ORCID Profile
0000-0003-0248-0258
Current Organisation
Garvan Institute of Medical Research
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Publisher: Springer Science and Business Media LLC
Date: 03-2006
DOI: 10.1038/NATURE04541
Abstract: The 9 + 2 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and ersity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 9 + 2 and 9 + 0 axonemes and other associated structures. How such unity and ersity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals. There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies. Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.
Publisher: Cold Spring Harbor Laboratory
Date: 12-06-2020
DOI: 10.1101/2020.06.09.140921
Abstract: Resistance to endocrine therapy is a major clinical challenge in the management of estrogen receptor (ER)-positive breast cancer. In this setting p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment resistant ER-positive breast cancer. We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate antitumor effects in p53 wildtype and p53 mutant ER positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and -resistant ER positive breast cancer. We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant resistant patient derived xenograft model. We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programs.
Publisher: Elsevier BV
Date: 09-2001
Publisher: Public Library of Science (PLoS)
Date: 26-10-2009
Publisher: American Society for Microbiology
Date: 07-2007
DOI: 10.1128/EC.00110-07
Abstract: We demonstrate that trypanosomes compromised in flagellar function are rapidly cleared from infected mice. Analysis of the PFR2 bloodstream RNA interference mutant revealed that defective cell motility occurred prior to cytokinesis failure. This validation provides a paradigm for the flagellum as a target for future assays and interventions against this human pathogen.
Publisher: Springer Science and Business Media LLC
Date: 11-2008
DOI: 10.1038/NRMICRO2009
Abstract: In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in eukaryotic flagella, modifications to the canonical assembly pathway and flagellum function in parasite virulence.
Publisher: Oxford University Press (OUP)
Date: 16-01-2014
Abstract: The alveolate microalga Chromera velia is an evolutionarily significant organism, representing the closest photosynthetic relative of the parasitic Apicomplexa. Chromera velia has been detected in and isolated from several stony corals and can be readily cultured in vitro under strictly autotrophic conditions. However, little is known about the ecology of this organism in the coral holobiont, an environment in which it could potentially access abundant organic carbon sources. To understand the response of C. velia to ecologically relevant organic compounds in vitro, we tested a mixotrophic culture strategy by supplementing inorganic f-medium with sugars, sugar-alcohols, organic acids and amino acids. For 15 of the 18 tested growth media, culture growth rate was significantly higher than that of strictly autotrophic cultures, and in three of these, a significant increase in maximum culture density was observed. In cultures supplemented with glutamate or glycine, the chlorophyll content per cell was up to 11-fold higher than cultures grown in standard inorganic media. Together, the in vitro culture growth and pigment responses demonstrate an ability to respond to nutritional resources when available. We propose that C. velia is a facultative opportunist in environments similarly enriched in such organic compounds, such as the coral holobiont.
Publisher: Cambridge University Press (CUP)
Date: 04-04-2012
DOI: 10.1017/S0031182012000443
Abstract: Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.
Publisher: The Company of Biologists
Date: 12-2005
DOI: 10.1242/JCS.02659
Abstract: Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Comparative genomics of flagellated and non-flagellated eukaryotes provides one way to identify new putative flagellar proteins. We identified the Parkin co-regulated gene, or PACRG, from such a screen. Male mice deficient in PACRG are sterile, but its function has been little explored. The flagellated protozoan parasite Trypanosoma brucei possesses two homologues of PACRG. We performed RNA interference knockdown experiments of the two genes independently and both together. Simultaneous ablation of both proteins produced slow growth and paralysis of the flagellum with consequent effects on organelle segregation. Moreover, using transmission electron microscopy, structural defects were seen in the axoneme, with microtubule doublets missing from the canonical 9+2 formation. The occurrence of missing doublets increased toward the distal end of the flagellum and sequential loss of doublets was observed along in idual axonemes. GFP fusion proteins of both PACRG homologues localised along the full length of the axoneme. Our results provide the first evidence for PACRG function within the axoneme, where we suggest that PACRG acts to maintain functional stability of the axonemal outer doublets of both motile and sensory cilia and flagella.
Publisher: Elsevier BV
Date: 02-2010
Publisher: Elsevier BV
Date: 02-2014
Abstract: Apicomplexa are an ancient group of single-celled pathogens of humans and animals that include the etiological agents of such devastating plagues as malaria, toxoplasmosis, and coccidiosis. The defining feature of the Apicomplexa is the apical complex, the invasion machinery used to gain access to host cells. Evidence gathered from apicomplexans and their closest relatives argues that the apical complex is an extreme ex le of flagellum adaptability. The value of non-apicomplexan models, such as Chromera velia, is considered in an effort to understand the modern apical complex. The origin of the apical complex is unknown, but recent evidence points to a remarkable contribution from the flagellum to its evolution.
Publisher: Bioscientifica
Date: 2019
DOI: 10.1530/ERC-18-0317
Abstract: Three inhibitors of CDK4/6 kinases were recently FDA approved for use in combination with endocrine therapy, and they significantly increase the progression-free survival of patients with advanced estrogen receptor-positive (ER+) breast cancer in the first-line treatment setting. As the new standard of care in some countries, there is the clinical emergence of patients with breast cancer that is both CDK4/6 inhibitor and endocrine therapy resistant. The strategies to combat these cancers with resistance to multiple treatments are not yet defined and represent the next major clinical challenge in ER+ breast cancer. In this review, we discuss how the molecular landscape of endocrine therapy resistance may affect the response to CDK4/6 inhibitors, and how this intersects with biomarkers of intrinsic insensitivity. We identify the handful of pre-clinical models of acquired resistance to CDK4/6 inhibitors and discuss whether the molecular changes in these models are likely to be relevant or modified in the context of endocrine therapy resistance. Finally, we consider the crucial question of how some of these changes are potentially amenable to therapy.
Publisher: Proceedings of the National Academy of Sciences
Date: 19-07-2017
Abstract: The distal end of the eukaryotic flagellum/cilium has critical functions, yet due to its small dimensions and association of tip structures with the axoneme is rather intractable to studying. We have developed biochemical approaches to identify a cohort of proteins specific for the flagellum tip structures. We sublocalized these proteins into in idual structures. Using functional studies, we elucidated how the identified proteins contribute to the function of the flagella connector, the mobile membrane junction at the tip of the trypanosome flagellum.
Publisher: Public Library of Science (PLoS)
Date: 03-11-2009
Publisher: Public Library of Science (PLoS)
Date: 02-09-2014
Publisher: Bioscientifica
Date: 12-2016
DOI: 10.1530/ERC-16-0427
Abstract: The estrogen receptor-α (herein called ER) is a nuclear sex steroid receptor (SSR) that is expressed in approximately 75% of breast cancers. Therapies that modulate ER action have substantially improved the survival of patients with ER-positive breast cancer, but resistance to treatment still remains a major clinical problem. Treating resistant breast cancer requires co-targeting of ER and alternate signalling pathways that contribute to resistance to improve the efficacy and benefit of currently available treatments. Emerging data have shown that other SSRs may regulate the sites at which ER binds to DNA in ways that can powerfully suppress the oncogenic activity of ER in breast cancer. This includes the progesterone receptor (PR) that was recently shown to reprogram the ER DNA binding landscape towards genes associated with a favourable outcome. Another attractive candidate is the androgen receptor (AR), which is expressed in the majority of breast cancers and inhibits growth of the normal breast and ER-positive tumours when activated by ligand. These findings have led to the initiation of breast cancer clinical trials evaluating therapies that selectively harness the ability of SSRs to ‘push’ ER towards anti-tumorigenic activity. Our review will focus on the established and emerging clinical evidence for activating PR or AR in ER-positive breast cancer to inhibit the tumour growth-promoting functions of ER.
Publisher: American Chemical Society (ACS)
Date: 02-2009
DOI: 10.1016/J.JASMS.2008.08.014
Abstract: The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD- or ETD-based tandem mass spectrometry alone.
Publisher: American Society for Microbiology
Date: 2014
DOI: 10.1128/EC.00155-13
Abstract: Since its first description, Chromera velia has attracted keen interest as the closest free-living relative of parasitic Apicomplexa. The life cycle of this unicellular alga is complex and involves a motile biflagellate form. Flagella are thought to be formed in the cytoplasm, a rare phenomenon shared with Plasmodium in which the canonical mode of flagellar assembly, intraflagellar transport, is dispensed with. Here we demonstrate the expression of intraflagellar transport components in C. velia , answering the question of whether this organism has the potential to assemble flagella via the canonical route. We have developed and characterized a culturing protocol that favors the generation of flagellate forms. From this, we have determined a marked shift in the mode of daughter cell production from two to four daughter cells per ision as a function of time after passage. We conduct an ultrastructural examination of the C. velia flagellate form by using serial TEM and show that flagellar biogenesis in C. velia occurs prior to cytokinesis. We demonstrate a close association of the flagellar apparatus with a complex system of apical structures, including a micropore, a conoid, and a complex endomembrane system reminiscent of the apical complex of parasitic apicomplexans. Recent work has begun to elucidate the possible flagellar origins of the apical complex, and we show that in C. velia these structures are contemporaneous within a single cell and share multiple connections. We propose that C. velia therefore represents a vital piece in the puzzle of the origins of the apical complex.
Publisher: Springer Science and Business Media LLC
Date: 12-08-2020
DOI: 10.1186/S13058-020-01318-2
Abstract: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer. We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer. We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model. We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.
Publisher: Springer Science and Business Media LLC
Date: 24-02-2022
Publisher: Elsevier BV
Date: 09-2021
DOI: 10.1016/J.CELREP.2021.109722
Abstract: DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.
Publisher: Elsevier BV
Date: 02-2009
Publisher: Bioscientifica
Date: 02-2019
DOI: 10.1530/ERC-18-0333
Abstract: The role of androgen receptor (AR) in endocrine-resistant breast cancer is controversial and clinical trials targeting AR with an AR antagonist (e.g., enzalutamide) have been initiated. Here, we investigated the consequence of AR antagonism using in vitro and in vivo models of endocrine resistance. AR antagonism in MCF7-derived tamoxifen-resistant (TamR) and long-term estrogen-deprived breast cancer cell lines were achieved using siRNA-mediated knockdown or pharmacological inhibition with enzalutamide. The efficacy of enzalutamide was further assessed in vivo in an estrogen-independent endocrine-resistant patient-derived xenograft (PDX) model. Knockdown of AR inhibited the growth of the endocrine-resistant cell line models. Microarray gene expression profiling of the TamR cells following AR knockdown revealed perturbations in proliferative signaling pathways upregulated in endocrine resistance. AR loss also increased some canonical ER signaling events and restored sensitivity of TamR cells to tamoxifen. In contrast, enzalutamide did not recapitulate the effect of AR knockdown in vitro , even though it inhibited canonical AR signaling, which suggests that it is the non-canonical AR activity that facilitated endocrine resistance. Enzalutamide had demonstrable efficacy in inhibiting AR activity in vivo but did not affect the growth of the endocrine-resistant PDX model. Our findings implicate non-canonical AR activity in facilitating an endocrine-resistant phenotype in breast cancer. Unlike canonical AR signaling which is inhibited by enzalutamide, non-canonical AR activity is not effectively antagonized by enzalutamide, and this has important implications in the design of future AR-targeted clinical trials in endocrine-resistant breast cancer.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Neil Portman.