Biology subjects

McClafferty, H.

Publications and source records attributed to McClafferty, H..

4 recordsLinked to original sources

bioRxiv2025

Adenylyl cyclase 9: fundamental change of regulation in vertebrates and gene sub-functionalization after teleost-specific whole-genome duplication

Adenosine 3:5 monophosphate (cAMP) is a ubiquitous signalling molecule generated by the adenylyl cyclase family of proteins which is encoded by ten genes. The biological significance of this diversity is not well understood. In mammals, transmembrane adenylyl cyclase 9 (AC9) is resistant to regulation by heterotrimeric G proteins. A major facet of this resistance is auto-inhibition -- in the presence of activated Gs, AC9 is inhibited by its C-terminal domain. Here, we examined the natural evolution of this seemingly paradoxical control mechanism. At the primary sequence level, the hallmarks of auto-inhibition are apparent in all vertebrates, none are found in invertebrates. Teleost-specific genome duplication (TGD) resulted in adcy9 ohnologs, one of which lacked the hallmarks of auto-inhibition. This was confirmed in functional assays upon cloning and heterologous expression of the requisite cDNAs. The tissue distributions of the adcy9 ohnologs in teleost species also pointed to their sub-functionalization. Above all, auto-inhibited adcy9 was largely restricted to the brain indicating a fundamental role in brain development or function. Our findings document a quantum leap of the regulation of the enzymatic activity of AC9 in vertebrates and the potency of TGD to meet an adaptational challenge through functionally diversified ohnologs.

evolutionary biology↗

bioRxiv2024

A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening and disease modelling. Here we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in the development of hematopoietic progenitors. We first identified nine candidate transcription factors that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these transcription factors that could also be detected during scRNAseq. iCRISPRa activation of these endogenous transcription factors resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2 and our analysis indicated that IGFBP2 is involved in the remodeling of metabolic activity during in vitro endothelial to hematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic cell fate and differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes controlling developmental events and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.

cell biology↗

bioRxiv2022

AMPK deficiency in smooth muscles causes persistent pulmonary hypertension after birth and premature death

We address a paradox, that AMPK may facilitate hypoxic pulmonary vasoconstriction and its deficiency precipitate pulmonary hypertension. Here we show that AMPK-1/2 deficiency in smooth muscles promotes persistent pulmonary hypertension of the newborn. Accordingly, dual AMPK-1/2 deletion in smooth muscles causes premature death of mice after birth, associated with increased muscularization and remodeling throughout the pulmonary arterial tree, reduced alveolar numbers and alveolar membrane thickening, but with no edema. Spectral Doppler ultrasound indicates pulmonary hypertension and attenuated hypoxic pulmonary vasoconstriction. Age-dependent right ventricular pressure elevation, dilation and reduced cardiac output was also evident. KV1.5 potassium currents of pulmonary arterial myocytes are markedly smaller under normoxia, which is known to facilitate pulmonary hypertension. Mitochondrial fragmentation and reactive oxygen species accumulation is also evident. Importantly, there is no evidence of systemic vasculopathy or hypertension in these mice. Moreover, hypoxic pulmonary vasoconstriction is attenuated by AMPK-1 or AMPK-2 deletion without triggering pulmonary hypertension.

pathology↗

bioRxiv2020

Dissection of the corticotroph transcriptome in a mouse model of glucocorticoid-induced suppression of the HPA axis

Glucocorticoids (GC) are prescribed for periods >3 months to 1-3% of the UK population; 10-50% of these patients develop hypothalamus-pituitary-adrenal (HPA) axis suppression, which may last over 6 months and is associated with morbidity and mortality. Recovery of higher nodes of the axis is necessary for recovery of adrenal function. We developed a mouse model of Dexamethasone (DEX)-induced HPA axis dysfunction in order to further explore recovery in the pituitary. Adult male C57BL6/J or those crossed with Pomc-eGFP mice were randomly assigned to receive DEX (~0.4 mg/kg bodyweight/day) or vehicle via drinking water for 4 weeks following which treatment was withdrawn. Tissues were harvested at 0, 1, and 4 weeks following withdrawal of treatment. Corticotrophs were isolated from Pomc-eGFP pituitaries using FACS, and RNA extracted for RNA-seq. DEX treatment suppressed corticosterone production, which remained partially suppressed at least 1 week following DEX withdrawal. In the adrenal, at time 0, Hsd3b2, Cyp11a1, and Mc2r mRNA levels were significantly reduced, with Mc2r and Cyp11a1 remaining reduced 1 week following DEX withdrawal. The corticotroph transcriptome was modified by DEX treatment with some differences between groups persisting 4 weeks following withdrawal. No genes supressed by DEX exhibited ongoing attenuation 1 and 4 weeks following withdrawal, whilst only 2 genes were upregulated and remained so following withdrawal. A pattern of rebound at 1 and 4 weeks was observed in 14 genes that increased following suppression, and 6 genes that were reduced by DEX and then increased. Chronic GC treatment may induce persistent changes in the pituitary that may influence future response to GC treatment or stress.

physiology↗