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.

Validation of the Cannabis Expectancy Questionnaire (CEQ) in adult cannabis users in treatment

Background Outcome expectancies are a key cognitive construct in the etiology, assessment and treatment of Substance Use Disorders. There is a research and clinical need for a cannabis expectancy measure validated in a clinical sample of cannabis users. Method The Cannabis Expectancy Questionnaire (CEQ) was subjected to exploratory (n = 501, mean age 27.45, 78% male) and confirmatory (n = 505, mean age 27.69, 78% male) factor analysis in two separate samples of cannabis users attending an outpatient cannabis treatment program. Weekly cannabis consumption was clinically assessed and patients completed the Severity of Dependence Scale-Cannabis (SDS-C) and the General Health Questionnaire (GHQ-28). Results Two factors representing Negative Cannabis Expectancies and Positive Cannabis Expectancies were identified. These provided a robust statistical and conceptual fit for the data. Internal reliabilities were high. Negative expectancies were associated with greater dependence severity (as measured by the SDS) and positive expectancies with higher consumption. The interaction of positive and negative expectancies was consistently significantly associated with self-reported functioning across all four GHQ-28 scales (Somatic Concerns, Anxiety, Social Dysfunction and Depression). Specifically, within the context of high positive cannabis expectancy, higher negative expectancy was predictive of more impaired functioning. By contrast, within the context of low positive cannabis expectancy, higher negative expectancy was predictive of better functioning. Conclusions The CEQ is the first cannabis expectancy measure to be validated in a sample of cannabis users in treatment. Negative and positive cannabis expectancy domains were uniquely associated with consumption, dependence severity and self-reported mental health functioning.

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.

Frequent activating FGFR2 mutations in endometrial carcinomas parallel germline mutations associated with craniosynostosis and skeletal dysplasia syndromes

Endometrial carcinoma is the most common gynecological malignancy in the United States. Although most women present with early disease confined to the uterus, the majority of persistent or recurrent tumors are refractory to current chemotherapies. We have identified a total of 11 different FGFR2 mutations in 3/10 (30%) of endometrial cell lines and 19/187 (10%) of primary uterine tumors. Mutations were seen primarily in tumors of the endometrioid histologic subtype (18/115 cases investigated, 16%). The majority of the somatic mutations identified were identical to germline activating mutations in FGFR2 and FGFR3 that cause Apert Syndrome, Beare-Stevenson Syndrome, hypochondroplasia, achondroplasia and SADDAN syndrome. The two most common somatic mutations identified were S252W (in eight tumors) and N550K (in five samples). Four novel mutations were identified, three of which are also likely to result in receptor gain-of-function. Extensive functional analyses have already been performed on many of these mutations, demonstrating they result in receptor activation through a variety of mechanisms. The discovery of activating FGFR2 mutations in endometrial carcinoma raises the possibility of employing anti-FGFR molecularly targeted therapies in patients with advanced or recurrent endometrial carcinoma.

The CDKN2A (p16) gene and human cancer

CDKN2A, the gene encoding the cell-cycle inhibitor p16CDKN2A, was first identified in 1994. Since then, somatic mutations have been observed in many cancers and germline alterations have been found in kindreds with familial atypical multiple mole/melanoma (FAMMM), also known as atypical mole syndrome. In this review we tabulate the known mutations in this gene and discuss specific aspects, particularly with respect to germline mutations and cancer predisposition.

A genome-based strategy uncovers frequent BRAF mutations in melanoma

Using a genome-scanning approach to search for oncogenes, a recent report identifies somatic mutations in the signaling gene BRAF that are particularly prevalent in melanoma.

PTEN inactivation is rare in melanoma tumours but occurs frequently in melanoma cell lines

Deletions detected in cytogenetic and loss of heterozygosity (LOH) studies indicate that at least one tumour suppressor gene maps to the long arm of chromosome 10. Previous deletion mapping studies have observed LOH on 10q in about 30% of melanomas analysed. The PTEN gene, mapping to chromosome band 10q23.3, encodes a protein with both lipid and protein phosphatase activity. Somatic mutations and deletions in have been detected in a variety of cell lines and tumours, including melanoma samples. We performed mutation analyses and extensive allelic loss studies to investigate the role this gene plays in melanoma pathogenesis. We found that a total of 34 out of 57 (60%) melanoma cell lines carried hemizygous deletions of chromosome 10q encompassing the PTEN locus. A further three cell lines carried smaller deletions excluding PTEN. Inactivation of both PTEN alleles by exon-specific homozygous deletion or mutation was observed in 13 out of 57 (23%) melanoma cell lines. The mutation spectrum observed does not indicate an important role for ultraviolet radiation in the genesis of these mutations, and evidence from three cell lines supports the acquisition of PTEN aberrations in culture. Ten out of 49 (20%) matched melanoma tumour/normal samples harboured hemizygous deletions of either the whole chromosome or most of the long arm. Mutations within were detected in only one of the 10 tumours demonstrating LOH at 10q23 that were analysed. These results suggest that PTEN inactivation may be important for the propagation of melanoma cells in culture, and that another chromosome 10 tumour suppressor gene may be important for melanoma pathogenesis.

Targeting mutant fibroblast growth factor receptors in cancer

Fibroblast growth factor receptors (FGFRs) play diverse roles in the control of cell proliferation, cell differentiation, angiogenesis and development. Activating the mutations of FGFRs in the germline has long been known to cause a variety of skeletal developmental disorders, but it is only recently that a similar spectrum of somatic FGFR mutations has been associated with human cancers. Many of these somatic mutations are gain-of-function and oncogenic and create dependencies in tumor cell lines harboring such mutations. A combination of knockdown studies and pharmaceutical inhibition in preclinical models has further substantiated genomically altered FGFR as a therapeutic target in cancer, and the oncology community is responding with clinical trials evaluating multikinase inhibitors with anti-FGFR activity and a new generation of specific pan-FGFR inhibitors.

Psychometric properties and health correlates of the Geriatric Anxiety Inventory in Australian community-residing older women

Abstract OBJECTIVE: To assess the psychometric properties and health correlates of the Geriatric Anxiety Inventory (GAI) in a cohort of Australian community-residing older women. METHOD: Cross-sectional study of a population-based cohort of women aged 60 years and over (N = 286). RESULTS: The GAI exhibited sound internal consistency and demonstrated good concurrent validity against the state half of the Spielberger State Trait Anxiety Inventory and the neuroticism domain of the NEO five-factor inventory. GAI score was significantly associated with self-reported sleep difficulties and perceived memory impairment, but not with age or cognitive function. Women with current DSM-IV Generalized Anxiety Disorder (GAD) had significantly higher GAI scores than women without such a history. In this cohort, the optimal cut-point to detect current GAD was 8/9. Although the GAI was designed to have few somatic items, women with a greater number of general medical problems or who rated their general health as worse had higher GAI scores. CONCLUSION: The GAI is a new scale designed specifically to measure anxiety in older people. In this Australian cohort of older women, the instrument had sound psychometric properties.

Cross-cultural comparisons of health-related quality of life in Australian and Japanese midlife women

Objective: The purpose of this study was to address (1) the existence of an association between menopausal status and the health-related quality of life (HRQOL) in Australian and Japanese women and (2) the relative contributions of menopausal status, modifiable lifestyle risk factors, health, and sociodemographic factors on HRQOL. Design: The Australian and Japanese Midlife Women's Health Study (AJMWHS) was a multisite, population-based study conducted in 2001 to 2002. Measures were conducted on data collected from a survey questionnaire used for a sample of women from Australia and Japan. HRQOL was assessed with seven subscales from the Short Form-36. Results: The differences seen in physical functioning, general health, and vitality are significant. The results support an effect of country of residence on physical functioning and general health. The impact of menopausal status on HRQOL was significantly associated with bodily pain and role-emotional. The country of residence did have a modifying effect on the relationship between menopausal status and physical functioning. After control for confounders, there was a significant difference between Australian and Japanese women for HRQOL. Menopausal status was not associated with HRQOL in the areas of general health and physical functioning. Modifiable lifestyle risk factors contributed more highly to HRQOL for the Australian women than for the Japanese women. If the women had a lowered body mass index, undertook physical activity, consumed dietary phytoestrogens, and used alcohol, their physical functioning seemed to be better. Differences were seen in the contributions to HRQOL in these areas, with lower body mass index in the Australian women and physical activity in the Japanese women being the highest predictors. Somatic and psychological symptoms seem to negatively affect both Japanese and Australian women's physical functioning, contributing more than sociodemographic factors, menopausal status, and behavioral determinants combined to general health and physical functioning. Conclusions: It is important that that consideration be given to incorporating the same tool within the cross-cultural design of studies so that comparisons between cultures and patterns of healthy aging can be made. The research suggests that there seems to be variations across Australian and Japanese midlife women in some areas of HRQOL and some factors that contribute to these areas.

Overview of methods used in cross-cultural comparisons of menopausal symptoms and their determinants : Guidelines for Strengthening the Reporting of Menopause and Aging (STROMA) studies

This paper reviews the methods used in cross-cultural studies of menopausal symptoms with the goal of formulating recommendations to facilitate comparisons of menopausal symptoms across cultures. It provides an overview of existing approaches and serves to introduce four separate reviews of vasomotor, psychological, somatic, and sexual symptoms at midlife. Building on an earlier review of cross-cultural studies of menopause covering time periods until 2004, these reviews are based on searches of Medline, PsycINFO, CINAHL and Google Scholar for English-language articles published from 2004 to 2010 using the terms “cross cultural comparison” and “menopause.” Two major criteria were used: a study had to include more than one culture, country, or ethnic group and to have asked about actual menopausal symptom experience. We found considerable variation across studies in age ranges, symptom lists, reference period for symptom recall, variables included in multivariate analyses, and the measurement of factors (e.g., menopausal status and hormonal factors, demographic, anthropometric, mental/physical health, and lifestyle measures) that influence vasomotor, psychological, somatic and sexual symptoms. Based on these reviews, we make recommendations for future research regarding age range, symptom lists, reference/recall periods, and measurement of menopausal status. Recommendations specific to the cross-cultural study of vasomotor, psychological, somatic, and sexual symptoms are found in the four reviews that follow this introduction.

Spin

“Spin” borrows idioms and metaphors from sports commentary and squeezes them into a single emotional rollercoaster. Accompanied by a driving soundtrack, text appears and disappears one word at a time. As the work progresses, multiple words fade in and out at the same time, filling the screen and testing our ability to read and assimilate these well-worn phrases. On the one hand, the work mimes some of what we enjoy about sport – its ability to take us to another place, to incite passion and emotion, and to enable us to share in common experiences, goals and desires. On the other hand, it plays up the hyperbolic language often associated with sports broadcasting. The very language that helps take us to another place, incite passion and make us feel part of something bigger than ourselves, is pushed to its extreme and starts to burst at the seams. This work was commissioned for “Kick Off: contemporary video art program” at Metricon Stadium, Gold Coast, and supported by Project Services, Department of Public Works, Queensland Government.

Do you see what I mean? charting changing representations and receptions of the disabled body in contemporary and pop cultural performance

The meaning of the body emerges through acts of seeing, looking and staring in daily and dramatic performances. Acts that are, as Maike Bleeker argues1, bound up with the scopic rules, regimes and narratives that apply in specific cultures at specific times. In Western culture, the disabled body has been seen as a sign of defect, deficiency, fear, shame or stigma. Disabled artists – Mat Fraser, Bill Shannon, Aaron Williamson, Katherine Araniello, Liz Crow and Ju Gosling – have attempted, via performances that co-opt conventional images of the disabled body, to challenge dominant ways of representing and responding such bodies from within. In this paper, I consider what happens when non-disabled artists co-opt images of the disabled body to draw attention to, affirm, and even exoticise, eroticise or beautify, other modalities of or desires for difference. As Carrie Sandahl has noted2, the signs, symbols and somatic idiosyncrasies of the disabled body are, today, transported or translated into theatre, film and television as a metaphor or "master trope" for every body’s experience of difference. This happens in performance art (Guillermo Gomez-Pena’s use of a wheelchair in Chamber of Confessions), performance (Marie Chouinard's use of crutches, canes and walkers to represent dancers’ experience of becoming different or mutant during training in bODY rEMIX /gOLDBERG vARIATIONS), and pop culture (characters in wheelchairs in Glee or Oz). In this paper, I chart changing representations and receptions of the disabled body in such contexts. I use analysis of this cultural shift as a starting point for a re-consideration of questions about whether a face-toface encounter with a disabled body is in fact a privileged site for the emergence of a politics, and whether co-opting disability as a metaphor for a range of difference differences reduces its currency as a category around which a specific group might mobilise a politics.

In vitro breeding strategies in the development of Australian native plants

Plant tissue culture is a technique that exploits the ability of many plant cells to revert to a meristematic state. Although originally developed for botanical research, plant tissue culture has now evolved into important commercial practices and has become a significant research tool in agriculture, horticulture and in many other areas of plant sciences. Plant tissue culture is the sterile culture of plant cells, tissues, or organs under aseptic conditions leading to cell multiplication or regeneration or organs and whole plants. The steps required to develop reliable systems for plant regeneration and their application in plant biotechnology are reviewed in countless books. Some of the major landmarks in the evolution of in vitro techniques are summarised in Table 5.1. In this chapter the current applications of this technology to agriculture, horticulture, forestry and plant breeding are briefly described with specific examples from Australian plants when applicable.