Biology subjects

Thompson, R. H.

Publications and source records attributed to Thompson, R. H..

3 recordsLinked to original sources

bioRxiv2022

Modification of Seurat v4 for the development of a phase assignment tool able to distinguish between G2 and Mitotic cells

Single cell RNA sequencing (scRNAseq) is a rapidly advancing field which allows for the characterization of the cellular heterogeneity of gene expression profiles within a population. Cell cycle phase is a major contributor to gene expression variance between cells and computational analysis tools have been developed to assign cell cycle phase to scRNAseq datasets. Whilst these tools can be extremely useful, all have the drawback that they classify cells as G1, S or G2/M. Discrete cell phase assignment tool are unable to differentiate between G2 and M and continuous phase assignments tools are unable to identify a region corresponding specifically to mitosis in a pseudo-timeline for continuous assignment along the cell cycle. Bulk RNA sequencing was used to identify differentially expressed genes between mitotic and interphase cells isolated based on phospho-histone H3 expression using fluorescence activated cell sorting. The gene lists were used to develop a Modified Seurat Mitotic Sort (MoSMiS) methodology which can distinguish G2 and M phase cells in single cell RNA sequencing data. The phase assignment tools present in Seurat were modified to allow for cell cycle phase assignment of all stages of the cell cycle identifying a mitotic specific cell population.

bioinformatics↗

bioRxiv2022

A SOD1-dependent mitotic DNA damage checkpoint

In the event of DNA damage, the cell cycle can be slowed or halted to allow for DNA repair. The mechanisms by which this occurs are well-characterised in interphase, although the mechanisms underpinning mitosis slowing in response to damage are unclear. Canonical checkpoints and DNA repair pathways are largely repressed in mitosis, and whilst there is some level of mitotic DNA synthesis and repair, the bulk of DNA damage is processed for post-mitotic repair. How the decision is made between mitotic DNA repair and post-mitotic DNA repair is not known. We have identified the antioxidant enzyme Superoxide Dismutase 1 (SOD1) as an essential factor mediating delayed mitotic progression in response to DNA damage and replication stress. Cells depleted of SOD1 no longer exhibit DNA damage dependent mitotic delay, and display increased levels of damaged centromeres and mitotic defects. Whilst reactive oxygen species (ROS)-inducing agents also lead to SOD1-dependent mitotic delay, intracellular ROS levels do not correlate with mitotic arrest. SOD1 appears to play an important role in DNA repair in interphase and is recruited to the nucleus in response to DNA damage. In addition to control of mitotic progression in response to genotoxic stress, SOD1 also plays a major role in mitotic DNA synthesis. SOD- depleted cells show reduced levels of mitotic EdU incorporation in response to either replication stress or DNA breaks, seemingly in tandem with Rad51 andSOD1-depletion induced mitotic progression in the presence of DNA breaks is Rad52-dependent. We suggest that there are two responses to DNA breaks in mitosis; either arrest and mitotic repair or progression and post-mitotic repair; and these two pathways exist in a fine balance, controlled by a signaling cascade involving SOD1.

molecular biology↗

bioRxiv2022

Glycemic challenge is associated with the rapid cellular activation of the locus ceruleus and nucleus of solitary tract: Circumscribed spatial analysis of phosphorylated MAP kinase immunoreactivity in relation to catecholaminergic and cholinergic chemoarchitecture

Rodent studies indicate that impaired glucose utilization or hypoglycemia is associated with cellular activation of neurons in the medulla (Winslow, 1733) (MY) believed to control feeding behavior and glucose counterregulation. However, such activation has been tracked primarily within hours of the challenge, rather than sooner, and has been poorly mapped within standardized brain atlases. Here, we report that within 15 min of receiving 2-deoxy-D-glucose (2-DG; 250 mg/kg, i.v.), which can trigger glucoprivic feeding behavior, marked elevations were observed in the numbers of rhombic brain (His, 1893) (RB) neuronal cell profiles immunoreactive for the cellular activation marker(s), phosphorylated p44/42 MAP kinases (phospho-ERK1/2), some of which were also catecholaminergic. We mapped their distributions within an open-access rat brain atlas and found that 2-DG-treated rats (compared to their saline-treated controls) displayed greater numbers of phospho-ERK1/2+ neurons in the locus ceruleus (Wenzel & Wenzel, 1812) (LC) and the nucleus of solitary tract (>1840) (NTS). Thus, 2-DG-activation of certain RB neurons is more rapid than perhaps previously realized, engaging neurons that serve multiple functional systems and are of varying cellular phenotypes. Mapping these populations within standardized brain atlas maps streamlines their targeting and/or comparable mapping in preclinical rodent models of disease.

neuroscience↗