Biology subjects

Singleton, M.

Publications and source records attributed to Singleton, M..

5 recordsLinked to original sources

bioRxiv2025

Shared molecular regulation of quiescence in neural and glioma stem cells reveals therapeutic vulnerabilities.

Quiescence, a reversible state of cell-cycle arrest, is an adaptive feature of many adult tissue stem cells, including those in the adult brain. In gliomas, brain tumour stem cells that reside in a quiescent state preferentially survive chemotherapy and radiotherapy, highlighting their critical role in therapy resistance and disease progression. To date, it remains unclear whether the molecular programs governing these states are functionally conserved between neural stem cells and brain tumour stem cells. Here, we establish novel in vitro models to study quiescence and find that glioma stem cells are markedly more resistant to entering quiescence than neural stem cells, suggesting that glioma stem cell quiescence more closely resembles a slow-cycling phenotype or shallow quiescence. Nonetheless, direct comparison of quiescent neural stem cells and quiescent/slow-cycling glioma stem cells, as they transition towards proliferation, reveals conserved gene expression trajectories, indicating shared molecular mechanisms. Furthermore, we find that pathways influencing quiescence in neural stem cells exert similar effects in glioma stem cells, underscoring the functional parallels between these populations. Finally, we identify that inhibition of TGF-{beta} signalling might provide an avenue to improve current standard-of-care treatments by targeting quiescent glioma stem cells. Note on version 2This version corrects two figure errors and expands the Methods. The conclusions of are unchanged. ImagesIn version 1, debris adjacent to the organoid had been digitally removed from the drug-treated image in Figure 6D. The unmodified original is restored here. Images within Figures 3A and 6D have each been processed identically to one another. Scattered cellular debris was present to a similar extent in control and drug-treated samples. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/634421v2_fig6.gif" ALT="Figure 6"> View larger version (51K): org.highwire.dtl.DTLVardef@19aef83org.highwire.dtl.DTLVardef@e2c1e1org.highwire.dtl.DTLVardef@126bf22org.highwire.dtl.DTLVardef@ce7acd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6:C_FLOATNO TGF--R1 inhibition drives GSCs out of a quiescent state. (A) QBC395 cells were cultured in BMP4 quiescence media plus inhibitors for three days. Graphs show percentage of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were normalised to mean of quiescence media alone. (B) QBC395 cells were cultured in palbociclib quiescence media plus inhibitors for three days. Graphs show proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were first normalised to vehicle control for each independent experiment and then quiescence media alone. (C) Schematic of glioblastoma organoid generation. Experiments began within 1-2 weeks of derivation to maximise cellular heterogeneity. Protocol adapted from Jacob and colleagues (60). (D) Image of glioblastoma organoid treated with 30 {micro}M LY-364397 or DMSO vehicle (control) for three days, stained for SOX2 (cyan) and Ki67 (magenta). Dashed lines demarcate organoid boundary. Scale bar: 100 {micro}m. Scattered cellular debris was observed in both control and drug-treated samples to a similar extent. (E) Proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+) after glioblastoma organoids were cultured in the presence of inhibitors. Data were normalised to mean of respective vehicle control. Graphs in (A, B, E) show mean {+/-} SEM. Experiments in (A, B) represent independent experiments on separate passages (n=3-4). Dots in (E) represent individual organoids. Statistics: one-way repeated measures ANOVA with Holm-Sidaks multiple comparisons test in (A, B). Two-way ANOVA in (E) with multiple comparisons t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Panel C created with BioRender (Agreement number: FY289ELRZ5). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/634421v2_fig3.gif" ALT="Figure 3"> View larger version (21K): org.highwire.dtl.DTLVardef@1e04d0corg.highwire.dtl.DTLVardef@e25e26org.highwire.dtl.DTLVardef@1934ab0org.highwire.dtl.DTLVardef@10c77fb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3:C_FLOATNO BMP4 and palbociclib treatment induces patient-derived GSCs into shallow versus deep states of quiescence, respectively. (A) Treatment with 16 ng/mL BMP4 markedly reduces the proportion of proliferating NSCs (SOX2+Ki67+) in primary adult mouse SVZ cultures. (B) Treatment with 16 ng/mL BMP4 modestly reduces the proportion of proliferating GSCs (SOX2+Ki67+) in primary patient-derived cultures (QBC395 cell line). (C) Concentration-response curve of mouse NSCs and patient-derived GSCs (QBC395 cell line) treated with BMP4, measuring proportion of proliferating stem cells (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. (D) Palbociclib treatment markedly reduces the proportion of proliferating GSCs (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. GSC graphs in (C, D) show mean {+/-} SEM of independent experiments conducted on separate passages (n=3-4). NSC graph in (C) shows mean {+/-} SEM of individual cell lines derived from SVZ of different mice (n=4). Scale bar: 15 {micro}m in (A, B). Cross in (D) represents concentration-response at 200 nM. Statistics in (C): two-way ANOVA reporting main effect of cell-line. C_FIG DataThe glioblastoma organoid data in Figure 6E have been updated following re-imaging and blinded re-analysis. Reported effect sizes have changed slightly; the conclusions are unchanged. Figure S2 has likewise been updated following re-imaging and re-analysis. MethodsThe Methods have been expanded to describe the analysis more fully, including data exclusion criteria, normalisation, and blinding. Minor changes to figure legends and the main text are not itemised.

cancer biology↗

bioRxiv2023

Evolutionary analyses of IDRs reveal widespread signals of conservation

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 8,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

bioinformatics↗

bioRxiv2023

A NRF2/beta3-adrenoreceptor axis drives a sustained antioxidant and metabolic rewiring through the pentose-phosphate pathway to alleviate cardiac stress

BackgroundCardiac {beta}3-adrenergic receptors ({beta}3AR) are upregulated in diseased hearts and mediate antithetic effects to those of {beta}1AR and {beta}2AR. {beta}3AR agonists were recently shown to protect from myocardial remodeling in preclinical studies and to improve systolic function in patients with severe heart failure. The underlying mechanisms, however, remain elusive. MethodsTo dissect functional, transcriptional and metabolic effects, hearts and isolated ventricular myocytes from mice harboring a moderate, cardiac-specific expression of a human ADRB3 transgene ({beta}3AR-Tg) and subjected to transverse aortic constriction (TAC) were assessed using echocardiography, RNAseq, PET scan, metabolomics, seahorse and metabolic flux analysis. Subsequently, signaling and metabolic pathways were investigated further in vivo in {beta}3AR-Tg and in vitro in neonatal rat ventricular myocytes adenovirally infected to express {beta}3AR and subjected to neurohormonal stress. These results were completed with an analysis of single nucleus RNAseq data from human cardiac myocytes from heart failure patients. ResultsCompared with WT littermate, {beta}3AR-Tg mice were protected from hypertrophy after transaortic constriction (TAC), while systolic function was preserved. {beta}3AR-expressing hearts displayed enhanced myocardial glucose uptake under stress in absence of increased lactate levels. Instead, metabolomic and metabolic flux analyses in stressed hearts revealed an increase in intermediates of the Pentose-Phosphate Pathway (PPP) in {beta}3AR-Tg, an alternative route of glucose utilization, paralleled with increased transcript levels of NADPH-producing and rate-limiting enzymes of the PPP, without fueling the hexosamine metabolism. The ensuing increased content of NADPH and of reduced glutathione decreased myocyte oxidant stress, while downstream oxidative metabolism assessed by oxygen consumption was preserved with higher glucose oxidation in {beta}3AR-Tg post-TAC compared to WT, together with increased mitochondrial biogenesis. Unbiased transcriptomics and pathway analysis identified NRF2 (NFE2L2) as upstream transcription factor which was functionally verified in {beta}3AR-expressing cardiac myocytes where its translocation and nuclear activity was dependent on {beta}3AR activation of nitric-oxide synthase (NOS) NO production. ConclusionModerate expression of cardiac {beta}3AR, at levels observed in human cardiac myocardium, exerts antioxidant effects through activation of the PPP and NRF2 pathway, thereby preserving myocardial oxidative metabolism, function and integrity under pathophysiological stress.

physiology↗

bioRxiv2023

Spatial transcriptomic analysis of Sonic Hedgehog Medulloblastoma identifies that the loss of heterogeneity and promotion of differentiation underlies the response to CDK4/6 inhibition

BackgroundMedulloblastoma (MB) is a malignant tumour of the cerebellum which can be classified into four major subgroups based on gene expression and genomic features. Single cell transcriptome studies have defined the cellular states underlying each MB subgroup, however the spatial organisation of these diverse cell states and how this impacts response to therapy remains to be determined. MethodsHere, we used spatially resolved transcriptomics to define the cellular diversity within a sonic hedgehog (SHH) patient-derived model of MB and identify how cells specific to a transcriptional state or spatial location are pivotal in responses to treatment with the CDK4/6 inhibitor, Palbociclib. We integrated spatial gene expression with histological annotation and single cell gene expression data from MB, developing a analysis strategy to spatially map cell type responses within the hybrid system of human and mouse cells and their interface within an intact brain tumour section. ResultsWe distinguish neoplastic and non-neoplastic cells within tumours and from the surrounding cerebellar tissue, further refining pathological annotation. We identify a regional response to Palbociclib, with reduced proliferation and induced neuronal differentiation in both treated tumours. Additionally, we resolve at a cellular resolution a distinct tumour interface where the tumour contacts neighbouring mouse brain tissue consisting of abundant astrocytes and microglia and continues to proliferate despite Palbociclib treatment. ConclusionsOur data highlight the power of using spatial transcriptomics to characterise the response of a tumour to a targeted therapy and provide further insights into the molecular and cellular basis underlying the response and resistance to CDK4/6 inhibitors in SHH MB.

genomics↗

bioRxiv2023

Leveraging genomic redundancy to improve inference and alignment of orthologous proteins

Identifying protein sequences with common ancestry is a core task in bioinformatics and evolutionary biology. However, methods for inferring and aligning such sequences in annotated genomes have not kept pace with the increasing scale and complexity of the available data. Thus, in this work we implemented several improvements to the traditional methodology that more fully leverage the redundancy of closely related genomes and the organization of their annotations. Two highlights include the application of the more flexible k-clique percolation algorithm for identifying clusters of orthologous proteins and the development of a novel technique for removing poorly supported regions of alignments with a phylogenetic HMM. In making the latter, we also wrote a fully documented Python package Homomorph that implements standard HMM algorithms and created a set of tutorials to promote its use by a wide audience. We applied the resulting pipeline to a set of 33 annotated Drosophila genomes, generating 22,813 orthologous groups and 8,566 high-quality alignments.

bioinformatics↗