Biology subjects

Probert, C.

Publications and source records attributed to Probert, C..

5 recordsLinked to original sources

bioRxiv2025

Hybrid Scaffolds Decouple Biochemical & Biophysical Regulation of Cell Phenotype

The extracellular matrix changes dramatically during the progression of diseases like cancer. These complex, tissue-specific changes are not adequately replicated by most current biomaterial disease models. Here we demonstrate, for the first time, a biomaterial system allowing combined, independent control over stiffness, composition and 3D collagen architecture. We demonstrate successful perfusion of defined hydrogel formulations into ice-templated collagen scaffolds, controlling the composition of these hybrid scaffolds at constant stiffness. The Youngs moduli of these hybrid scaffolds can also be tuned independently of composition via chemical crosslinking. Encapsulation of human dermal fibroblasts reveals that fibroblast morphology depends on hybrid scaffold composition and on viscoelasticity, highlighting the importance of a system that decouples biophysical from biochemical properties. Finally, we demonstrate application of this system to exert combined control over biochemical and biophysical drivers of cell growth and invasion, focussing on breast cancer as proof-of-concept. We show that collagen fibre patterning enhances breast cancer cell proliferation, also directing the invasion of patient-derived breast cancer cells. These hybrid scaffolds are therefore promising new tools for dissecting the diverse but complementary roles played by the extracellular matrix in regulating cell phenotype, in a range of healthcare applications.

bioengineering↗

bioRxiv2023

EmbedGEM: A framework to evaluate the utility of embeddings for genetic discovery

Machine learning (ML)-derived embeddings are a compressed representation of high content data modalities. Embeddings can capture detailed information about disease states and have been qualitatively shown to be useful in genetic discovery. Despite their promise, embeddings have a major limitation: it is unclear if genetic variants associated with embeddings are relevant to the disease or trait of interest. In this work we describe EmbedGEM (Embedding Genetic Evaluation Methods), a framework to systematically evaluate the utility of embeddings in genetic discovery. EmbedGEM focuses on comparing embeddings along two axes: heritability and disease relevance. As measures of heritability, we consider the number of genome-wide significant associations and the mean{chi} 2 statistic at significant loci. For disease relevance, we compute polygenic risk scores for each embedding principal component, then evaluate their association with high-confidence disease or trait labels in a held-out evaluation patient set. While our development of EmbedGEM is motivated by embeddings, the approach is generally applicable to multivariate traits, and can readily be extended to accommodate additional metrics along the evaluation axes. We demonstrate EmbedGEMs utility by evaluating embeddings and multivariate traits in two separate datasets: i) a synthetic dataset simulated to demonstrate the ability of the framework to correctly rank traits based on their heritability and disease relevance, and ii) a real data from the UK Biobank including metabolic and liver-related traits. Importantly, we show that greater disease relevance does not automatically follow from greater heritability.

bioinformatics↗

bioRxiv2023

CTCF is essential for proper mitotic spindle structure and anaphase segregation

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.

cell biology↗

bioRxiv2019

Macrophage-specific NF-kappa B activation dynamics can segregate inflammatory bowel disease patients

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-{kappa}B transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-{kappa}B/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohns patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohns donors. Using a similar lentivirus approach, NF-{kappa}B-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-{kappa}B-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohns patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-{kappa}B-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohns patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-{kappa}B activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy. Significance statementThis manuscript describes two dynamic assays of NF-{kappa}B activation in blood-derived macrophages that can segregate IBD patients into groups with different phenotypes. For the first time we introduce the use of dynamic measurements of a transcription factor activation as a method to stratify patients and we are confident that our approach will lead in future to early patient stratification and prediction of treatment outcome.

immunology↗

bioRxiv2019

Identification of a novel therapeutic agent for Inflammatory Bowel Disease guided by systems medicine

ObjectiveInflammatory bowel diseases cause significant morbidity and mortality. Aberrant NF-{kappa}B signalling is strongly associated with these conditions, and several established drugs influence the NF-{kappa}B signalling network to exert their effect. This study aimed to identify drugs which alter NF-{kappa}B signalling and may be repositioned for use in inflammatory bowel disease. DesignThe SysmedIBD consortium established a novel drug-repurposing pipeline based on a combination of in-silico drug discovery and biological assays targeted at demonstrating an impact on NF-kappaB signalling, and a murine model of IBD. ResultsThe drug discovery algorithm identified several drugs already established in IBD, including corticosteroids. The highest-ranked drug was the macrolide antibiotic Clarithromycin, which has previously been reported to have anti-inflammatory effects in aseptic conditions. Clarithromycins effects were validated in several experiments: it influenced NF-{kappa}B mediated transcription in murine peritoneal macrophages and intestinal enteroids; it suppressed NF-{kappa}B protein shuttling in murine reporter enteroids; it suppressed NF-{kappa}B (p65) DNA binding in the small intestine of mice exposed to LPS, and it reduced the severity of dextran sulphate sodium-induced colitis in C57BL/6 mice. Clarithromycin also suppressed NF-{kappa}B (p65) nuclear translocation in human intestinal enteroids. ConclusionsThese findings demonstrate that in-silico drug repositioning algorithms can viably be allied to laboratory validation assays in the context of inflammatory bowel disease; and that further clinical assessment of clarithromycin in the management of inflammatory bowel disease is required.

systems biology↗