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Rodger E.J.,University of Otago | Rodger E.J.,Maurice Wilkins Center for Molecular Biodiscovery | Chatterjee A.,University of Otago | Chatterjee A.,Maurice Wilkins Center for Molecular Biodiscovery
Clinical Epigenetics | Year: 2017

A report of the 6th Epigenomics of Common Diseases Conference held at the Wellcome Genome Campus in Hinxton, Cambridge, UK, on 1–4 November 2016. © 2017, The Author(s).


PubMed | University of Otago, Maurice Wilkins Center for Molecular Biodiscovery, Capital and Coast District Health Board, Pathlab Bay of Plenty and 2 more.
Type: Journal Article | Journal: Oncotarget | Year: 2016

Melanoma, the most aggressive skin cancer type, is responsible for 75% of skin cancer related deaths worldwide. Given that New Zealand (NZ) has the worlds highest melanoma incidence, we sought to determine the frequency of mutations in NZ melanomas in recurrently mutated genes. NZ melanomas were from localities distributed between North (35S-42S) and South Islands (41S-47S). A total of 529 melanomas were analyzed for BRAF exon 15 mutations by Sanger sequencing, and also by Sequenom MelaCarta MassARRAY. While, a relatively low incidence of BRAFV600E mutations (23.4%) was observed overall in NZ melanomas, the incidence of NRAS mutations in South Island melanomas was high compared to North Island melanomas (38.3% vs. 21.9%, P=0.0005), and to The Cancer Genome Atlas database (TCGA) (38.3% vs. 22%, P=0.0004). In contrast, the incidence of EPHB6G404S mutations was 0% in South Island melanomas, and was 7.8% in North Island (P=0.0002). Overall, these data suggest that melanomas from geographically different regions in NZ have markedly different mutation frequencies, in particular in the NRAS and EPHB6 genes, when compared to TCGA or other populations. These data have implications for the causation and treatment of malignant melanoma in NZ.


Young S.W.,University of Auckland | Young S.W.,North Shore Hospital | Roberts T.,North Shore Hospital | Johnson S.,University of Auckland | And 5 more authors.
Clinical Orthopaedics and Related Research | Year: 2015

Background: In human TKA studies, intraosseous regional administration (IORA) of prophylactic antibiotics achieves local tissue antibiotic concentrations 10 times greater than systemic administration. However, it is unclear if such high concentrations provide more effective prophylaxis. Questions/purposes: We asked: (1) What prophylaxis dosage and route (intravenous [IV] versus IORA of prophylactic antibiotics) produce less in vivo bacterial burden compared with no-antibiotic controls? (2) Compared with controls, what prophylaxis dosage and route yield fewer colony-forming units (CFUs) in euthanized animals in a model of TKA? (3) Is prophylactic IORA of antibiotics more effective than same-dose IV antibiotic administration in reducing CFUs? Methods: Mice (six to nine per group) were block randomized to one of six prophylaxis regimens: control, systemic cefazolin (C100IV), IORA of cefazolin (C100IORA), systemic vancomycin (V110IV), low-dose systemic vancomycin (V25IV), and low-dose IORA of vancomycin (V25IORA). Surgery involved placement of an intraarticular knee prosthesis, followed by an inoculum of bioluminescent Staphylococcus aureus strain Xen36. Biophotonic imaging assessed in vivo bacterial loads, and after 4 days bacterial load was quantified using culture-based techniques. Comparisons were made for each prophylactic regimen to controls and between same-dose IV and IORA of prophylactic antibiotic regimens. Results: Mice treated with systemic high-dose vancomycin, IORA of vancomycin, or IORA of cefazolin had lower in vivo Staphylococcus aureus burdens (median area under curve, Control: 5.0 × 106; V110IV: 1.5 × 106, difference of medians 3.5 × 106, p = 0.003; V25IV: 1.94 × 106, difference 3.07 × 106, p = 0.49; V25IORA: 1.51 × 106, difference 3.5 × 106, p = 0.0011; C100IORA: 1.55 × 106, difference 3.46 × 106, p = 0.0016; C100IV: 2.35 × 106, difference 2.66 × 106, p = 0.23.) Similar findings were seen with culture-based techniques on recovered implants. IORA of prophylactic antibiotics was more effective than same-dose IV administration in reducing bacterial load on recovered implants (median CFUs < 7.0 × 100 vs 2.83 × 102, p = 0.0183). Conclusions: IORA of prophylactic cefazolin and vancomycin was more effective than the same dose of antibiotic given systemically. The effectiveness of vancomycin in particular was enhanced by IORA of prophylactic antibiotics despite using a lower dose. Clinical relevance: Our study supports previous studies of IORA of prophylactic antibiotics in humans and suggests this novel form of administration has the potential to enhance the effectiveness of prophylaxis in TKA. Because of concerns regarding antibiotic stewardship, IORA of prophylactic vancomycin may be more appropriately restricted to patients having TKA who are at greater risk of infection, and clinical trials are in progress. © 2015, The Author(s).


Watkins H.A.,Maurice Wilkins Center for Molecular Biodiscovery | Baker E.N.,Maurice Wilkins Center for Molecular Biodiscovery
Journal of Bacteriology | Year: 2010

The open reading frame Rv2228c from Mycobacterium tuberculosis is predicted to encode a protein composed of two domains, each with individual functions, annotated through sequence similarity searches. The N-terminal domain is homologous with prokaryotic and eukaryotic RNase H domains and the C-terminal domain with α-ribazole phosphatase (CobC). The N-terminal domain of Rv2228c (Rv2228c/N) and the full-length protein were expressed as fusions with maltose binding protein (MBP). Rv2228c/N was shown to have RNase H activity with a hybrid RNA/DNA substrate as well as double-stranded RNase activity. The full-length protein was shown to have additional CobC activity. The crystal structure of the MBP-Rv2228c/N fusion protein was solved by molecular replacement and refined at 2.25-Å resolution (R = 0.182; Rfree = 0.238). The protein is monomeric in solution but associates in the crystal to form a dimer. The Rv2228c/N domain has the classic RNase H fold and catalytic machinery but lacks several surface features that play important roles in the cleavage of RNA/DNA hybrids by other RNases H. The absence of either the basic protrusion of some RNases H or the hybrid binding domain of others appears to be compensated by the C-terminal CobC domain in full-length Rv2228c. The double-stranded-RNase activity of Rv2228c/N contrasts with classical RNases H and is attributed to the absence in Rv2228c/N of a key phosphate binding pocket. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Chatterjee A.,University of Otago | Chatterjee A.,Maurice Wilkins Center for Molecular Biodiscovery | Eccles M.R.,University of Otago | Eccles M.R.,Maurice Wilkins Center for Molecular Biodiscovery
Genome Biology | Year: 2015

A report of the Keystone Symposia joint meetings on DNA Methylation and Epigenomics held in Keystone, Colorado, USA, 29 March to 3 April, 2015. © 2015 Chatterjee and Eccles et al.; licensee BioMed Central.


Chatterjee A.,University of Otago | Chatterjee A.,Maurice Wilkins Center for Molecular Biodiscovery | Stockwell P.A.,University of Otago | Rodger E.J.,University of Otago | And 4 more authors.
Epigenomics | Year: 2016

Aim: The Cancer Genome Atlas contains multiple levels of genomic data (mutation, gene expression, DNA methylation, copy number variation) for 33 cancer types for almost 11,000 patients. However, a dearth of appropriate software tools makes it difficult for bench scientists to use these data effectively. Materials & methods: Here, we present a suite of flexible, fast and command line-based scripts that will allow retrieval and analysis of DNA methylation (tool: scan-tcga-methylation.awk), mRNA (tool: scan-tcga-mRNA.awk) and miRNA expression (tool: scan-tcga-miRNAs.awk) from cancer genome atlas network level 3 data. Results: We demonstrate the utility of these tools by analyzing DNA methylation and mRNA expression signatures of 60 frequently deregulated cancer genes and also of 30 miRNAs in primary (n = 102) and metastatic melanoma patients (n = 367). Conclusion: Our analysis illustrates the validity of the scan-tcga tools and reveals the epigenomic signatures and importance of identifying smaller patient subgroups with distinct molecular profiles. © 2016 Aniruddha Chatterjee.


Evans A.,University of Auckland | Jamieson S.M.F.,University of Auckland | Jamieson S.M.F.,Maurice Wilkins Center for Molecular Biodiscovery | Liu D.-X.,University of Auckland | And 4 more authors.
Cancer Letters | Year: 2016

Human GH expression is associated with poor survival outcomes for endometrial cancer patients, enhanced oncogenicity of endometrial cancer cells and reduced sensitivity to ionising radiation in vitro, suggesting that GH is a potential target for anticancer therapy. However, whether GH receptor inhibition sensitises to radiotherapy in vivo has not been tested. In the current study, we evaluated whether the GH receptor antagonist, pegvisomant (Pfizer), sensitises to radiotherapy in vivo in an endometrial tumour xenograft model. Subcutaneous administration of pegvisomant (20 or 100 mg/kg/day, s.c.) reduced serum IGF1 levels by 23% and 68%, respectively, compared to vehicle treated controls. RL95-2 xenografts grown in immunodeficient NIH-III mice were treated with vehicle or pegvisomant (100 mg/kg/day), with or without fractionated gamma radiation (10 × 2.5 Gy over 5 days). When combined with radiation, pegvisomant significantly increased the median time tumours took to reach 3× the pre-radiation treatment volume (49 days versus 72 days; p = 0.001). Immunohistochemistry studies demonstrated that 100 mg/kg pegvisomant every second day was sufficient to abrogate MAP Kinase signalling throughout the tumour. In addition, treatment with pegvisomant increased hypoxic regions in irradiated tumours, as determined by immunohistochemical detection of pimonidazole adducts, and decreased the area of CD31 labelling in unirradiated tumours, suggesting an anti-vascular effect. Pegvisomant did not affect intratumoral staining for HIF1α, VEGF-A, CD11b, or phospho-EGFR. Our results suggest that blockade of the human GH receptor may improve the response of GH and/or IGF1-responsive endometrial tumours to radiation. © 2016 Elsevier Ireland Ltd.


Grattan D.R.,University of Otago | Grattan D.R.,Maurice Wilkins Center for Molecular Biodiscovery
Journal of Endocrinology | Year: 2015

The hypothalamic control of prolactin secretion is different from other anterior pituitary hormones, in that it is predominantly inhibitory, by means of dopamine from the tuberoinfundibular dopamine neurons. In addition, prolactin does not have an endocrine target tissue, and therefore lacks the classical feedback pathway to regulate its secretion. Instead, it is regulated by short loop feedback, whereby prolactin itself acts in the brain to stimulate production of dopamine and thereby inhibit its own secretion. Finally, despite its relatively simplename,prolactinhas a broadrange offunctions inthebody, inadditiontoitsdefiningrole in promoting lactation. As such, the hypothalamo-prolactin axis has many characteristics that are quite distinct from other hypothalamo-pituitary systems. This review will provide a brief overview of our current understanding of the neuroendocrine control of prolactin secretion, in particular focusing on the plasticity evident in this system, which keeps prolactin secretion at low levels most of the time, but enables extended periods of hyperprolactinemia when necessary for lactation. Key prolactin functions beyond milk production will be discussed, particularly focusing on the role of prolactin in inducing adaptive responses in multiple different systems to facilitate lactation, and the consequences if prolactin action is impaired. A feature of this pleiotropic activity is that functions thatmay be adaptive in the lactating state might bemaladaptive if prolactin levels are elevated inappropriately. Overall,my goal is to give aflavourofboth the history and current state of thefield ofprolactin neuroendocrinology, and identify some exciting new areas of research development. © 2015 Society for Endocrinology.


PubMed | University of Auckland and Maurice Wilkins Center for Molecular Biodiscovery
Type: | Journal: The Journal of antimicrobial chemotherapy | Year: 2016

Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the worlds population, resulting in 1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise.We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery.We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro.We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.


Cognard E.,University of Auckland | Dargaville C.G.,University of Auckland | Hay D.L.,Maurice Wilkins Center for Molecular Biodiscovery | Hay D.L.,University of Auckland | And 2 more authors.
Biochemical Journal | Year: 2013

Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we showthat β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells. © The Authors Journal compilation © 2013 Biochemical Society.

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