Characterizing the temporal dynamics of gene expression in single cells with sci-fate, bioRxiv, 2019-06-11

AbstractGene expression programs are dynamic, e.g. the cell cycle, response to stimuli, normal differentiation and development, etc. However, nearly all techniques for profiling gene expression in single cells fail to directly capture the dynamics of transcriptional programs, which limits the scope of biology that can be effectively investigated. Towards addressing this, we developed sci-fate, a new technique that combines S4U labeling of newly synthesized mRNA with single cell combinatorial indexing (sci-), in order to concurrently profile the whole and newly synthesized transcriptome in each of many single cells. As a proof-of-concept, we applied sci-fate to a model system of cortisol response and characterized expression dynamics in over 6,000 single cells. From these data, we quantify the dynamics of the cell cycle and glucocorticoid receptor activation, while also exploring their intersection. We furthermore use these data to develop a framework for inferring the distribution of cell state transitions. We anticipate sci-fate will be broadly applicable to quantitatively characterize transcriptional dynamics in diverse systems.

biorxiv genomics 100-200-users 2019

A robust benchmark for germline structural variant detection, bioRxiv, 2019-06-10

AbstractNew technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls ≥50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

biorxiv genomics 100-200-users 2019

A Single Cell Transcriptomic Atlas Characterizes Aging Tissues in the Mouse, bioRxiv, 2019-06-08

AbstractAging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death 1. Despite rapid advances over recent years 2, many of the molecular and cellular processes which underlie progressive loss of healthy physiology are poorly understood. To gain a better insight into these processes we have created a single cell transcriptomic atlas across the life span of Mus musculus which includes data from 18 tissues and organs. We discovered cell-specific changes occurring across multiple cell types and organs, as well as age related changes in the cellular composition of different organs. Using single-cell transcriptomic data we were able to assess cell type specific manifestations of different hallmarks of aging, such as senescence 3, changes in the activity of metabolic pathways 4, depletion of stem-cell populations 5, genomic instability 6 and the role of inflammation as well as other changes in the organism’s immune system2. This Tabula Muris Senis provides a wealth of new molecular information about how the most significant hallmarks of aging are reflected in a broad range of tissues and cell types.

biorxiv cell-biology 100-200-users 2019

Effectiveness of treatments for firework fears in dogs, bioRxiv, 2019-06-07

AbstractAn online questionnaire survey investigated (1) management and (2) treatment methods for firework fears in dogs employed by dog owners and their (perceived) effectiveness. A PCA on data from 1225 respondents revealed four management strategies (i.e. interventions during firework exposure) the principal components “Environmental modification” (e.g. providing a hiding place, keeping windows and blinds closed, and playing music), “FeedPlay” (providing the dog with chews, play and food during fireworks in general, as well as contingent on loud bangs), “Alternative” (use of calming neutraceuticals, pheromones, herbal products, homeopathic products, Bach flowers, and essential oils) and “Interaction” (allowing body contact, petting and talking to the dog when loud bangs occurred). To explore possible effects of these management methods on fear development, the components were correlated with a score for fear progression. Of the four components, only “FeedPlay” was statistically associated with an improvement in fear responses to fireworks. To evaluate the effectiveness of various treatment strategies, owners were asked to select from a range of options which interventions they had used and whether they considered them as effective. With prescription medication (N=202), improvements were noted by 70% of owners. While individual products were not evaluated, the reported success rates for the categories “pheromones” (N=316), “herbal products” (N=282), “neutraceuticals” (N=211), “essential oils” (N=183), “homeopathic remedies” (N=250) and “Bach flowers” (N=281) were all in the range of 27-35%, which is not higher than would be expected based on a placebo effect. 44% of respondents (N=300) deemed pressure vests as effective. Counterconditioning (providing desirable stimuli after the occurrence of noises) was the most successful training technique according to the owners (N=694), with a reported effectiveness of over 70%. Relaxation training (N=433) was reported to be almost as successful at 69%, while noise CDs (N=377) were effective in 55% of cases. Thus, counterconditioining, relaxation training and psychoactive medication appear to be the most effective strategies in the treatment of firework fears in dogs. On this basis it is recommended that ad-hoc counterconditioning and relaxation training should complement the standard behavioral technique of desensitization counterconditioning with noise recordings.Highlights<jatslist list-type=bullet><jatslist-item>An online survey on treatment options for firework fears in dogs was performed<jatslist-item><jatslist-item>Feeding or playing with dogs during fireworks was associated with fear improvement<jatslist-item><jatslist-item>Success was highest for ad-hoc counterconditioning, relaxation training and medication<jatslist-item><jatslist-item>Success was similar for pheromones, neutraceuticals and other alternative products<jatslist-item><jatslist-item>Success rates for these alternative products are consistent with a placebo effect<jatslist-item>

biorxiv animal-behavior-and-cognition 100-200-users 2019

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin de-condensation and nuclear envelope rupture, bioRxiv, 2019-06-07

AbstractNeutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high resolution time-lapse microscopy of mouse and human neutrophils (PMN) and differentiated HL-60 neutrophil-like cells (dHL-60) labelled with fluorescent markers of organelles and stimulated with ionomycin or lipopolysaccharides to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin de-condensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin de-condensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a step-wise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.Significance StatementNeutrophils are white blood cells specialized as the first line of host defense in the immune system. One way they protect organisms is through NETosis, in which they expel their DNA to form a web-like trap that ensnares pathogens and promotes clotting. However, NETs also mediate sterile inflammation, causing damage to the body. We used high-resolution live-cell microscopy to perform the first systematic characterization of the timing of dynamic cellular events leading to NETosis in human and mouse neutrophils and a neutrophil-like cell line. We discovered that NETosis proceeds by a step-wise sequence of cellular events that is conserved across species, and requires the activity of the PAD4 enzyme for DNA to be released from the nucleus and cell membrane.

biorxiv immunology 100-200-users 2019

RNA velocity and protein acceleration from single-cell multiomics experiments, bioRxiv, 2019-06-07

AbstractThe simultaneous quantification of protein and RNA makes possible the inference of past, present and future cell states from single experimental snapshots. To enable such temporal analysis from multimodal single-cell experiments, we introduce an extension of the RNA velocity method that leverages estimates of unprocessed transcript and protein abundances to extrapolate cell states. We apply the model to four datasets and demonstrate consistency among landscapes and phase portraits.

biorxiv bioinformatics 100-200-users 2019