Biology subjects

Wilkinson, M.

Publications and source records attributed to Wilkinson, M..

21 records · Page 2Linked to original sources

bioRxiv2019

Selective labelling of arginine residues engaged in binding sulfatedglycosaminoglycans

IThe activities of hundreds of proteins in the extracellular space are regulated by binding to the glycosaminoglycan heparan sulfate (HS). These interactions are driven by ionic bonds between sulfate and carboxylate groups on the polysaccharide and the side chains of basic residues in the protein. Here we develop a method to selectively label the guanidino side chains of arginine residues in proteins that engage the anionic groups in the sugar. The protein is bound to heparin (a common experimental proxy for HS) on an affinity column. Arginine side chains that are exposed to solvent, and thus involved in binding, are protected by reaction with the dicarbonyl phenylgyoxal (PGO). Elution of the bound proteins then exposes arginine side chains that had directly engaged with anionic groups on the polysaccharide. These are reacted with hydroxyl-phenylglyoxal (HPG). PGO was found to generate three products: a 1:1 product, the 1:1 water condensed product and a 2:1 PGO:arginine product. These three reaction products and that of HPG had distinct masses. Scripts were written to analyse the mass spectra and so identify HPG labelled arginine residues. Proof of principle was acquired on model peptides. The method was then applied to the identification of heparin binding arginine residues in fibroblast growth factors (FGF) 1 and 2. The data demonstrate that four out of eleven arginine residues on FGF2 and five out of six arginine residues of FGF1 engage heparin. Our approach provides a rapid and reliable means to identify arginines involved in functional complexes such as those of proteins with heparin

biochemistry↗

bioRxiv2017

Differential Brd4-bound enhancers drive critical sex differences in glioblastoma

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma, we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited opposing responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis, while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET inhibitors than are female cells. Thus, for the first time, Brd4 activity is revealed to drive a sex differences in stem cell and tumorigenic phenotype, resulting in diametrically opposite responses to BET inhibition in male and female GBM cells. This has important implications for the clinical evaluation and use of BET inhibitors. SignificanceConsistent sex differences in incidence and outcome have been reported in numerous cancers including brain tumors. GBM, the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity, including that of GBM. We report that sex-biased Brd4 activity drive sex differences in GBM and render male and female tumor cells differentially sensitive to BET inhibitors. The observed sex differences in BETi treatment strongly indicate that sex differences in disease biology translate into sex differences in therapeutic responses. This has critical implications for clinical use of BET inhibitors further affirming the importance of inclusion of sex as a biological variable.

genomics↗

bioRxiv2016

Influenza C incidence and herd immunity in Lancaster, UK, in the winter of 2014-2015

Influenza C is not included in the annual seasonal influenza vaccine, and has historically been regarded as a minor respiratory pathogen. However, recent work has highlighted its potential role as a cause of pneumonia in infants. We performed nasopharyngeal or nasal swabbing and/or serum sampling (n=148) in Lancaster, UK, over the winter of 2014-2015. Using enzyme-linked immunosorbent assay (ELISA), we estimated a seropositivity of 77%. By contrast, only 2 individuals, both asymptomatic adults, were influenza C-positive by polymerase chain reaction (PCR). Deep sequencing of nasopharyngeal samples produced partial sequences for 4 genome segments in one of these patients. Bayesian phylogenetic analysis demonstrated that the influenza C genome from this individual is evolutionarily distant to those sampled in recent years and represents a novel genome constellation, indicating that it is a product of a decades-old reassortment event. Although we find no evidence that influenza C was a significant respiratory pathogen during the winter of 2014-2015 in Lancaster, we confirm previous observations of seropositivity in the majority of the population. We calculate that this level of herd immunity would be sufficient to suppress epidemics of influenza C and restricts the virus to sporadic endemic spread.

microbiology↗