Biology subjects

Donaldson, I. J.

Publications and source records attributed to Donaldson, I. J..

4 recordsLinked to original sources

bioRxiv2025

Human pluripotent stem cell model of multiple epiphyseal dysplasia with MATN3 mutation identifies altered Matrix organisation and upregulation of the cholesterol biosynthesis pathway

Multiple epiphyseal dysplasia (MED), caused by mutations in MATN3, is a chondrodysplasia affecting the cartilage growth plate and is characterised by delayed epiphyseal ossification, short stature, and early onset osteoarthritis. Here we generated an in vitro human pluripotent stem cell (hPSC) model of cartilage growth-plate development to identify pathogenic mechanisms underlying MED. hPSCs were differentiated to chondrocytes via a mesenchymal intermediate, followed by TGF{beta}3+BMP2 induced chondrogenic pellet culture. MATN3-mutant hPSCs were generated by reprogramming MED patient PBMCs or by CRISPR-Cas9 gene editing to introduce a MATN3 mutation in a hESC line. RNAseq was used to assess chondrogenesis and identify MED pathogenic mechanisms. Transmission electron microscopy (TEM) was used to assess extracellular matrix assembly. The resultant hPSC-derived cartilage pellets displayed a typical cartilage morphology and strongly expressed cartilage matrix markers, e.g., collagen II and matrilin-3. Matrilin-3 protein was detected within both the matrix and cells of heterozygous mutant hPSC-cartilage pellets. RNAseq of mutant hPSC-cartilage pellets revealed significant enrichment for ECM organisation and cholesterol biosynthesis pathway genes as well as sightly increased expression of some unfolded protein response (UPR) marker genes. MATN3 mutant hPSC-derived cartilage pellets displayed abnormal matrix assembly, distended ER, accumulation of lipid droplets, and increased cholesterol content. Our model revealed mutant matrilin-3 induces cholesterol biosynthesis pathway upregulation and abnormal matrix assembly during MED pathogenesis. This study provides new insights into the molecular mechanisms underlying MED and highlights potential therapeutic targets.

cell biology↗

bioRxiv2025

Epigenome and transcriptome changes in KMT2D-related Kabuki syndrome Type 1 iPSCs, neuronal progenitors and cortical neurons

Kabuki syndrome type 1 (KS1) is a neurodevelopmental disorder caused by loss-of-function variants in KMT2D which encodes a H3K4 methyltransferase. The mechanisms underlying neurodevelopmental problems in KS1 are still largely unknown. Here, we track the epigenome and transcriptome across three stages of neuronal differentiation using patient-derived induced pluripotent stem cells (iPSCs) to gain insights into the disease mechanism of KS1. In KS1 iPSCs we detected significantly lower levels of functional KMT2D transcript and KMT2D protein, and lower global H3K4me1 and H3K4me2 levels. We identify loss of thousands of H3K4me1 peaks in iPSCs, neuronal progenitors (NPs) and early cortical neurons (CNs) in KS1. We show that the number of lost peaks increase as differentiation progresses. We also identify hundreds of differentially expressed genes (DEGs) in iPSCs, NPs and CNs in KS1. In contrast with the epigenomic changes, the number of DEGs decrease as differentiation progresses. Our analysis reveals significant enrichment of differentially downregulated genes in areas containing putative enhancer regions with H3K4me1 loss. We also identify a set of distinct transcription factor binding sites in differentially methylated regions and a set of DEGs related to KS1 phenotypes. We find that genes regulated by SUZ12, a subunit of Polycomb Repressive complex 2, are over-represented in KS1 DEGs at early stages of differentiation. In conclusion, we present a disease-relevant human cellular model for KS1 that provides mechanistic insights for the disorder and could be used for high throughput drug screening for KS1.

genetics↗

bioRxiv2022

Aspergillus fumigatus can exhibit persistence to the fungicidal drug voriconazole

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung-diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non- mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A sub-population of the persister isolates can survive for extended periods and even grow at slow rates in the presence of supra-MIC (Minimum Inhibitory Concentration) of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seems to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCEThe phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge about antifungal tolerance and, particularly, persistence are still very scarce. In this study, we have characterised the response of the prominent fungal pathogen Aspergillus fumigatus to the first line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and identify various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.

microbiology↗

bioRxiv2019

TALE and NF-Y co-occupancy marks enhancers of developmental control genes during zygotic genome activation in zebrafish

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts control to the embryo at early blastula stages. ZGA is thought to be mediated by specialized maternally deposited transcription factors (TFs), but here we demonstrate that NF-Y and TALE - TFs with known later roles in embryogenesis - co-occupy unique genomic elements at zebrafish ZGA. We show that these elements are selectively associated with early-expressed genes involved in transcriptional regulation and possess enhancer activity in vivo. In contrast, we find that elements individually occupied by either NF-Y or TALE are associated with genes acting later in development - such that NF-Y controls a cilia gene expression program while TALE TFs control expression of hox genes. We conclude that NF-Y and TALE have a shared role at ZGA, but separate roles later during development, demonstrating that combinations of known TFs can regulate subsets of key developmental genes at vertebrate ZGA.

developmental biology↗