David Simpson

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Papers by David Simpson

Comparison of SARS-CoV-2 Evolution in Paediatric Primary Airway Epithelial Cell Cultures Compared with Vero-Derived Cell Lines

Viruses, 2022

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality p... more SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly...

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A Multi-Omics Approach Identifies Key Regulatory Pathways Induced by Long-Term Zinc Supplementation in Human Primary Retinal Pigment Epithelium

Nutrients, 2020

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation... more In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation...

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Machine learning approaches to supporting the identification of photoreceptor-enriched genes based on expression data

BMC bioinformatics, Jan 8, 2006

Retinal photoreceptors are highly specialised cells, which detect light and are central to mammal... more Retinal photoreceptors are highly specialised cells, which detect light and are central to mammalian vision. Many retinal diseases occur as a result of inherited dysfunction of the rod and cone photoreceptor cells. Development and maintenance of photoreceptors requires appropriate regulation of the many genes specifically or highly expressed in these cells. Over the last decades, different experimental approaches have been developed to identify photoreceptor enriched genes. Recent progress in RNA analysis technology has generated large amounts of gene expression data relevant to retinal development. This paper assesses a machine learning methodology for supporting the identification of photoreceptor enriched genes based on expression data. Based on the analysis of publicly-available gene expression data from the developing mouse retina generated by serial analysis of gene expression (SAGE), this paper presents a predictive methodology comprising several in silico models for detectin...

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Individual mRNA expression profiles reveal the effects of specific microRNAs

Genome Biology, 2008

MicroRNA target expression <p>The effect of a microRNA on the levels of its target mRNAs can be m... more

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A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma

BMC Genomics, 2019

RESEARCH ARTICLE Open Access Prediction of microRNAs affecting mRNA

Background: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to... more Background: MicroRNAs (miRNAs) are small RNA molecules (~22 nucleotides) which have been shown to play an important role both in development and in maintenance of adult tissue. Conditional inactivation of miRNAs in the eye causes loss of visual function and progressive retinal degeneration. In addition to inhibiting translation, miRNAs can mediate degradation of targeted mRNAs. We have previously shown that candidate miRNAs affecting transcript levels in a tissue can be deduced from mRNA microarray expression profiles. The purpose of this study was to predict miRNAs which affect mRNA levels in developing and adult retinal tissue and to confirm their expression. Results: Microarray expression data from ciliary epithelial retinal stem cells (CE-RSCs), developing and adult mouse retina were generated or downloaded from public repositories. Analysis of gene expression profiles detected the effects of multiple miRNAs in CE-RSCs and retina. The expression of 20 selected miRNAs was confirm...

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Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties

Molecular Vision, 2020

Purpose To better characterize retinal endothelial barrier properties through analysis of individ... more Purpose To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs). Methods Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses. Results Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells ...

format_quoteIdentified cell cycle variation as the primary source of heterogeneity in cultured RMECs, alongside distinct transcriptomic clusters.format_quote

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Development of a combined RT-PCR: NGS assay for microRNA biomarkers

Results and Discussion qPCR cycling conditions and limit of detection qPCR analysis of miR-21 sh... more Results and Discussion qPCR cycling conditions and limit of detection qPCR analysis of miR-21 showed PCR efficiency of 101% and r 2 of 0.9961 across a 7-log dilution series.  Compatible with Ion Torrent and Illumina sequencing platforms. End-point PCR cycling conditions for library construction  Assay end-point PCR protocols were optimised for library production. Sanger sequencing of libraries  Conventional sequencing confirmed specific amplification of target miRNAs from a pool of total cellular RNA. NGS analysis using the Ion PGM  Detection of miR-21isomiRs expressed in cells derived from circulating endothelial progenitors  IsomiR levels correlated with Illumina small RNA-Seq Bioinformatics interface  Automated analysis of raw IzoSeq sequencing data (FastQ) to generate isomiR abundance.  GUI developed

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MicroRNA-containing extracellular vesicles released from endothelial colony-forming cells modulate angiogenesis during ischaemic retinopathy

Journal of cellular and molecular medicine, Jan 20, 2017

Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that mo... more Endothelial colony-forming cells (ECFCs) are a defined subtype of endothelial progenitors that modulate vascular repair and promote perfusion in ischaemic tissues. Their paracrine activity on resident vasculature is ill-defined, but mediated, at least in part, by the transfer of extracellular vesicles (EVs). To evaluate the potential of isolated EVs to provide an alternative to cell-based therapies, we first performed a physical and molecular characterization of those released by ECFCs. Their effects upon endothelial cells in vitro and angiogenesis in vivo in a model of proliferative retinopathy were assessed. The EVs expressed typical markers CD9 and CD63 and formed a heterogeneous population ranging in size from ~60 to 1500 nm by electron microscopy. ECFC EVs were taken up by endothelial cells and increased cell migration. This was reflected by microarray analyses which showed significant changes in expression of genes associated with angiogenesis. Sequencing of small RNAs in ECFC...

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TRPV2 Channels Contribute to Stretch-Activated Cation Currents and Myogenic Constriction in Retinal Arterioles

Investigative ophthalmology & visual science, 2016

Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been... more Activation of the transient receptor potential channels, TRPC6, TRPM4, and TRPP1 (PKD2), has been shown to contribute to the myogenic constriction of cerebral arteries. In the present study we sought to determine the potential role of various mechanosensitive TRP channels to myogenic signaling in arterioles of the rat retina. Rat retinal arterioles were isolated for RT-PCR, Fura-2 Ca2+ microfluorimetry, patch-clamp electrophysiology, and pressure myography studies. In some experiments, confocal immunolabeling of wholemount preparations was used to examine the localization of specific mechanosensitive TRP channels in retinal vascular smooth muscle cells (VSMCs). Reverse transcription-polymerase chain reaction analysis demonstrated mRNA expression for TRPC1, M7, V1, V2, V4, and P1, but not TRPC6 or M4, in isolated retinal arterioles. Immunolabeling revealed plasma membrane, cytosolic and nuclear expression of TRPC1, M7, V1, V2, V4, and P1 in retinal VSMCs. Hypoosmotic stretch-induced ...

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