Shake-it-off A simple ultrasonic cryo-EM specimen preparation device, bioRxiv, 2019-05-10
AbstractAlthough microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments.SynopsisA method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.
biorxiv biophysics 0-100-users 2019Stress-driven transposable element de-repression dynamics in a fungal pathogen, bioRxiv, 2019-05-10
AbstractTransposable elements (TEs) are drivers of genome evolution and affect the expression landscape of the host genome. Stress is a major factor inducing TE activity, however the regulatory mechanisms underlying de-repression are poorly understood. Key unresolved questions are whether different types of stress differentially induce TE activity and whether different TEs respond differently to the same stress. Plant pathogens are excellent models to dissect the impact of stress on TEs, because lifestyle transitions on and off the host impose exposure to a variety of stress conditions. We analyzed the TE expression landscape of four well-characterized strains of the major wheat pathogen Zymoseptoria tritici. We experimentally exposed strains to nutrient starvation and host infection stress. Contrary to expectations, we show that the two distinct conditions induce the expression of different sets of TEs. In particular, the most highly expressed TEs, including MITE and LTR-Gypsy elements, show highly distinct de-repression across stress conditions. Both the genomic context of TEs and the genetic background stress (i.e. different strains harboring the same TEs) were major predictors of de-repression dynamics under stress. Genomic defenses inducing point mutations in repetitive regions were largely ineffective to prevent TE de-repression. Consistent with TE de-repression being governed by epigenetic effects, we found that gene expression profiles under stress varied significantly depending on the proximity to the closest TEs. The unexpected complexity in TE responsiveness to stress across genetic backgrounds and genomic locations shows that species harbor substantial genetic variation to control TEs.
biorxiv genomics 0-100-users 2019Systematic comparative analysis of single cell RNA-sequencing methods, bioRxiv, 2019-05-10
ABSTRACTA multitude of single-cell RNA sequencing methods have been developed in recent years, with dramatic advances in scale and power, and enabling major discoveries and large scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single cell andor single nucleus profiling from three types of samples – cell lines, peripheral blood mononuclear cells and brain tissue – generating 36 libraries in six separate experiments in a single center. To analyze these datasets, we developed and applied scumi, a flexible computational pipeline that can be used for any scRNA-seq method. We evaluated the methods for both basic performance and for their ability to recover known biological information in the samples. Our study will help guide experiments with the methods in this study as well as serve as a benchmark for future studies and for computational algorithm development.
biorxiv genomics 100-200-users 2019A megaplasmid family responsible for dissemination of multidrug resistance in Pseudomonas, bioRxiv, 2019-05-08
AbstractMultidrug resistance (MDR) represents a global threat to health. Although plasmids can play an important role in the dissemination of MDR, they have not been commonly linked to the emergence of antimicrobial resistance in the pathogen Pseudomonas aeruginosa. We used whole genome sequencing to characterize a collection of P. aeruginosa clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicated that 1) these large plasmids comprise a family present in different members of the Pseudomonas genus and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the pangenome of the megaplasmid family is highly flexible and diverse, comprising a substantial core genome (average of 48% of plasmid genes), but with individual members carrying large numbers of unique genes. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.FundingThis work was supported by the International Pseudomonas Genomics Consortium, funded by Cystic Fibrosis Canada [RCL]; and the Secretaría de Educación, Ciencia, Tecnología e Innovación (SECTEI), Mexico [AC].
biorxiv microbiology 0-100-users 2019Single-cell RNA-sequencing of differentiating iPS cells reveals dynamic genetic effects on gene expression, bioRxiv, 2019-05-08
AbstractRecent developments in stem cell biology have enabled the study of cell fate decisions in early human development that are impossible to study in vivo. However, understanding how development varies across individuals and, in particular, the influence of common genetic variants during this process has not been characterised. Here, we exploit human iPS cell lines from 125 donors, a pooled experimental design, and single-cell RNA-sequencing to study population variation of endoderm differentiation. We identify molecular markers that are predictive of differentiation efficiency, and utilise heterogeneity in the genetic background across individuals to map hundreds of expression quantitative trait loci that influence expression dynamically during differentiation and across cellular contexts.
biorxiv genomics 100-200-users 2019The neural basis of tadpole transport in poison frogs, bioRxiv, 2019-05-08
AbstractParental care has evolved repeatedly and independently across animals. While the ecological and evolutionary significance of parental behaviour is well recognized, underlying mechanisms remain poorly understood. We took advantage of behavioural diversity across closely related species of South American poison frogs (Family Dendrobatidae) to identify neural correlates of parental behaviour shared across sexes and species. We characterized differences in neural induction, gene expression in active neurons, and activity of specific neuronal types in three species with distinct parental care patterns male uniparental, female uniparental, and biparental. We identified the medial pallium and preoptic area as core brain regions associated with parental care, independent of sex and species. Identification of neurons active during parental care confirms a role for neuropeptides associated with care in other vertebrates as well as identifying novel candidates. Our work is the first to explore neural and molecular mechanisms of parental care in amphibians and highlights the potential for mechanistic studies in closely related but behaviourally variable species to build a more complete understanding of how shared principles and species-specific diversity govern parental care and other social behaviour.
biorxiv animal-behavior-and-cognition 0-100-users 2019