School of Biomedical Sciences - Research Publications
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ItemCell cycle-regulated PLEIADE/AtMAP65-3 links membrane and microtubule dynamics during plant cytokinesis(WILEY-BLACKWELL, 2016-11)Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between cell cycle cues, membrane trafficking and microtubule dynamics. Plant cytokinesis occurs within a transient membrane compartment known as the cell plate, to which vesicles are delivered by a plant-specific microtubule array, the phragmoplast. While membrane proteins required for cytokinesis are known, how these are coordinated with microtubule dynamics and regulated by cell cycle cues remains unclear. Here, we document physical and genetic interactions between Transport Protein Particle II (TRAPPII) tethering factors and microtubule-associated proteins of the PLEIADE/AtMAP65 family. These interactions do not specifically affect the recruitment of either TRAPPII or MAP65 proteins to the cell plate or midzone. Rather, and based on single versus double mutant phenotypes, it appears that they are required to coordinate cytokinesis with the nuclear division cycle. As MAP65 family members are known to be targets of cell cycle-regulated kinases, our results provide a conceptual framework for how membrane and microtubule dynamics may be coordinated with each other and with the nuclear cycle during plant cytokinesis.
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ItemWhole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana(PUBLIC LIBRARY SCIENCE, 2015-03)Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.
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ItemCooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen(PUBLIC LIBRARY SCIENCE, 2016-06)Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.
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ItemDose planning objectives in anal canal cancer IMRT: the TROG ANROTAT experience(WILEY-BLACKWELL, 2015-06)INTRODUCTION: Intensity modulated radiotherapy (IMRT) is ideal for anal canal cancer (ACC), delivering high doses to irregular tumour volumes whilst minimising dose to surrounding normal tissues. Establishing achievable dose objectives is a challenge. The purpose of this paper was to utilise data collected in the Assessment of New Radiation Oncology Treatments and Technologies (ANROTAT) project to evaluate the feasibility of ACC IMRT dose planning objectives employed in the Australian situation. METHODS: Ten Australian centres were randomly allocated three data sets from 15 non-identifiable computed tomography data sets representing a range of disease stages and gender. Each data set was planned by two different centres, producing 30 plans. All tumour and organ at risk (OAR) contours, prescription and dose constraint details were provided. Dose-volume histograms (DVHs) for each plan were analysed to evaluate the feasibility of dose planning objectives provided. RESULTS: All dose planning objectives for the bone marrow (BM) and femoral heads were achieved. Median planned doses exceeded one or more objectives for bowel, external genitalia and bladder. This reached statistical significance for bowel V30 (P = 0.04), V45 (P < 0.001), V50 (P < 0.001), external genitalia V20 (P < 0.001) and bladder V35 (P < 0.001), V40 (P = 0.01). Gender was found to be the only significant factor in the likelihood of achieving the bowel V50 (P = 0.03) and BM V30 constraints (P = 0.04). CONCLUSION: The dose planning objectives used in the ANROTAT project provide a good starting point for ACC IMRT planning. To facilitate clinical implementation, it is important to prioritise OAR objectives and recognise factors that affect the achievability of these objectives.
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ItemFT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy(PUBLIC LIBRARY SCIENCE, 2015-07-29)Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 μM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 μM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.
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ItemBetter Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants(FRONTIERS MEDIA SA, 2016-09-27)In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools.
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ItemVEGF-D promotes pulmonary oedema in hyperoxic acute lung injury(WILEY-BLACKWELL, 2016-06)
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ItemIFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid(PUBLIC LIBRARY SCIENCE, 2016-02)Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.
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ItemInhibition of NOX1/4 with GKT137831: a potential novel treatment to attenuate neuroglial cell inflammation in the retina(BIOMED CENTRAL LTD, 2015-07-30)BACKGROUND: Inflammation and the excess production of reactive oxygen species (ROS) contribute significantly to the pathogenesis of ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity. We hypothesized that GKT137831, a dual inhibitor of NADPH oxidases (NOX) 1 and NOX4, reduces inflammation in the ischemic retina by dampening the pro-inflammatory phenotype of retinal immune cells as well as macroglial Müller cells and neurons. METHODS: Ischemic retinopathy was induced in Sprague-Dawley rats by exposure to 80 % O2 cycled with 21 % O2 for 3 h per day from postnatal day (P) 0 to P11, followed by room air (P12 to P18). GKT137831 was administered P12 to P18 (60 mg/kg, subcutaneous) and comparisons were to room air controls. Retinal inflammation was examined by measuring leukocyte adherence to the retinal vasculature, ionized calcium-binding adaptor protein-1-positive microglia/macrophages, and the mRNA and protein levels of key inflammatory factors involved in retinal disease. Damage to Müller cells was evaluated by quantitating glial fibrillary acidic protein-positive cells and vascular leakage with an albumin ELISA. To verify the anti-inflammatory actions of GKT137831 on glia and neurons involved in ischemic retinopathy, primary cultures of rat retinal microglia, Müller cells, and ganglion cells were exposed to the in vitro counterpart of ischemia, hypoxia (0.5 %), and treated with GKT137831 for up to 72 h. ROS levels were evaluated with dihydroethidium and the protein and gene expression of inflammatory factors with quantitative PCR, ELISA, and a protein cytokine array. RESULTS: In the ischemic retina, GKT137831 reduced the increased leukocyte adherence to the vasculature, the pro-inflammatory phenotype of microglia and macroglia, the increased gene and protein expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and leukocyte adhesion molecules as well as vascular leakage. In all cultured cell types, GKT137831 reduced the hypoxia-induced increase in ROS levels and protein expression of various inflammatory mediators. CONCLUSIONS: NOX1/4 enzyme inhibition with GKT137831 has potent anti-inflammatory effects in the retina, indicating its potential as a treatment for a variety of vision-threatening retinopathies.
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ItemInhibition of cathepsin proteases attenuates migration and sensitizes aggressive N-Myc amplified human neuroblastoma cells to doxorubicin(IMPACT JOURNALS LLC, 2015-05-10)Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.