The association between MC1R genotype and BRAF mutation status in cutaneous melanoma : findings from an Australian population

There is increasing epidemiological and molecular evidence that cutaneous melanomas arise through multiple causal pathways. The purpose of this study was to explore the relationship between germline and somatic mutations in a population-based series of melanoma patients to reshape and refine the divergent pathway model for melanoma. Melanomas collected from 123 Australian patients were analyzed for melanocortin-1 receptor (MC1R) variants and mutations in the BRAF and NRAS genes. Detailed phenotypic and sun exposure data were systematically collected from all patients. We found that BRAF-mutant melanomas were significantly more likely from younger patients and those with high nevus counts, and were more likely in melanomas with adjacent neval remnants. Conversely, BRAF-mutant melanomas were significantly less likely in people with high levels of lifetime sun exposure. We observed no association between germline MC1R status and somatic BRAF mutations in melanomas from this population. BRAF-mutant melanomas have different origins from other cutaneous melanomas. These data support the divergent pathways hypothesis for melanoma, which may require a reappraisal of targeted cancer prevention activities.

Polybrominated diphenyl ether (PBDE) concentrations decrease with age : analysis of pooled human blood serum in the Australian population

Introduction Polybrominated diphenyl ethers (PBDEs) are considered to be a cost effective and efficient way to reduce the possibility of product ignition and inhibit the spread of fire, thereby limiting harm caused by fires. PBDEs are incorporated into a wide variety of manufactured products and are now considered an ubiquitous contaminant found worldwide in biological and environmental samples1 . In comparison to “traditional” persistent organic pollutants (POPs), the exposure modes of PBDEs in humans are less well defined, although dietary sources, inhalation (air/particulate matter) and dust ingestion have been reported 2-4. Limited investigations of population specific factors such as age or gender and PBDE concentrations report: no conclusive correlation by age in adults; higher concentrations in children ; similar concentrations in maternal and cord blood; and no gender differences. After preliminary findings of higher PBDE concentrations in children than in adults in Australia11 we sought to investigate at what age the PBDE concentrations peaked in an effort to focus exposure studies. This investigation involved the collection of blood samples from young age groups and the development of a simple model to predict PBDE concentrations by age in Australia.

Alexithymia, craving and attachment in a heavy drinking population

Up to fifty per cent of individuals with Alcohol use disorders (AUD) also have alexithymia a personality construct hypothesized to be related to attachment difficulties. The relationship between alexithymia, craving, anxious attachment and alcohol-dependence severity was examined in 254 patients participating in a Cognitive-Behavioral Therapy (CBT) program for alcohol-dependence. Participants completed the Toronto Alexithymia Scale (TAS-20), the Obsessive Compulsive Drinking Scale (OCDS), the Revised Adult Attachment Anxiety Subscale (RAAS-Anxiety) and the Alcohol Use Disorder Identification Test (AUDIT). MANOVA indicated that individuals with alexithymia reported significantly higher levels of total OCDS, obsessive thoughts about alcohol, and compulsive drinking urges and behavior, compared to the non-alexithymic group. Regression analyses found that anxious attachment partially mediated the relationship between alexithymia and craving. Anxious attachment may be a potential treatment target to reduce alcohol consumption in those with alcohol-dependence and alexithymia. Research Highlights ► There were significant relationships of alexithymia, craving and anxious attachment. ► Alexithymic alcoholics reported higher levels of craving and alcoholism severity. ► Anxious attachment partially mediated the relationship of alexithymia and craving.

Queensland Government’s people’s question time : the challenges and opportunities of population growth in Queensland

As AITPM National President, I was invited by Queensland’s Premier, Hon. Anna Bligh MP, as an audience guest to People’s Question Time on Wednesday 24 March 2010, which focused on ‘The Challenges and Opportunities of Population Growth in Queensland’. On the panel were: Premier and Minister for the Arts, Anna Bligh; Minister for Climate Change and Sustainability, Kate Jones; Minister for Infrastructure and Planning, Stirling Hinchliffe; Michael Rayner – Growth Management Summit Advisory Panel, Principal Director, Cox Rayner Architects; and Greg Hallam – Executive Director, Local Government Association of Queensland. The moderator for this session was Law Academic Erin O’Brien, of Queensland University of Technology.

Investigation into the introduction of clonal strains of Pseudomonas aeruginosa to the cystic fibrosis population by consumption of raw salad vegetables

Most salad vegetables are eaten fresh by consumers. However, raw vegetables may pose a risk of transmitting opportunistic bacteria to immunocompromised people, including cystic fibrosis (CF) patients. In particular, CF patients are vulnerable to chronic Pseudomonas aeruginosa lung infections and this organism is the primary cause of morbidity and mortality in this group. Clonal variants of P. aeruginosa have been identified as emerging threats to people afflicted with CF; however it has not yet been proven from where these clones originate or how they are transmitted. Due to the organisms‟ aquatic environmental niche, it was hypothesised that vegetables may be a source of these clones. To test this hypothesis, lettuce, tomatoes, mushrooms and bean sprout packages (n = 150) were analysed from a green grocer, supermarket and farmers‟ market within the Brisbane region, availability permitting. The internal and external surfaces of the vegetables were separately analysed for the presence of clonal strains of P. aeruginosa using washings and homogenisation techniques, respectively. This separation was in an attempt to establish which surface was contaminated, so that recommendations could be made to decrease or eliminate P. aeruginosa from these foods prior to consumption. Soil and water samples (n = 17) from local farms were also analysed for the presence of P. aeruginosa. Presumptive identification of isolates recovered from these environmental samples was made based on growth on Cetrimide agar at 42°C, presence of the cytochrome-oxidase enzyme and inability to ferment lactose. P. aeruginosa duplex real-time polymerase chain reaction assay (PAduplex) was performed on all bacterial isolates presumptively identified as P. aeruginosa. Enterobacterial repetitive intergenic consensus strain typing PCR (ERIC-PCR) was subsequently performed on confirmed bacterial isolates. Although 72 P. aeruginosa were isolated, none of these proved to be clonal strains. The significance of these findings is that vegetables may pose a risk of transmitting sporadic strains of P. aeruginosa to people afflicted with CF and possibly, other immunocompromised people.

The molecular mechanisms utilised by mesenchymal stem cells in migration to acute myocardial infarction

Heart damage caused by acute myocardial infarction (AMI) is a leading cause of death and disability in Australia. Novel therapies are still required for the treatment of this condition due to the poor reparative ability of the heart. As such, cellular therapies that assist in the recovery of heart muscle are of great current interest. Culture expanded mesenchymal stem cells (MSC) represent a stem and progenitor cell population that has been shown to promote tissue recovery in pre-clinical studies of AMI. For MSC-based therapies in the clinic, an intravenous route of administration would ideally be used due to the low cost, ease of delivery and relative safety. The study of MSC migration is therefore clinically relevant for a minimally invasive cell therapy to promote regeneration of damaged tissue. C57BL/6, UBI-GFP-BL/6 and CD44-/-/GFP+/+ mice were utilised to investigate mMSC migration. To assist in murine models of MSC migration, a novel method was used for the isolation of murine MSC (mMSC). These mMSC were then expanded in culture and putative mMSC were positive for Sca-1, CD90.2, and CD44 and were negative for CD45 and CD11b. Furthermore, mMSC from C57BL/6 and UBI-GFP-BL/6 mice were shown to differentiate into cells of the mesodermal lineage. Cells from CD44-/-/GFP+/+ mice were positive for Sca-1 and CD90.2, and negative for CD44, CD45 and CD11b however, these cells were unable to differentiate into adipocytes and chondrocytes and express lineage specific genes, PLIN and ACAN. Analysis of mMSC chemokine receptor (CR) expression showed that although mMSC do express chemokine receptors, (including those specific for chemokines released after AMI), these were low or undetectable by mRNA. However, protein expression could be detected, which was predominantly cytoplasmic. It was further shown that in both healthy (unperturbed) and inflamed tissues, mMSC had very little specific migration and engraftment after intravenous injection. To determine if poor mMSC migration was due to the inability of mMSC to respond to chemotactic stimuli, chemokine expression in bone marrow, skin injury and hearts (healthy and after AMI) was analysed at various time points by quantitative real-time PCR (qRT PCR). Many chemokines were up-regulated after skin biopsy and AMI, but the highest acute levels were found for CXCL12 and CCL7. Due to their high expression in infarcted hearts, the chemokines CXCL12 and CCL7 were tested for their effect on mMSC migration. Despite CR expression at both protein and mRNA levels, migration in response to CXCL12 and CCL7 was low in mMSC cultured on Nunclon plastic. A novel tissue culture plastic technology (UpCellTM) was then used that allowed gentle non-enzymatic dissociation of mMSC, thus preserving surface expression of the CRs. Despite this the in vitro data indicated that CXCL12 fails to induce significant migration ability of mMSC, while CCL7 induces significant, but low-level migration. We speculated this may be because of low levels of surface expression of chemokine receptors. In a strategy to increase cell surface expression of mMSC chemokine receptors and enhance their in vitro and in vivo migration capacity, mMSC were pre-treated with pro-inflammatory cytokines. Increased levels of both mRNA and surface protein expression were found for CRs by pre-treating mMSC with pro-inflammatory cytokines including TNF-á, IFN-ã, IL-1á and IL-6. Furthermore, the chemotactic response of mMSC to CXCL12 and CCL7 was significantly higher with these pretreated cells. Finally, the effectiveness of this type of cell manipulation was demonstrated in vivo, where mMSC pre-treated with TNF-á and IFN-ã showed significantly increased migration in skin injury and AMI models. Therefore this thesis has demonstrated, using in vitro and in vivo models, the potential for prior manipulation of MSC as a possible means for increasing the utility of intravenously delivery for MSC-based cellular therapies.

Spatial population genetic structure reveals strong natal site fidelity in Echinocladius martini (Diptera: Chironomidae) in northeast Queensland, Australia

1. A diverse array of patterns has been reported regarding the spatial extent of population genetic structure and effective dispersal in freshwater macroinvertebrates. In river systems, the movements of many taxa can be restricted to varying degrees by the natural stream channel hierarchy. 2. In this study, we sampled populations of the non-biting freshwater midge Echinocladius martini in the Paluma bioregion of tropical northeast Queensland to investigate fine scale patterns of within- and among-stream dispersal and gene flow within a purported historical refuge. We amplified a 639 bp fragment of mitochondrial COI and analysed genetic structure using pairwise ΦST, hierarchical AMOVA, Mantel tests and a parsimony network. Genetic variation was partitioned among stream sections using Streamtree to investigate the effect of potential instream dispersal barriers. 3. The data revealed strong natal site fidelity and significant differentiation among neighbouring, geographically proximate streams. We found evidence for only episodic adult flight among sites on separate stream reaches. Overall, however, our data suggested that both larval and adult dispersal was largely limited to within a stream channel. 4. This may arise from a combination of the high density of riparian vegetation physically restricting dispersal and from the joint effects of habitat stability and large population sizes. Together these may mitigate the requirement for movement among streams to avoid inbreeding and local extinction due to habitat change and may thus enable persistence of upstream populations in the absence of regular compensatory upstream flight. Taken together, these data suggest that dispersal of E. martini is highly restricted, to the scale of only a few kilometres, and hence occurs predominantly within the natal stream.

Laser doppler imaging in a paediatric burns population

Objective Laser Doppler imaging (LDI) was compared to wound outcomes in children's burns, to determine if the technology could be used to predict these outcomes. Methods Forty-eight patients with a total of 85 burns were included in the study. Patient median age was 4 years 10 months and scans were taken 0–186 h post-burn using the fast, low-resolution setting on the Moor LDI2 laser Doppler imager. Wounds were managed by standard practice, without taking into account the scan results. Time until complete re-epithelialisation and whether or not grafting and scar management were required were recorded for each wound. If wounds were treated with Silvazine™ or Acticoat™ prior to the scan, this was also recorded. Results The predominant colour of the scan was found to be significantly related to the re-epithelialisation, grafting and scar management outcomes and could be used to predict those outcomes. The prior use of Acticoat™ did not affect the scan relationship to outcomes, however, the use of Silvazine™ did complicate the relationship for light blue and green scanned partial thickness wounds. Scans taken within the 24-h window after-burn also appeared to be accurate predictors of wound outcome. Conclusion Laser Doppler imaging is accurate and effective in a paediatric population with a low-resolution fast-scan.

Psychometric properties of the parents' fever management scale in a Turkish population

Aim: This paper reports a study designed to assess the psychometric properties (validity and reliability) of a Turkish version of the Australian Parents’ Fever Management Scale (PFMS). Background: Little is known about childhood fever management among Turkish parents. No scales to measure parents’ fever management practices in Turkey are available. Design: This is a methodological study. Methods: Eighty parents, of febrile children aged six months to five years, were randomly selected from the paedaitric hospital and two community family health centers in Sakarya, Turkey. The PFMS was back translated; language equivalence and content validity were validated. PFMS and socio-demographic data were collected in 2009. Means and standard deviations were calculated for interval level data and p values greater than 0.05 were considered statistically significant. Unrotated principal component analysis was used to determine construct validity and Cronbach’s coefficient alpha determined the internal consistency reliability. Results: The PFMS was psychometrically sound in this population. Construct validity, confirmed by confirmatory factor analysis [KMO 0.812, Bartlett’s Specificity (χ² = 182.799, df=28, P < 0·001)] revealed the Turkish version to be comprised of the eight original PFMS items. Internal consistency reliability coefficient was 0.80 and the scale’s total-item correlation coefficients ranged from 0.15 to 0.66 and were significant (p<0.001). Interestingly parents reported high scores on the PFMS 34.52±4.60 (range 8-40 with 40 indicating a high burden of care for febrile children). Conclusion: The PFMS was as psychometrically robust in a Turkish population as in an Australian population and is, therefore, a useful tool for health professionals to identify parents’ practices, provide targeted education thereby in reducing the unnecessary burden of care they place on themselves when caring for a febrile child. Relevance to clinical practice. Testing in different populations, cultures and healthcare systems will further assist in reporting the PFMS usefulness in clinical practice and research.

Use of first nucleotide change technology to determine the frequency of factor V Leiden in a population of Australian blood donors

Activated protein C resistance (APCR), the most common risk factor for venous thrombosis, is the result of a G to A base substitution at nucleotide 1691 (R506Q) in the factor V gene. Current techniques to detect the factor V Leiden mutation, such as determination of restriction length polymorphisms, do not have the capacity to screen large numbers of samples in a rapid, cost- effective test. The aim of this study was to apply the first nucleotide change (FNC) technology, to the detection of the factor V Leiden mutation. After preliminary amplification of genomic DNA by polymerase chain reaction (PCR), an allele-specific primer was hybridised to the PCR product and extended using fluorescent terminating dideoxynucleotides which were detected by colorimetric assay. Using this ELISA-based assay, the prevalence of the factor V Leiden mutation was determined in an Australian blood donor population (n = 500). A total of 18 heterozygotes were identified (3.6%) and all of these were confirmed with conventional MnlI restriction digest. No homozygotes for the variant allele were detected. We conclude from this study that the frequency of 3.6% is compatible with others published for Caucasian populations. In addition, the FNC technology shows promise as the basis for a rapid, automated DNA based test for factor V Leiden.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.