Biology subjects

Tolomeo, D.

Publications and source records attributed to Tolomeo, D..

3 recordsLinked to original sources

bioRxiv2024

Generative Modelling of Oncogene-carrying Extrachromosomal Circular DNA Biogenesis and Dynamics in Cells

Extrachromosomal circular DNAs (ecDNA) are focal gene amplifications frequently associated with cancer development and often indicating a poor prognosis. To understand the early dynamics of oncogene-carrying ecDNAs, we previously developed CRISPR-C, a tool for precise ecDNA generation by deleting specific chromosomal regions. Here, we adapted CRISPR-C to recreate tumor ecDNAs. This method also allowed us to enhance ecDNA generation efficiency by directly delivering Cas9 protein and sgRNAs as a ribonucleoprotein complex. By using the modified CRISPR-C, we successfully generated ecDNAs carrying oncogenes (EGFR, CDK4, MDM2, MYC, MYCN, FGFR2, ABCB1, and DHFR) in various human cell types. Furthermore, we demonstrated that our method could generate chimeric ecDNAs composed of target sequences from distant intra or inter-chromosomal regions. Using these generative ecDNA cell models, we studied the oncogene ecDNA expression and stability. The MDM2 expression was increased after CRISPR-C, while CDK4 was decreased indicating genomic-context dependent effect. The copy number of CRISPR-C generated CDK4 was ecDNA increased in cells after a long period of treatment with the CDK4 inhibitor palbociclib. Unlike CDK4, the CRISPR-C generated ABCB1 ecDNA was unstable in cells under normal growth conditions, but is stably retained when the cells were treated with colcemid, a recognized substrate for ABCB1. We thus provide valuable tools and an attractive platform for studying ecDNA biogenesisy and in vitro drug screening on ecDNA stability.

cancer biology↗

bioRxiv2024

A unique subset of pericystic endothelium associates with aberrant microvascular remodelling and impaired blood perfusion early in polycystic kidney disease

Hallmarks of autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary kidney anomaly, include expanding fluid-filled epithelial cysts, inflammation, and fibrosis. Despite previous work showing the potential of vascular-based therapies, renal microvascular alterations in ADPKD, and their timing, are poorly understood. Using single-cell transcriptomics of human kidney microvasculature, we identify a population of endothelial cells adjacent to cysts in ADPKD. This pericystic endothelium, distinguishable by its expression of osteopontin (SPP1), has a distinct molecular profile compared to the common endothelial cell injury signature in other kidney diseases. SPP1+ pericystic endothelium was also present in an orthologous mouse model of ADPKD before overt kidney functional decline. By interrogating geometric, topological and fractal properties from three-dimensional imaging of early ADPKD mouse kidneys, we show that pericystic endothelium associates with disorganisation and non-uniformity of the renal cortical microvasculature. Concurrently, we detected region-specific reductions in cortical blood flow within ADPKD murine kidneys using arterial spin labelling. We conclude that ADPKD kidneys contain a unique subset of endothelium manifesting with aberrant remodelling and impaired blood perfusion. Its detection, prior to renal functional decline, advocates the vasculature as a therapeutic target to modulate or preserve renal function in early ADPKD.

pathology↗

bioRxiv2018

A systems-level analysis highlights microglial activation as a modifying factor in common forms of human epilepsy

The common human epilepsies are associated with distinct patterns of reduced cortical thickness, detectable on neuroimaging, with important clinical consequences. To explore underlying mechanisms, we layered MRI-based cortical structural maps from a large-scale epilepsy neuroimaging study onto highly spatially-resolved human brain gene expression data, identifying >2,500 genes overexpressed in regions of reduced cortical thickness, compared to relatively-protected regions. The resulting set of differentially-expressed genes shows enrichment for microglial markers, and in particular, activated microglial states. Parallel analyses of cell-specific eQTLs show enrichment in human genetic signatures of epilepsy severity, but not epilepsy causation. Post mortem brain tissue from humans with epilepsy shows excess activated microglia. In an experimental model, depletion of activated microglia prevents cortical thinning, but not the development of chronic seizures. These convergent data strongly implicate activated microglia in cortical thinning, representing a new dimension for concern and disease modification in the epilepsies, potentially distinct from seizure control.

neuroscience↗