Biology subjects

Marioni, J.

Publications and source records attributed to Marioni, J..

28 records · Page 2Linked to original sources

bioRxiv2021

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

genetics↗

bioRxiv2021

geneBasis: an iterative approach for unsupervised selection of targeted gene panels from scRNA-seq.

The problem of selecting targeted gene panels that capture maximum variability encoded in scRNA-sequencing data has become of great practical importance. scRNA-seq datasets are increasingly being used to identify gene panels that can be probed using alternative molecular technologies, such as spatial transcriptomics. In this context, the number of genes that can be probed is an important limiting factor, so choosing the best subset of genes is vital. Existing methods for this task are limited by either a reliance on pre-existing cell type labels or by difficulties in identifying markers of rare cell types. We resolve this by introducing an iterative approach, geneBasis, for selecting an optimal gene panel, where each newly added gene captures the maximum distance between the true manifold and the manifold constructed using the currently selected gene panel. We demonstrate, using a variety of metrics and diverse datasets, that our approach outperforms existing strategies, and can not only resolve cell types but also more subtle cell state differences. Our approach is available as an open source, easy-to-use, documented R package (https://github.com/MarioniLab/geneBasisR).

bioinformatics↗

bioRxiv2021

Cells of the human intestinal tract mapped across space and time

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

cell biology↗

bioRxiv2020

Time-resolved single-cell RNAseq profiling identifies a novel Fabp5-expressing subpopulation of inflammatory myeloid cells in chronic spinal cord injury

Innate immune responses following spinal cord injury (SCI) participate in early secondary pathogenesis and wound healing events. Here, we used time-resolved scRNAseq to map transcriptional profiles of SC tissue-resident and infiltrating myeloid cells post-SCI. Our work identifies a novel subpopulation of Fabp5+ inflammatory myeloid cells, comprising both resident and infiltrating cells and displaying a delayed cytotoxic profile at the lesion epicentre, which may serve as a target for future therapeutics.

neuroscience↗

bioRxiv2020

First report of antiviral activity of nordihydroguaiaretic acid against Fort Sherman-like virus (Orthobunyavirus)

The genus Orthobunyavirus are a group of viruses within arbovirus, with a zoonotic cycle, some of which could lead to human infection. A characteristic of these viruses is their lack of antiviral treatment or vaccine for its prevention. The objective of this work was to study the in vitro antiviral activity of nordihydroguaiaretic acid (NDGA), the most important active compound of Larrea divaricata Cav. (Zigophyllaceae), against Fort Sherman-like virus (FSV-like) as a model of Orthobunyavirus genus. At the same time, the effect of NDGA as a lipolytic agent on the cell cycle of this viral model was assessed. The method of reducing plaque forming units on LLC-MK2 cells was used to detect the action of NDGA on CbaAr426 and SFCrEq231 isolates of FSV-like. NDGA did not show virucidal effect, but it had antiviral activity with a similar inhibition in both isolates, which was dose dependent. It was established that the NDGA has a better inhibition one-hour post infection (p.i.), showing a different behavior in each isolate, which was dependent upon the time p.i. Since virus multiplication is dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the sterol regulatory element-binding proteins (SREBP) pathway and the 5-lipoxigenase (5-LOX). We determined by using caffeic acid, a 5-LOX inhibitor, that the inhibition of this enzyme negatively affected the FSV-like replication; and by the use of resveratrol, a SREBP1 inhibitor, it was showed that the negative regulation of this pathway only had action on the SFCrEq231 reduction. In addition, it was proved that the NDGA acts intracellularly, since it showed the ability to incorporate into LLC-MK2 cells. The information provided in this work converts the NDGA in a good antiviral candidate, especially for Orthobunyavirus infections, and a useful tool for the biochemical study of FSV-like that causes an infection poorly studied and potentially dangerous.

microbiology↗

bioRxiv2020

Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs

Absence of a specialised wound epidermis is hypothesised to block limb regeneration in higher vertebrates. To elucidate the cellular and molecular determinants of this tissue, we performed single-cell transcriptomics in regeneration-competent, -restricted, and -incompetent Xenopus tadpoles. We identified apical-ectodermal-ridge (AER) cells as the specialised wound epidermis, and found that their abundance on the amputation plane correlates with regeneration potential and injury-induced mesenchymal plasticity. By using ex vivo regenerating limb cultures, we demonstrate that extrinsic cues produced during limb development block AER cell formation. We identify Noggin, a morphogen expressed in cartilage/bone progenitor cells, as one of the key inhibitors of AER cell formation in regeneration-incompetent tadpoles. Extrinsic inhibitory cues can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. Together, these results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates. One Sentence SummaryExtrinsic cues associated with chondrogenic progression inhibit AER cell formation and restrict limb regeneration potential.

developmental biology↗

bioRxiv2019

Resolving the fibrotic niche of human liver cirrhosis using single-cell transcriptomics

Currently there are no effective antifibrotic therapies for liver cirrhosis, a major killer worldwide. To obtain a cellular resolution of directly-relevant pathogenesis and to inform therapeutic design, we profile the transcriptomes of over 100,000 primary human single cells, yielding molecular definitions for the major non-parenchymal cell types present in healthy and cirrhotic human liver. We uncover a novel scar-associated TREM2+CD9+ macrophage subpopulation with a fibrogenic phenotype, that has a distinct differentiation trajectory from circulating monocytes. In the endothelial compartment, we show that newly-defined ACKR1+ and PLVAP+ endothelial cells expand in cirrhosis and are topographically located in the fibrotic septae. Multi-lineage ligand-receptor modelling of specific interactions between the novel scar-associated macrophages, endothelial cells and collagen-producing myofibroblasts in the fibrotic niche, reveals intra-scar activity of several major pathways which promote hepatic fibrosis. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides the conceptual framework required to discover rational therapeutic targets in liver cirrhosis.

immunology↗

bioRxiv2018

Distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data

Droplet-based single-cell RNA sequencing protocols have dramatically increased the throughput and efficiency of single-cell transcriptomics studies. A key computational challenge when processing these data is to distinguish libraries for real cells from empty droplets. Existing methods for cell calling set a minimum threshold on the total unique molecular identifier (UMI) count for each library, which indiscriminately discards cell libraries with low UMI counts. Here, we describe a new statistical method for calling cells from droplet-based data, based on detecting significant deviations from the expression profile of the ambient solution. Using simulations, we demonstrate that our method has greater power than existing approaches for detecting cell libraries with low UMI counts, while controlling the false discovery rate among detected cells. We also apply our method to real data, where we show that the use of our method results in the retention of distinct cell types that would otherwise have been discarded.

bioinformatics↗

bioRxiv2017

Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.

genomics↗

bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗