Biology subjects

Chappell, J.

Publications and source records attributed to Chappell, J..

28 records · Page 2Linked to original sources

bioRxiv2020

Primed smooth muscle cells acting as first responder cells in disease

RationaleVascular smooth muscle cell (VSMC) dysregulation is a hallmark of vascular disease, including atherosclerosis. In particular, the majority of cells within atherosclerotic lesions are generated from pre-existing VSMCs and a clonal nature has been documented for VSMC-derived cells in multiple disease models. However, the mechanisms underlying the generation of oligoclonal lesions and the phenotype of proliferating VSMCs are unknown. ObjectiveTo understand the cellular mechanisms underlying clonal VSMC expansion in disease. Methods and ResultsHere we analyse clonal dynamics in multi-color lineage-traced animals over time after vessel injury. We demonstrate that VSMC proliferation is initiated in a small fraction of VSMCs that initially expand clonally in the medial layer and then migrate to form the oligoclonal neointima. Selective activation of VSMC proliferation also occurs in vitro, suggesting that this is a cell-autonomous feature. Mapping of VSMC trajectories using single-cell RNA-sequencing reveals a continuum of cellular states after injury and suggests that VSMC proliferation initiates in cells that have downregulated the contractile phenotype and show evidence of pronounced phenotypic switching. We show that proliferation is associated with induced expression of stem cell antigen 1 (SCA1) and the expression signature previously identified in SCA1+ VSMCs in healthy arteries. A remarkably increased proliferation of SCA1+ VSMCs, directly validated in functional assays, indicates that SCA1+ VSMCs act as "first responders" in vascular injury. Early atherosclerotic lesions also had clonal VSMC contribution and we show that the proliferation-associated injury response is conserved in plaque VSMCs, extending these findings to atherosclerosis. Finally, we identify VSMCs in healthy human arteries that correspond to the SCA1+ state in mouse VSMCs and show that genes identified as differentially expressed in this human VSMC subpopulation are enriched for genes showing genetic association with cardiovascular disease. ConclusionsWe show that cell-intrinsic, selective VSMC activation drives clonal proliferation after injury and in atherosclerosis. Our study suggests that healthy mouse and human arteries contain VSMCs characterised by expression of disease-associated genes that are predisposed for proliferation. Targeting such "first responder" cells in patients undergoing vascular surgery could effectively prevent injury-associated VSMC activation and neoatherosclerosis.

cell biology↗

bioRxiv2020

The dynamic, combinatorial cis-regulatory lexicon of epidermal differentiation

Transcription factors (TFs) bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multi-omic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep learning framework to model the cis-regulatory logic of chromatin accessibility. This identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach reveals the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code in dynamic gene regulation. HIGHLIGHTSO_LIAn integrative multi-omic resource profiling chromatin and expression dynamics across keratinocyte differentiation C_LIO_LIPredictive deep learning models of chromatin dynamics reveal a high-resolution cis-regulatory DNA motif lexicon of epidermal differentiation C_LIO_LIModel interpretation enables discovery of combinatorial cis-regulatory logic of homotypic and heterotypic motif combinations C_LIO_LIMassively parallel reporter experiments validate temporal dynamics and cis-regulatory logic of the combinatorial motif lexicon C_LI

genomics↗

bioRxiv2020

Revealing the Key Regulators of Cell Identity in the Human Adult Pancreas

Cellular identity during development is under the control of transcription factors that form gene regulatory networks. However, the transcription factors and gene regulatory networks underlying cellular identity in the human adult pancreas remain largely unexplored. Here, we integrate multiple single-cell RNA-sequencing datasets of the human adult pancreas, totaling 7393 cells, and comprehensively reconstruct gene regulatory networks. We show that a network of 142 transcription factors forms distinct regulatory modules that characterize pancreatic cell types. We present evidence that our approach identifies regulators of cell identity in the human adult pancreas. We predict that HEYL, BHLHE41 and JUND are active in acinar, beta and alpha cells, respectively, and show that these proteins are present in the human adult pancreas as well as in human induced pluripotent stem cell (hiPSC)-derived islet cells. Using single-cell transcriptomics, we found that JUND represses beta cell genes in hiPSC-alpha cells. Both BHLHE41 and JUND depletion seemed to increase the number of sc-enterochromaffin cells in hiPSC-derived islets. The comprehensive gene regulatory network atlas can be explored interactively online. We anticipate our analysis to be the starting point for a more sophisticated dissection of how transcription factors regulate cell identity in the human adult pancreas. Furthermore, given that transcription factors are major regulators of embryo development and are often perturbed in diseases, a comprehensive understanding of how transcription factors work will be relevant in development and disease. HIGHLIGHTSO_LIReconstruction of gene regulatory networks for human adult pancreatic cell types C_LIO_LIAn interactive resource to explore and visualize gene expression and regulatory states C_LIO_LIPrediction of putative transcription factors that drive pancreatic cell identity C_LIO_LIBHLHE41 depletion in primary islets induces apoptosis C_LI

bioinformatics↗

bioRxiv2020

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 and multiple endemic, epidemic and bat coronavirus

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog {beta}-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.

microbiology↗

bioRxiv2020

Identification of a molecular fingerprint for synaptic glia

The inability to specifically identify and manipulate synaptic glial cells remains a major obstacle to understanding fundamental aspects of synapse formation, stability and repair. Using a combinatorial gene expression approach, we discovered molecular markers that allow us to specifically label perisynaptic Schwann cells (PSCs), glial cells at neuromuscular synapses. Using these markers, we demonstrate that PSCs fully-differentiate postnatally and have a unique molecular signature that includes genes predicted and known to play critical roles at synapses. These findings will serve as a springboard for unprecedented approaches for studying molecular determinants of PSC differentiation and function at neuromuscular synapses and possibly synapse-associated glia throughout the CNS.

neuroscience↗

bioRxiv2019

Pericyte Bridges in Homeostasis and Hyperglycemia: Reconsidering Pericyte Dropout and Microvascular Structures

Diabetic retinopathy threatens the vision of a third of diabetic patients. Progression of the disease is attributed to the dropout of pericytes, a cell type that enwraps and stabilizes the microvasculature. In tandem with this presumptive pericyte dropout, there is enriched formation of structures assumed to be remnants of collapsed or regressed vessels, previously classified as acellular capillaries, string vessels, and basement membrane bridges. Instead of endothelial cells, we show that pericytes colocalize with basement membrane bridges, and both bridging structures are enriched by cell-specific knockout of KLF4 and reversibly enriched with elevation of Ang-2, PDGF-BB, and blood sugar. Our data suggests that pericyte counts from retinal digests have misclassified pericyte bridges as endothelial structures and have exaggerated the role of pericyte loss in DR progression. In vivo imaging of corneal limbal vessels demonstrates pericyte migration off-vessel, implicating pericyte movement in formation of pericyte bridges and pathogenesis of diabetic retinopathy.

cell biology↗

bioRxiv2018

PLANT-Dx: A Molecular Diagnostic for Point of Use Detection of Plant Pathogens

Synthetic biology based diagnostic technologies have improved upon gold standard diagnostic methodologies by decreasing cost, increasing accuracy, and enhancing portability. However there has been little effort in adapting these technologies towards applications related to point-of-use monitoring of plant and crop health. Here, we take a step towards this vision by developing an approach that couples isothermal amplification of specific plant pathogen genomic sequences with customizable synthetic RNA regulators that are designed to trigger the production of a colorimetric output in cell-free gene expression reactions. We demonstrate our system can sense viral derived sequences with high-sensitivity and specificity, and can be utilized to directly detect viruses from infected plant material. Furthermore, we demonstrate that the entire system can operate using only body heat and naked-eye visual analysis of outputs. We anticipate these strategies to be important components of user-friendly and deployable diagnostic systems that can be configured to detect a range of important plant pathogens.

synthetic biology↗

bioRxiv2018

Synthetic pluripotent bacterial stem cells

A defining property of stem cells is their ability to differentiate via asymmetric cell division, in which a stem cell creates a differentiated daughter cell but retains its own phenotype. Here, we describe a synthetic genetic circuit for controlling asymmetrical cell division in Escherichia coli. Specifically, we engineered an inducible system that can bind and segregate plasmid DNA to a single position in the cell. Upon division, the co-localized plasmids are kept by one and only one of the daughter cells. The other daughter cell receives no plasmid DNA and is hence irreversibly differentiated from its sibling. In this way, we achieved asymmetric cell division though asymmetric plasmid partitioning. We also characterized an orthogonal inducible circuit that enables the simultaneous asymmetric partitioning of two plasmid species - resulting in pluripotent cells that have four distinct differentiated states. These results point the way towards engineering multicellular systems from prokaryotic hosts.

synthetic biology↗

bioRxiv2018

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

synthetic biology↗

bioRxiv2017

Computational design of Small Transcription Activating RNAs (STARs) for versatile and dynamic gene regulation

A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this goal is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design - allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to [~]9000-fold gene activation. We then demonstrate the versatility of RNA-based transcription control by showing the broad utility of STARs - from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.

synthetic biology↗