Biology subjects

Clarke, A. R.

Publications and source records attributed to Clarke, A. R..

8 recordsLinked to original sources

bioRxiv2024

Therapeutic window for treatment of prion disease defined by timed depletion of Prion protein

There is no effective treatment preventing the progression of neurodegenerative diseases such as prion and Alzheimers diseases. These fatal diseases of the central nervous system, involve progressive accumulation of a misfolded protein long before overt clinical signs of disease. Removal of prion protein early in the pathological process appears to halt the progression however, it is not known whether intervention at later disease stages could be effective. We investigated the potential for intervention throughout the course of prion disease, by developing a mouse model in which Prnp expression can be manipulated in a tissue specific and time dependent manner. Depleting Prnp from neuronal populations in CNS throughout the preclinical phase substantially prolonged incubation. The pathology was dramatically altered to a pattern of astrocytic associated prion deposition. However once overt clinical symptoms of disease were apparent Prnp depletion did not alter disease progression. This study establishes a wide window for intervention, and suggests timely treatment could delay the onset of clinical disease potentially well beyond the lifetime of an individual.

neuroscience↗

bioRxiv2023

Lineage maintenance due to assortative mating but not ecological divergence in a sympatric sibling species pair

The combination of differential adaptation to ecological niches and the development of reproductive barriers are considered helpful for maintaining co-existing species. In the absence of one of these elements, species boundaries are expected to breakdown. The tephritid fruit fly species pair, Bactrocera tryoni and B. neohumeralis, have significant overlap in geographic range and host use, with time of day of male mating the only known difference in their mating systems. Using a combination of ecological (seasonal abundance, host use and habitat use) and genomic data, we tested the differing roles of competition and assortative mating on the maintenance of the species boundaries in this species pair. Genome-wide SNP analyses found strong genetic differentiation between the species with no evidence for hybridization in the field. Most outlier SNPs were restricted to narrow regions towards the centromeres and telomeres of chromosomes and high nucleotide diversity rates were observed throughout the chromosomes of both species. Enrichment of annotation terms indicated an overabundance of genes with the abnormal neuroanatomy term. Terms of interest associated with sleep and circadian rhythm, potentially important to the allochronic reproductive barrier, were non-enriched. Ecological data found limited evidence for ecological differentiation between the two species based on significant positive correlations between species numbers trapped at different times of the year, trapped in different habitats within a region, or when reared from fruit. Our study highlights the significance of assortative mating over ecological differentiation for sympatric maintenance of the B. tryoni/B. neohumeralis sibling pair.

evolutionary biology↗

bioRxiv2023

Alanine replacements in the structured C-terminal domain of the prion protein reveal conformationally variable regions as major determinants for prion propagation

Mutational analysis of the cellular prion protein (PrPC) has revealed various regions of the protein that modulate prion propagation. However, most approaches involve deletions, insertions, or replacements in the presence of the wild-type cellular protein, which may mask the true phenotype. Here, site-directed alanine mutagenesis of PrPC was conducted to identify sites particularly a surface patch of the protein pertinent to prion propagation in the absence of the wild-type prion protein. Mutations were targeted to the helical, sheet and loop regions of PrPC, or a combination thereof and the mutated proteins expressed in PK1 cells in which endogenous PrPC had been silenced. PK1 cells are a clone of mouse neuroblastoma cells that are highly susceptible to Rocky Mountain Laboratory mouse prions. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titres of mouse prions, we found that all mutations within the structured 121-230 domain, irrespective of secondary structure, severely reduced prion propagation. The reduction was most pronounced for mutations within conformationally variable regions of the protein (G123A.L124A.G125A and V188A.T191A.T192A) and those neighbouring or within helix 1 (S134A.R135A.M153A and H139A.G141A.D146A). While mutations G123A and G125A would likely disrupt the structure of the prion fibril, the other mutations are unlikely to cause disruption. Our data therefore suggests that conformationally variable regions within the structured domain of PrPC are the major determinants of prion propagation efficacy.

biochemistry↗

bioRxiv2022

Divergent east-west lineages in an Australian fruit fly associated with the Carpentaria basin divide

Bactrocera jarvisi is an endemic Australian fruit fly species (Diptera: Tephritidae). It occurs commonly across tropical and subtropical coastal Australia, from far-northern Western Australia, across the Top End of the Northern Territory, and then down the Queensland east coast. Across this range, its distribution crosses several well documented biogeographic barriers. In order to better understand factors leading to the divergence of Australian fruit fly lineages, we carried out a population genetic study of B. jarvisi from across its range using genome-wide SNP analysis, utilising adult specimens gained from trapping and fruit rearing. Populations from the Northern Territory (NT) and Western Australia were genetically similar to each other, but divergent from the genetically uniform east-coast (=Queensland, QLD) population. Phylogenetic analysis demonstrated that the NT population derived from the QLD population. We infer a role for the Carpentaria Basin as a biogeographic barrier restricting east-west gene flow. The QLD populations were largely panmictic and recognised east-coast biogeographic barriers play no part in north-south population structuring. While the NT and QLD populations were genetically distinct, there was evidence for the historically recent translocation of flies from each region to the other. Flies reared from different host fruits collected in the same location showed no genetic divergence. While a role for the Carpentaria Basin as a barrier to gene flow for Australian fruit flies agrees with existing work on the related B. tryoni, the reason(s) for population panmixia for B. jarvisi (and B. tryoni) over the entire Queensland east coast, a linear north-south distance of >2000km, remains unknown.

genetics↗

bioRxiv2022

Prion propagation is dependent on key amino acids in Charge cluster 2 within the prion protein

Prions consist of assemblies of aberrantly folded cellular prion protein (PrPC) upon template-assisted conversion and propagation of disease-associated PrP. To dissect the N-terminal residues critical for efficient prion propagation, we generated a library of point, double, or triple alanine replacements within residues 23-111 of PrP, stably expressed them in cells silenced for endogenous mouse PrPC and challenged the reconstituted cells with four mouse prion strains. Amino acids (aa) 105-111 of Charge Cluster 2 (CC2), which is disordered in PrPC, were required for propagation of all four prion strains; other residues had no effect or exhibited strain-specific effects. Replacements in CC2, including aa105-111, dominantly inhibited prion propagation in the presence of endogenous wild type PrPC whilst other changes were not inhibitory. Single alanine replacements within aa105-111 identified leucine 108 and valine 111 or the cluster of lysine 105, threonine 106 and asparagine 107 as critical for prion propagation. These residues mediate specific ordering of CC2 in the prion fibrils from Rocky Mountain Laboratory (RML) and ME7 mouse prion strains.

molecular biology↗

bioRxiv2022

Metalation calculators for E. coli strain JM109 (DE3): Aerobic, anaerobic and hydrogen peroxide exposed cells cultured in LB media

Three web-based calculators, and three analogous spreadsheets, have been generated that predict in vivo metal occupancies of proteins based on known metal affinities. The calculations exploit estimates of the availabilities of the labile buffered pools of different metals inside a cell. Here, metal availabilities have been estimated for a strain of E. coli that is commonly used in molecular biology and biochemistry research, for example in the production of recombinant proteins. Metal availabilities have been examined for cells grown in LB medium aerobically, anaerobically and in response to H2O2 by monitoring the abundance of a selected set of metal-responsive transcripts by qPCR. The selected genes are regulated by DNA-binding metal sensors that have been thermodynamically characterised in related bacterial cells enabling gene expression to be read-out as a function of intracellular metal availabilities expressed as free energies for forming metal complexes. The calculators compare these values with the free energies for forming complexes with the protein of interest, derived from metal affinities, to estimate how effectively the protein can compete with exchangeable binding sites in the intracellular milieu. The calculators then inter-compete the different metals, limiting total occupancy of the site to a maximum stoichiometry of 1, to output percentage occupancies with each metal. In addition to making these new and conditional calculators available, an original purpose of this article was to provide a tutorial which discusses constraints of this approach and presents ways in which such calculators might be exploited in basic and applied research, and in next-generation manufacturing.

cell biology↗

bioRxiv2020

Plexin-B1 mutation drives prostate cancer metastasis

Prostate cancer mortality is associated with the metastatic spread of tumour cells. A better understanding of the mechanisms which allow a locally advanced tumour to disseminate around the body will identify new therapeutic targets to block this process. One of set of genes implicated in metastasis are plexins, which can promote or suppress tumour progression depending on cancer type and cellular context. We have taken a mouse genetics approach to gain insight into the role of Plexin-B1 in prostate cancer progression in vivo. We show here that genetic deletion of Plexin-B1 in PbCre+Ptenfl/flKrasG12V and PbCre+Ptenfl/flp53fl/fl mouse prostate cancer models significantly decreased metastasis. High levels of prostate epithelial cell-specific expression of wild-type Plexin-B1 in knock-in mice with a PbCre+Ptenfl/flKrasG12V background also significantly decreased metastasis. In contrast, expression of a Plexin-B1 mutant (P1597L; identified from metastatic deposits in prostate cancer patients) in prostate epithelial cells in PbCre+Ptenfl/flKrasG12V and PbCre+Ptenfl/flp53fl/fl mice significantly increased metastasis, in particular metastasis to distant sites. In line with these findings, both deletion and overexpression of wild-type Plexin-B1 reduced invasion of tumour cells into the prostate stroma, while overexpression of mutant Plexin-B1 significantly increased invasion, suggesting that Plexin-B1 has a role in the initial stages of metastasis. Invasion and metastasis also correlated with phosphorylation of myosin light chain, suggesting that Plexin-B1 signals via the Rho/ROCK pathway to promote metastasis. Our results demonstrate that mutant Plexin-B1 promotes metastasis in prostate cancer and represents a new therapeutic target to suppress tumour spread.

cancer biology↗

bioRxiv2019

3D imaging of colorectal cancer organoids identifies responses to Tankyrase inhibitors

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

cancer biology↗