The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in ...resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays.
The RNA interference (RNAi) triggered by short small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool ...to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome-assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations.
Abstract
The Molecular Evolutionary Genetics Analysis (MEGA) software enables comparative analysis of molecular sequences in phylogenetics and evolutionary medicine. Here, we introduce the macOS ...version of the MEGA software. This new version eliminates the need for virtualization and emulation programs previously required to use MEGA on Apple computers. MEGA for macOS utilizes memory and computing resources efficiently for conducting evolutionary analyses on macOS. It has a native Cocoa graphical user interface that is programmed to provide a consistent user experience across macOS, Windows, and Linux. MEGA for macOS is available from www.megasoftware.net free of charge.
The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable ...cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
Abstract
We implement two measures for quantifying genealogical concordance in phylogenomic data sets: the gene concordance factor (gCF) and the novel site concordance factor (sCF). For every branch ...of a reference tree, gCF is defined as the percentage of “decisive” gene trees containing that branch. This measure is already in wide usage, but here we introduce a package that calculates it while accounting for variable taxon coverage among gene trees. sCF is a new measure defined as the percentage of decisive sites supporting a branch in the reference tree. gCF and sCF complement classical measures of branch support in phylogenetics by providing a full description of underlying disagreement among loci and sites. An easy to use implementation and tutorial is freely available in the IQ-TREE software package (http://www.iqtree.org/doc/Concordance-Factor, last accessed May 13, 2020).
Ggtree is a comprehensive R package for visualizing and annotating phylogenetic trees with associated data. It can also map and visualize associated external data on phylogenies with two general ...methods. Method 1 allows external data to be mapped on the tree structure and used as visual characteristic in tree and data visualization. Method 2 plots the data with the tree side by side using different geometric functions after reordering the data based on the tree structure. These two methods integrate data with phylogeny for further exploration and comparison in the evolutionary biology context. Ggtree is available from http://www.bioconductor.org/packages/ggtree.
Abstract
HYpothesis testing using PHYlogenies (HyPhy) is a scriptable, open-source package for fitting a broad range of evolutionary models to multiple sequence alignments, and for conducting ...subsequent parameter estimation and hypothesis testing, primarily in the maximum likelihood statistical framework. It has become a popular choice for characterizing various aspects of the evolutionary process: natural selection, evolutionary rates, recombination, and coevolution. The 2.5 release (available from www.hyphy.org) includes a completely re-engineered computational core and analysis library that introduces new classes of evolutionary models and statistical tests, delivers substantial performance and stability enhancements, improves usability, streamlines end-to-end analysis workflows, makes it easier to develop custom analyses, and is mostly backward compatible with previous HyPhy releases.
Abstract
ModelTest-NG is a reimplementation from scratch of jModelTest and ProtTest, two popular tools for selecting the best-fit nucleotide and amino acid substitution models, respectively. ...ModelTest-NG is one to two orders of magnitude faster than jModelTest and ProtTest but equally accurate and introduces several new features, such as ascertainment bias correction, mixture, and free-rate models, or the automatic processing of single partitions. ModelTest-NG is available under a GNU GPL3 license at https://github.com/ddarriba/modeltest , last accessed September 2, 2019.
While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We ...present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
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•CRISPRi and CRISPRa provide complementary information for mapping complex pathways•CRISPRi/a expression series (up to ∼1,000-fold) reveal how gene dose controls function•CRISPRi provides strong (typically 90%–99%) knockdown with minimal off-target effects•Genome-scale screens elucidate pathways controlling cholera/diphtheria toxicity
Genome-scale-specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9 have been applied to growth and toxin-resistance screens, establishing CRISPRi and CRISPRa as powerful tools that provide rich and complementary information.
RNA sequencing using the latest single-molecule sequencing instruments produces reads that are thousands of nucleotides long. The ability to assemble these long reads can greatly improve the ...sensitivity of long-read analyses. Here we present StringTie2, a reference-guided transcriptome assembler that works with both short and long reads. StringTie2 includes new methods to handle the high error rate of long reads and offers the ability to work with full-length super-reads assembled from short reads, which further improves the quality of short-read assemblies. StringTie2 is more accurate and faster and uses less memory than all comparable short-read and long-read analysis tools.
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