Biology subjects

Warren, D. T.

Publications and source records attributed to Warren, D. T..

3 recordsLinked to original sources

bioRxiv2025

P3ANUT: An enhanced DNA sequencing analysis platform for uncovering and correcting errors in peptide phage display library screening

Display technologies are used extensively in the discovery of peptides and antibodies towards the development of new medicines and diagnostic tools. Phage display technology enables the filtering of <1010 random peptides/antibodies down to and enriched pool of candidates with favourable binding affinities. In recent years, next-generation DNA sequencing technologies have increased the precision and accuracy with which the peptide sequences of phage display library clones are identified. Inaccuracies in DNA sequencing such as substitutions, insertions and deletions in the library oligonucleotide region have the potential to result in the identification of erroneous candidate sequences. Here, we describe a Python Pipeline for Phage Analysis through a Normative Unified Toolset (P3ANUT) which employs Levenshtein distance, k-mer approaches and a novel encoding scheme on paired-end sequencing outputs to correct sequencing errors from next-generation sequencing outputs of display library screens. We introduce an easy-to-use and highly customisable computational tool with graphical user- and command line interfaces to process entire datasets within a single input, as well as visualisation tools for candidate analysis and data generation. P3ANUT shows significant improvements in read recovery, overall read quality, and runtime compared to a previously published pipeline. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/648809v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@15e1172org.highwire.dtl.DTLVardef@cb9097org.highwire.dtl.DTLVardef@81cabcorg.highwire.dtl.DTLVardef@1253865_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

A hydrogel-based model of aortic stiffness reveals that microtubules are novel regulators of smooth muscle cell hypertrophy

During ageing, the extracellular matrix of the aortic wall becomes more rigid. In response, VSMCs generate enhanced contractile forces. Our previous findings demonstrate that VSMC volume is enhanced in response to increased matrix rigidity, but our understanding of mechanisms regulating this process remain incomplete. In this current study, we show that microtubule stability in VSMCs is reduced in response to enhanced matrix rigidity via piezo1-mediated Ca2+ influx. Moreover, VSMC volume and Ca2+ flux was regulated by microtubule dynamics; microtubule stabilising agents reduced both VSMC volume and Ca2+ flux on rigid hydrogels, whereas microtubule destabilising agents increased VSMC volume and Ca2+ flux on pliable hydrogels. Finally, we show that disruption of the microtubule deacetylase HDAC6 uncoupled these processes and increased K40 alpha tubulin acetylation, VSMC volume and Ca2+ flux on pliable hydrogels, but did not alter VSMC microtubule stability. These findings uncover a microtubule stability switch that controls VSMC volume by regulating Ca2+ flux. Together, these data demonstrate that manipulation of microtubule stability can modify VSMC matrix rigidity response.

cell biology↗

bioRxiv2021

Using polyacrylamide hydrogels to model physiological aortic stiffness reveals that microtubules are critical regulators of isolated smooth muscle cell morphology and contractility.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aortic wall and normally exist in a quiescent, contractile phenotype where actomyosin-derived contractile forces maintain vascular tone. However, VSMCs are not terminally differentiated and can dedifferentiate into a proliferative, synthetic phenotype. Actomyosin force generation is essential for the function of both phenotypes. Whilst much is already known about the mechanisms of VSMC actomyosin force generation, existing assays are either low throughput and time consuming, or qualitative and inconsistent. In this study, we use polyacrylamide hydrogels, tuned to mimic the physiological stiffness of the aortic wall, in a VSMC contractility assay. Isolated VSMC area decreases following stimulation with the contractile agonists angiotensin II or carbachol. Importantly, the angiotensin II induced reduction in cell area correlated with increased traction stress generation. Inhibition of actomyosin activity using blebbistatin or Y-27632 prevented angiotensin II mediated changes in VSMC morphology, suggesting that changes in VSMC morphology and actomyosin activity are core components of the contractile response. Furthermore, we show that microtubule stability is an essential regulator of isolated VSMC contractility. Treatment with either colchicine or paclitaxel uncoupled the morphological and/or traction stress responses of angiotensin II stimulated VSMCs. Our findings support the tensegrity model and we demonstrate that microtubules act to balance the actomyosin-derived traction stress generation and regulate the morphological responses of VSMCs.

cell biology↗