Probing the Architecture of the Budding Yeast Inner Kinetochore

Chromosome segregation must occur with fidelity to maintain genome stability and prevent aneuploid disorders. During segregation, the kinetochore is assembled onto centromeric DNA and achieves the attachment of microtubule plus ends, which provide the force to physically segregate chromosomes to opposite poles of the cell. The kinetochore is a macromolecular protein/DNA machine composed of 8-9 biochemical complexes, the DNA-binding components of which define the inner kinetochore, and the microtubule-binding components of which define the outer kinetochore. The 3D protein architecture of the inner kinetochore in living cells remains poorly understood due to the resolution limits of live-cell imaging techniques. Here, we use pairwise, in vivo fluorescence microscopy to determine the position of kinetochore components relative to the centromere specific histone H3 variant, Cse4, with nanometer resolution. In addition, we complement kinetochore focus analysis with DNA dynamics simulations to propose a connection between chromatin dynamics and the observed anisotropy of the inner kinetochore proteins, Ame1 and Cse4, relative to the outer kinetochore proteins, Ndc80 and Nuf2.Bachelor of Scienc

Identification of a selective G1-phase benzimidazolone inhibitor by a senescence-targeted virtual screen using artificial neural networks

Cellular senescence is a barrier to tumorigenesis in normal cells and tumour cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning-based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~2M lead-like compounds. 147 virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase (SA-β-gal) assays. Among the found hits a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced SA-β-gal activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1 and CDC25C. Additionally, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long term treatments. Preliminary structure-activity and structure clustering analyses are reported and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor-like profile in normal cells, with different pathways affected in cancer cells

Structure-based design, synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies

Low impact sampling of speleothems – reconciling scientific study with cave conservation

Speleothems are increasingly valued as important paleoclimate archives and yet the removal of samples from caves can come at a cost to natural heritage, impacting delicate environments with limited mechanisms for repair. Conservation of cave environments is a key responsibility for scientists and, with this in mind, we are working to develop and implement techniques that allow us to extract valuable scientific data, with minimal impact. In this study, we demonstrate the utility of low-impact reconnaissance dating surveys on caves in southern Tasmania and southwest Western Australia as a precursor to the removal of stalagmites for paleoclimate reconstruction. Small flakes of calcite were discretely extracted from the base and tip of fallen stalagmites and dated using U-Th techniques. We specifically targeted stalagmites that have naturally fallen or been previously broken by human interference, to further reduce our impact on the caves. This approach provides maximum and minimum age constraints for each stalagmite and valuable information of growth frequencies without the need to remove whole samples from the cave. Selecting the most appropriate samples to analyze based on reconnaissance ages greatly reduces the quantity of speleothem material to be removed from a cave to locate a desired interval of past time, mitigating the impacts of the research. Moreover, the reconnaissance age data enable us to build an archive of speleothem ages from the cave for future scientific research and to provide information on the age and nature of cave development, useful for cave management purposes and other studies. To assess the accuracy of this method we compared the reconnaissance age with the results of a detailed age evaluation on a small number of stalagmites removed from the caves. We have found this method to be effective and has allowed us to successfully identify several stalagmites suitable for our scientific objectives

Identification of a selective G1-phase benzimidazolone inhibitor by a senescence-targeted virtual screen using artificial neural networks

Microarray data for associated publication. Accession number in the Gene Expression Omnibus is GSE72621

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Palaeoenvironmental change in tropical Australasia over the last 30 000 years - a synthesis by the OZ-INTIMATE group

The tropics are the major source of heat and moisture for the Australasian region. Determining the tropics' response over time to changes in climate forcing mechanisms, such as summer insolation, and the effects of relative sea level on exposed continent