species, O. ochengi. The long association between filarial nematodes and Wolbachia symbionts may permit more frequent genetic exchange between their genomes

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants

Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in &gt;100 mutants, and sensitive, identifying and monitoring sites over a &gt;10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that &gt;4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

BAckground: Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. Results: We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. Conclusion: We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material

Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology.

Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.Health Innovation Challenge Fund (WT098600, HICF-T5-342) (Department of Health, Wellcome Trust)This is the final version of the article. It first appeared from the Microbiology Society via http://dx.doi.org/10.1099/mgen.0.00008

Efficient depletion of host DNA contamination in malaria clinical sequencing.

The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci