Biology subjects

MacPherson, H.

Publications and source records attributed to MacPherson, H..

4 recordsLinked to original sources

bioRxiv2025

Single cell proteomic analysis defines discrete neutrophil functional states in human glioblastoma

Neutrophils are vital innate immune cells shown to infiltrate glioblastomas, however we currently lack the molecular understanding of their functional states within the tumour niche. Given that neutrophils are known to display a prominent discordance between mRNA and protein abundance, we developed ultra-sensitive mini-bulk and single cell proteomic (SCP) workflows to study the heterogeneity of peripheral blood and tumour associated neutrophils (TAN) from patients with glioblastoma. Mini-bulk analysis enabled a deeper protein coverage of circulating immature, mature and TAN populations, defining signatures of maturity and demonstrating that TANs resemble mature circulating neutrophils. Analysis of the SCP data resulted in the detection of >1,100 proteins from a single TAN providing a detailed characterization of neutrophil subsets in glioblastoma. Our approach shows evidence of pathogenic and anti-tumorigenic clusters and discovers cell states invisible to scRNAseq, opening new opportunities to selectively target pro-tumoural neutrophil states.

immunology↗

bioRxiv2023

In vitro modelling of anterior primitive streak patterning with hESC reveals the dynamic of WNT and NODAL signalling required to specify notochord progenitors

Notochord progenitors (NotoPs) represent a scarce yet crucial embryonic cell population, playing important roles in embryo patterning and eventually giving rise to the cells that form and maintain intervertebral discs. The mechanisms regulating NotoPs emergence are unclear. This knowledge gap persists due to the inherent complexity of cell fate patterning during gastrulation, particularly within the anterior primitive streak (APS), where NotoPs first arise alongside other important progenitors including neuro-mesodermal and endodermal progenitors. To gain insights into this process, we use micropatterning together with FGF and the WNT pathway activator CHIR9901, to guide the development of human embryonic stem cells into reproducible patterns of APS cell fates. We show that small variations in CHIR9901 dosage dictate the downstream dynamics of endogenous TGFbeta signalling which in turn controls cell fate decisions. We show that sustained NODAL signalling induces endoderm while NODAL inhibition is needed for NMP specification. Furthermore, we unveil a crosstalk between TGFbeta and WNT signaling pathways, wherein TGFbeta inhibition enhances WNT activity. Finally, we demonstrate that the timely inhibition of TGFbeta signalling is imperative for the emergence of NotoPs. Our work elucidates the signalling regimes underpinning NotoPs emergence and provides novel insights into the regulatory mechanisms controlling the balance of APS cell fates during gastrulation.

developmental biology↗

bioRxiv2022

Pseudogenes limit the identification of novel common transcripts generated by their parent genes

The human genome contains numerous duplicated regions, such as parent-pseudogene pairs, causing sequencing reads to align equally well to either gene. The extent to which this ambiguity complicates transcriptomic analyses is currently unknown. This is concerning as many parent genes have been linked to disease, including GBA1, causally linked to both Parkinsons and Gaucher disease. We find that most of the short sequencing reads that map to GBA1, also map to its pseudogene, GBAP1. Using long-read RNA-sequencing in human brain, where all reads mapped uniquely, we demonstrate significant differences in expression compared to short-read data. We identify novel transcripts from both GBA1 and GBAP1, including protein-coding transcripts that are translated in vitro and detected in proteomic data, but that lack GCase activity. By combining long-read with single-nuclear RNA-sequencing to analyse brain-relevant cell types we demonstrate that transcript expression varies by brain region with cell-type-selectivity. Taken together, these results suggest a non-lysosomal function for both GBA1 and GBAP1 in brain. Finally, we demonstrate that inaccuracies in annotation are widespread among parent genes, with implications for many human diseases.

genetics↗

bioRxiv2017

Evaluating the Role of Cell-free Fetal DNA in Inflammation and Spontaneous Preterm Birth.

Preterm birth is the leading cause of neonatal mortality. While spontaneous preterm birth (sPTB) is the cause of over 70% of PTB, the pathogenesis behind sPTB remains unclear. Cell-free fetal DNA (cff-DNA) originates from the placenta and is increased in women who develop PTB. It has been demonstrated that fetal DNA is hypomethylated and is pro-inflammatory. The pro-inflammatory properties of placental-derived DNA, the effects of placental inflammation on the production of cff-DNA, and its significance in the pathogenesis of PTB are unknown.\n\nUsing a human placental explant model, we analysed the effect of lipopolysaccharide (LPS) stimulation on cff-DNA production, and used the cff-DNA generated by these explants to examine the methylation profile and in-vitro pro-inflammatory properties of cff-DNA. LPS caused significant production of TNF- from placental explants, but did not significantly increase the cff-DNA production. Placental-derived cff-DNA, was found to have a small proportion of unmethylated CpG motifs, but was more similar to adult DNA than to more highly unmethylated E-coli DNA. However, cff-DNA did not elicit production of inflammatory cytokines (IL-6, IL-8, TNF- and CXCL10) by peripheral blood mononuclear cells from pregnant women. Furthermore, in contrast to LPS, intra-uterine injections of mouse placental DNA did not decrease time to delivery in an in-vivo mouse PTB model compared to control animals.\n\nThis study demonstrates that placental inflammation does not increase the production of cff-DNA in placental explants, and cff-DNA alone is not sufficient to elicit an inflammatory response in human PBMC cultures ex-vivo. It also shows that mouse placental DNA does not cause PTB in-vivo. This suggests that cff-DNA might be predominantly an effect of parturition and not a principal causative agent.

immunology↗