Biology subjects

Marshall, R.

Publications and source records attributed to Marshall, R..

6 recordsLinked to original sources

bioRxiv2020

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis

Fibroblastic foci (FF) represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling un-manipulated myo/fibroblasts within FF in situ by laser capture microdissection. The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures which correlate with fibrillar collagen gene expression. WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that TGF-{beta}1, RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-{beta}1-induced mTORC1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identifies the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions. What is the key question?Can we identify a transcriptional signature associated with collagen gene expression in the fibrotic focus, the cardinal fibrotic lesion in IPF? What is the bottom line?We herein define the major transcriptional programmes involved in fibrogenesis in IPF by profiling myo/fibroblasts within FF in situ by laser capture microdissection. Why read on?The data provide strong support for a human tissue-based approach to identify critical transcriptional nodes associated with fibrogenesis in situ and further identifies the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

cell biology↗

bioRxiv2019

In vitro implementation of robust gene regulation in a synthetic biomolecular integral controller

Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Motivated by the need to tune the dynamics and improve the robustness of synthetic gene circuits, biological engineers have proposed various designs that mimic natural molecular feedback control mechanisms. However, practical and predictable implementations have proved challenging because of the complexity of synthesis and analysis of complex biomolecular networks. Here, we analyze and experimentally validate a first synthetic biomolecular controller executed in vitro. The controller is based on the interaction between a sigma and an anti-sigma factor, which ensures that gene expression tracks an externally imposed reference level, and achieves this goal even in the presence of disturbances. Our design relies upon an analog of the well-known principle of integral feedback in control theory. We implement the controller in an Escherichia coli cell-free transcription-translation (TXTL) system, a platform that allows rapid prototyping and implementation. Modeling and theory guide experimental implementation of the controller with well-defined operational predictability.

synthetic biology↗

bioRxiv2018

Biomarkers of collagen synthesis predict progression in the PROFILE idiopathic pulmonary fibrosis cohort

Idiopathic pulmonary fibrosis (IPF) is characterised by excessive extracellular matrix (ECM) deposition and remodelling. Measuring this activity provides an opportunity to develop tools capable of identifying individuals at-risk of progression. Longitudinal change in markers of ECM synthesis was assessed in 145 newly-diagnosed individuals with IPF. Serum levels of collagen synthesis neoepitopes, PRO-C3 and PRO-C6 (collagen type 3 and 6), were elevated in IPF compared with controls at baseline, and progressive disease versus stable disease during follow up, (PRO-C3 p<0.001; PRO-C6 p=0.029). Assessment of rate of change in neoepitope levels from baseline to 3 months (defined as slope to month 3: HIGH slope, slope > 0 vs. LOW slope, slope <=0) demonstrated no relationship with mortality for these markers (PRO-C3 (HR 1.62, p=0.080); PINP (HR 0.76, p=0.309); PRO-C6 (HR 1.14, p=0.628)). As previously reported, rising concentrations of collagen degradation markers C1M, C3M, C6M and CRPM were associated with an increased risk of overall mortality (HR=1.84, CI 1.03 - 3.27, p=0.038, HR=2.44, CI 1.39-4.31, p=0.002; HR= 2.19, CI 1.25-3.82, p=0.006; HR= 2.13 CI 1.21-3.75, p=0.009 respectively). Elevated levels of PRO-C3 and PRO-C6 associate with IPF disease progression. Collagen synthesis and degradation biomarkers have the potential to enhance clinical trials in IPF and may inform prognostic assessment and therapeutic decision making in the clinic.

clinical trials↗

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

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

synthetic biology↗

bioRxiv2017

Tuning of recombinant protein expression in Escherichia coli by manipulating transcription, translation initiation rates and incorporation of non-canonical amino acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. Based on the results, we developed a biophysical model, which suggests that the density of co-transcriptional translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

synthetic biology↗