Tunability of DNA polymerase stability during eukaryotic DNA replication, bioRxiv, 2019-04-08
SummaryStructural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.
biorxiv biophysics 100-200-users 2019Measuring shared responses across subjects using intersubject correlation, bioRxiv, 2019-04-06
AbstractOur capacity to jointly represent information about the world underpins our social experience. By leveraging one individual’s brain activity to model another’s, we can measure shared information across brains—even in dynamic, naturalistic scenarios where an explicit response model may be unobtainable. Introducing experimental manipulations allows us to measure, for example, shared responses between speakers and listeners, or between perception and recall. In this tutorial, we develop the logic of intersubject correlation (ISC) analysis and discuss the family of neuroscientific questions that stem from this approach. We also extend this logic to spatially distributed response patterns and functional network estimation. We provide a thorough and accessible treatment of methodological considerations specific to ISC analysis, and outline best practices.
biorxiv neuroscience 100-200-users 2019PIRATE A fast and scalable pangenomics toolbox for clustering diverged orthologues in bacteria, bioRxiv, 2019-04-05
AbstractCataloguing the distribution of genes within natural bacterial populations is essential for understanding evolutionary processes and the genetic basis of adaptation. Here we present a pangenomics toolbox, PIRATE (Pangenome Iterative Refinement And Threshold Evaluation), which identifies and classifies orthologous gene families in bacterial pangenomes over a wide range of sequence similarity thresholds. PIRATE builds upon recent scalable software developments to allow for the rapid interrogation of thousands of isolates. PIRATE clusters genes (or other annotated features) over a wide range of amino-acid or nucleotide identity thresholds and uses the clustering information to rapidly classify paralogous gene families into either putative fissionfusion events or gene duplications. Furthermore, PIRATE orders the pangenome using a directed graph, provides a measure of allelic variation and estimates sequence divergence for each gene family. We demonstrate that PIRATE scales linearly with both number of samples and computation resources, allowing for analysis of large genomic datasets, and compares favorably to other popular tools. PIRATE provides a robust framework for analysing bacterial pangenomes, from largely clonal to panmictic species.AvailabilityPIRATE is implemented in Perl and is freely available under an GNU GPL 3 open source license from <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comSionBaylissPIRATE>httpsgithub.comSionBaylissPIRATE<jatsext-link>.<jatssec sec-type=supplementary-material>Supplementary InformationSupplementary data is available online.
biorxiv bioinformatics 100-200-users 2019Single-cell transcriptomics of the naked mole-rat reveals unexpected features of mammalian immunity, bioRxiv, 2019-04-05
AbstractUsing single-cell transcriptional profiling we mapped the immune system of the naked mole-rat (Heterocephalus glaber), a small but long-lived and cancer-resistant subterranean rodent. Both splenic and circulating immune cells were examined in healthy young animals and following an infection-mimicking lipopolysaccharide challenge. Our study revealed that the naked mole-rat immune system is characterized by a high myeloid to lymphoid cell ratio that includes a novel, lipopolysaccharide responsive, granulocyte cell subset not found in the mouse. Conversely, we find that naked mole-rats do not have a cell subset that corresponds to natural killer cells as defined in other well-characterized mammalian species. Supporting this finding, we show that the naked mole-rat genome has not expanded any of the gene families encoding diverse natural killer cell receptors, which are the genomic hallmarks of species in which natural killer cells have been described. These unusual features suggest an atypical mode of immunosurveillance and a greater reliance on myeloid-biased innate immunity.
biorxiv immunology 100-200-users 2019Muscle fiber hypertrophy in response to 6 weeks of high-volume resistance training in trained young men is largely attributed to sarcoplasmic hypertrophy, bioRxiv, 2019-04-02
ABSTRACTCellular adaptations that occur during skeletal muscle hypertrophy in response to high-volume resistance training are not well-characterized. Therefore, we sought to explore how actin, myosin, sarcoplasmic protein, mitochondrial, and glycogen concentrations were altered in individuals that exhibited mean skeletal muscle fiber cross-sectional area (fCSA) hypertrophy following 6 weeks of high-volume resistance training. Thirty-one previously resistance-trained, college-aged males (mean ± standard deviation 21±2 years, 5±3 training years) had vastus lateralis (VL) muscle biopsies obtained prior to training (PRE), at week 3 (W3), and at week 6 (W6). Muscle tissue from 15 subjects exhibiting PRE to W6 VL mean fCSA increases ranging from 320-1600 μm2 was further interrogated using various biochemical and histological assays as well as proteomic analysis. Seven of these individuals donated a VL biopsy after refraining from training 8 days following the last training session (W7) to determine how deloading affected biomarkers. The 15 fCSA hypertrophic responders experienced a +23% increase in mean fCSA from PRE to W6 (p<0.001) and, while muscle glycogen concentrations remained unaltered, citrate synthase activity levels decreased by 24% (p<0.001) suggesting mitochondrial volume decreased. Interestingly, both myosin and actin concentrations decreased ~30% from PRE to W6 (p<0.05). Phalloidin-actin staining similarly revealed actin concentrations per fiber decreased from PRE to W6. Proteomic analysis of the sarcoplasmic fraction from PRE to W6 indicated 40 proteins were up-regulated (p<0.05), KEGG analysis indicated that the glycolysisgluconeogenesis pathway was upregulated (FDR sig. <0.001), and DAVID indicated that the following functionally-annotated pathways were upregulated (FDR value <0.05) a) glycolysis (8 proteins), b) acetylation (23 proteins), c) gluconeogenesis (5 proteins) and d) cytoplasm (20 proteins). At W7, sarcoplasmic protein concentrations remained higher than PRE (+66%, p<0.05), and both actin and myosin concentrations remained lower than PRE (~−50%, p<0.05). These data suggest that short-term high-volume resistance training may a) reduce muscle fiber actin and myosin protein concentrations in spite of increasing fCSA, and b) promote sarcoplasmic expansion coincident with a coordinated up-regulation of sarcoplasmic proteins involved in glycolysis and other metabolic processes related to ATP generation. Interestingly, these effects seem to persist up to 8 days following training.
biorxiv molecular-biology 100-200-users 2019MARGO (Massively Automated Real-time GUI for Object-tracking), a platform for high-throughput ethology, bioRxiv, 2019-03-30
AbstractFast object tracking in real time allows convenient tracking of very large numbers of animals and closed-loop experiments that control stimuli for multiple animals in parallel. We developed MARGO, a real-time animal tracking suite for custom behavioral experiments. We demonstrated that MARGO can rapidly and accurately track large numbers of animals in parallel over very long timescales. We incorporated control of peripheral hardware, and implemented a flexible software architecture for defining new experimental routines. These features enable closed-loop delivery of stimuli to many individuals simultaneously. We highlight MARGO’s ability to coordinate tracking and hardware control with two custom behavioral assays (measuring phototaxis and optomotor response) and one optogenetic operant conditioning assay. There are currently several open source animal trackers. MARGO’s strengths are 1) robustness, 2) high throughput, 3) flexible control of hardware and 4) real-time closed-loop control of sensory and optogenetic stimuli, all of which are optimized for large-scale experimentation.
biorxiv neuroscience 100-200-users 2019