XIAP downregulation accompanies mebendazole growth inhibition in melanoma xenografts

Mebendazole (MBZ) was identified as a promising therapeutic on the basis of its ability to induce apoptosis in melanoma cell lines through a B-cell lymphoma 2 (BCL2)-dependent mechanism. We now show that in a human xenograft melanoma model, oral MBZ is as effective as the current standard of care temozolomide in reducing tumor growth. Inhibition of melanoma growth in vivo is accompanied by phosphorylation of BCL2 and decreased levels of X-linked inhibitor of apoptosis (XIAP). Reduced expression of XIAP on treatment with MBZ is partially mediated by its proteasomal degradation. Furthermore, exposure of melanoma cells to MBZ promotes the interaction of SMAC/DIABLO with XIAP, thereby alleviating XIAP's inhibition on apoptosis. XIAP expression on exposure to MBZ is indicative of sensitivity to MBZ as MBZ-resistant cells do not show reduced levels of XIAP after treatment. Resistance to MBZ can be reversed partially by siRNA knockdown of cellular levels of XIAP. Our data indicate that MBZ is a promising antimelanoma agent on the basis of its effects on key antiapoptotic proteins.

Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status

BACKGROUND: Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. RESULTS: The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1. CONCLUSIONS: Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

HIV-1 Pr55 Gag virus-like particles (VLPs) are strong immunogens with potential as candidate HIV vaccines. VLP immunogenicity can be broadened by making chimaeric Gag molecules: however, VLPs incorporating polypeptides longer than 200 aa fused in frame with Gag have not yet been reported. We constructed a range of gag-derived genes encoding in-frame C-terminal fusions of myristoylation-competent native Pr55Gag and p6-truncated Gag (Pr50Gag) to test the effects of polypeptide length and sequence on VLP formation and morphology, in an insect cell expression system. Fused sequences included a modified reverse transcriptase-Tat-Nef fusion polypeptide (RTTN, 778 aa), and truncated versions of RTTN ranging from 113 aa to 450 aa. Baculovirus-expressed chimaeric proteins were examined by western blot and electron microscopy. All chimaeras formed VLPs which could be purified by sucrose gradient centrifugation. VLP diameter increased with protein MW, from ∼100 nm for Pr55Gag to ∼250 nm for GagRTTN. The presence or absence of the Gag p6 region did not obviously affect VLP formation or appearance. GagRT chimaeric particles were successfully used in mice to boost T-cell responses to Gag and RT that were elicited by a DNA vaccine encoding a GagRTTN polypeptide, indicating the potential of such chimaeras to be used as candidate HIV vaccines. © 2008 Elsevier B.V. All rights reserved.

A qualitative approach to assessing the validity of the posttraumatic growth inventory

The Posttraumatic Growth Inventory (PTGI; Tedeschi & Calhoun, 1996) is the most commonly used measure of positive psychological change that can result from negotiating a traumatic experience. Whilst the PTGI has strong internal reliability, validity studies are still sparse. The presented research details trauma survivors’ understanding of items comprising the PTGI in order to qualitatively assess content validity. Participants were 14 trauma survivors who completed the PTGI and participated in a semi-structured interview. Thematic Analysis was conducted on participant’s transcribed interviews. One latent theme was identified reflecting that questions were consistently understood. A relationship was found between the constituent themes identified and the five factors of the PTGI. Participants answered the PTGI statements in a way that is consistent with the purpose of the instrument with only a small discrepancy found when some participants used the PTGI scale to indicate when a decrease in an element of the inventory had been experienced. Overall results supported the content validity of the PTGI.

Coal seam gas waters - their geochemical signature and potential environmental implications

Coal Seam Gas (CSG) is a form of natural gas (mainly methane) sorbed in underground coal beds. To mine this gas, wells are drilled directly into an underground coal seam and groundwater (CSG water) is pumped out to the surface. This lowers the downhole piezometric pressure and enables gas desporption from the coal matrix. In the United States, this gas has been extracted commercially since the 1980s. The economic success of US CSG projects has inspired exploration and development in Australia and New Zealand. In Australia, Queensland’s Bowen and Surat basins have been the subject of increased CSG development over the last decade. CSG growth in other Australian basins has not matured to the same level but exploration and development are taking place at an accelerated pace in the Sydney Basin (Illawarra and the Hunter Valley, NSW) and in the Gunnedah Basin. Similarly, CSG exploration in New Zealand has focused in the Waikato region (Maramarua and Huntly), in the West Coast region (Buller, Reefton, and Greymouth), and in Southland (Kaitangata, Mataura, and Ohai). Figure 1 shows a Shcoeller diagram with CSG samples from selected basins in Australia, New Zealand, and the USA. CSG water from all of these basins exhibit the same geochemical signature – low calcium, low magnesium, high bicarbonate, low sulphate and, sometimes, high chloride. This water quality is a direct result of specific biological and geological processes that have taken part in the formation of CSG. In general, these processes include the weathering of rocks (carbonates, dolomite, and halite), cation exchange with clays (responsible for enhanced sodium and depleted calcium and magnesium), and biogenic processes (accounting for the presence of high bicarbonate concentrations). The salinity of CSG waters tends to be brackish (TDS < 30000 mg/l) with a fairly neutral pH. These particular characteristics need to be taken into consideration when assessing water management and disposal alternatives. Environmental issues associated with CSG water disposal have been prominent in developed basins such as the Powder River Basin (PRB) in the United States. When disposed on the land or used for irrigation, water having a high dissolved salts content may reduce water availability to crops thus affecting crop yield. In addition, the high sodium, low calcium and low magnesium concentrations increase the potential to disperse soils and significantly reduce the water infiltration rate. Therefore, CSG waters need to be properly characterised, treated, and disposed to safeguard the environment without compromising other natural resources.

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Comparative effects of posterior eye cup tissues from Myopic and Hyperopic chick eyes on cultured scleral fibroblasts

The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and -smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or -15D lenses (lens-induced myopia, LIM) for 3 days (post hatch day 5 to 8) (n=6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from -15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9±2.6 µg and 8.4±1.1 µg, p=0.12; GAG 11.2±0.6 µg and 10.1±1.0 µg, p=0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2±1.7 µg versus 23.2±1.5 µg, p=0.21; GAG 28.1±1.7 µg versus. 28.7±1.2 µg, p=0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA, LIM 46.9±6.4 versus LIH 51.5±4.7 µg, p=0.31), whereas GAG content was higher for cells in co-culture with choroid from LIH eyes (GAG 32.5±0.7 µg versus 18.9±1.2 µg, F1,6=9.210, p=0.0002). In contrast, fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4±6.3 µg versus Empty basket: 46.03±1.0 µg; F1,6=69.99, p=0.0005 and LIH: 27.9±2.3 µg versus empty basket: 46.03±1.0 µg; p=0.0004). GAG content was higher with RPE from LIH eyes (LIH: 33.7±1.9 µg versus empty basket: 29.5±0.8 µg, F1,6=13.99, p=0.010) and lower with RPE from LIM eyes (LIM: 27.7±0.9 µg versus empty basket: 29.5±0.8 µg, p=0.021). GAG content of cells in co-culture with choroid from LIH eyes was higher compared to co-culture with choroid from LIM eyes (32.5±0.7 µg versus 18.9±1.2 µg respectively, F1,6=9.210, p=0.0002). In conclusion, these experiments provide evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.

Economic growth and inequality patterns in the presence of costly technology adoption and uncertainty

We develop a stochastic endogenous growth model to explain the diversity in growth and inequality patterns and the non-convergence of incomes in transitional economies where an underdeveloped financial sector imposes an implicit, fixed cost on the diversification of idiosyncratic risk. In the model endogenous growth occurs through physical and human capital deepening, with the latter being the more dominant element. We interpret the fixed cost as a ‘learning by doing’ cost for entrepreneurs who undertake risk in the absence of well developed financial markets and institutions that help diversify such risk. As such, this cost may be interpreted as the implicit returns foregone due to the lack of diversification opportunities that would otherwise have been available, had such institutions been present. The analytical and numerical results of the model suggest three growth outcomes depending on the productivity differences between the projects and the fixed cost associated with the more productive project. We label these outcomes as poverty trap, dual economy and balanced growth. Further analysis of these three outcomes highlights the existence of a diversity within diversity. Specifically, within the ‘poverty trap’ and ‘dual economy’ scenarios growth and inequality patterns differ, depending on the initial conditions. This additional diversity allows the model to capture a richer range of outcomes that are consistent with the empirical experience of several transitional economies.

Scaffolds for growth factor delivery as applied to bone tissue engineering

There remains a substantial shortfall in treatment of severe skeletal injuries. The current gold standard of autologous bone grafting from the same patient, has many undesirable side effects associated such as donor site morbidity. Tissue engineering seeks to offer a solution to this problem. The primary requirements for tissue engineered scaffolds have already been well established, and many materials, such as polyesters, present themselves as potential candidates for bone defects; they have comparable structural features, but they often lack the required osteoconductivity to promote adequate bone regeneration. By combining these materials with biological growth factors; which promote the infiltration of cells into the scaffold as well as the differentiation into the specific cell and tissue type, it is possible to increase the formation of new bone. However cost and potential complications associated with growth factors means controlled release is an important consideration in the design of new bone tissue engineering strategies. This review will cover recent research in the area of encapsulation and release of growth factors within a variety of different polymeric scaffolds.

Emergency Health Services (EHS) : demand and service delivery models. [Monograph 2 : Queensland EHS users’ profile]

Emergency health is a critical component of Australia’s health system and one which is increasingly congested from growing demand and blocked access to inpatient beds. The Emergency Health Services Queensland (EHSQ) study aims to identify the factors driving increased demand for emergency health and to evaluate strategies which may safely reduce the future demand growth. This monograph addresses the characteristics of users of emergency health services with an aim to identify those that appear to contribute to demand growth. This study utilises data on patients treated by Emergency Departments (ED) and Queensland Ambulance Service (QAS) across Queensland. ED data was derived from the Emergency Department Information System (EDIS) for the period 2001-02 through to 2010-11. Ambulance data was extracted from the QAS’ Ambulance Information Management System (AIMS) and electronic Ambulance Report Form (eARF) for the period 2001-02 through to 2009-10. Due to discrepancies and comparability issues for ED data, this monograph compares data from the 2003-04 time period with 2010-11 data for 21 of the reporting EDs. Also a snapshot of users for the 2010-11 financial year for 31 reporting EDs is used to describe the characteristics of users and to compare those characteristics with population demographics. For QAS data, the 2002-03 and 2009-10 time periods were selected for detailed analyses to identify trends. • Demand for emergency health care services is increasing, representing both increased population and increased relative utilisation. Per capita demand for ED attention has increased by 2% per annum over the last decade and for ambulance attention by 3.7% per annum. • The growth in ED demand is prominent in more urgent triage categories with actual decline in less urgent patients. An estimated 55% of patients attend hospital EDs outside of normal working hours. There is no evidence that patients presenting out of hours are significantly different to those presenting within working hours; they have similar triage assessments and outcomes. • Patients suffering from injuries and poisoning comprise 28% of the ED workload (an increase of 65% in the study period), whilst declines of 32% in cardiovascular and circulatory conditions, and musculoskeletal problems have been observed. • 25.6% of patients attending EDs are admitted to hospital. 19% of admitted patients and 7% of patients who die in the ED are triage category 4 or 5 on arrival. • The average age of ED patients is 35.6 years. Demand has grown in all age groups and amongst both men and women. Men have higher utilisation rates for ED in all age groups. The only group where the growth rate in women has exceeded men is in the 20-29 age group; this growth is particularly in the injury and poisoning categories. • Considerable attention has been paid publicly to ED performance criteria. It is worth noting that 50% of all patients were treated within 33 minutes of arrival. • Patients from lower socioeconomic areas appear to have higher utilisation rates and the utilisation rate for indigenous people appears to exceed those of European and other backgrounds. The utilisation rates for immigrant people is generally less than that of Australian born however it has not been possible to eliminate the confounding impact of different age and socioeconomic profiles. • Demand for ambulance service is also increasing at a rate that exceeds population growth. Utilisation rates have increased by an average of 5% per annum in Queensland compared to 3.6% nationally, and the utilisation rate in Queensland is 27% higher than the national average. • The growth in ambulance utilisation has also been amongst the more urgent categories of dispatch and utilisation rates are higher in rural and regional areas than in the metropolitan area. The demand for ambulance increases with age but the growth in demand for ambulance service has been more prominent in younger age groups. These findings contribute significantly to an understanding of the growth in demand for emergency health. It shows that the growth is amongst patients in genuine need of emergency healthcare and public rhetoric that the congestion of emergency health services is due to inappropriate attendees is unable to be substantiated. The consistency of the growth in demand over the last decade reflects not only the changing demographics of the Australian population but also the changes in health status, standards of acute health care and other social factors. The growth is also amongst patients with acute injury and poisoning which is inconsistent with rates of chronic disease as a fundamental driver. We have also interviewed patients in regard to their decision making choices for acute health care and the factors that influence these decisions and this will be the subject of a third Monograph and publications.

Analytical electron microscopy of Mg-SiO smokes; a comparison with infrared and XRD studies

Experimentally obtained Mg.SiO smokes were studied by analytical electron microscopy using the same samples that had been previously characterized by repeated infrared spectroscopy. Analytical electron microscopy shows that unannealed smokes contain some degree of microcrystallinity which increases with increased annealing for up to 30 hr. An SiO2 polymorph (tridymite) and MgO may form contemporaneously as a result of growth of forsterite (Mg2SiO4) microcrystallites in the initially nonstoichiometric smokes. After 4 hr annealing, forsterite and tridymite react to enstatite (MgSiO3). We suggest that infrared spectroscopy and X-ray diffraction analysis should be complemented by detailed analytical electron microscopy to detect budding crystallinity in vapor phase condensates.

Experimental transformation of kaolinite to halloysite

A well-characterized kaolinite has been hydrated in order to test the hypothesis that platey kaolinite will roll upon hydration. Kaolinite hydrates are prepared by repeated intercalation of kaolinite with potassium acetate and subsequent washing with water. On hydration, kaolinite plates roll along the major crystallographic directions to form tubes identical to proper tubular halloysite. Most tubes are elongated along the b crystallographic axis, while some are elongated along the a axis. Overall, the tubes exhibit a range of crystallinity. Well-ordered examples show a 2-layer structure, while poorly ordered tubes show little or no 3-dimensional order. Cross-sectional views of the formed tubes show both smoothly curved layers and planar faces. These characteristics of the experimentally formed tubes are shared by natural halloysites. Therefore, it is proposed that planar kaolinite can transform to tubular halloysite.

Psychosocial experience of cancer : surpassing mere survival and recognising the potential for posttraumatic growth as well as distress.

The publication of the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV; American Psychiatric Association, 1994) introduced the notion that a life-threatening illness can be a stressor and catalyst for Posttraumatic Stress Disorder (PTSD). Since then a solid body of research has been established investigating the post-diagnosis experience of cancer. These studies have identified a number of short and long-term life changes resulting from a diagnosis of cancer and associated treatments. In this chapter, we discuss the psychosocial response to the cancer experience and the potential for cancer-related distress. Cancer can represent a life-threatening diagnosis that may be associated with aggressive treatments and result in physical and psychological changes. The potential for future trauma through the lasting effects of the disease and treatment, and the possibility of recurrence, can be a source of continued psychological distress. In addition to the documented adverse repercussions of cancer, we also outline the recent shift that has occurred in the psycho-oncology literature regarding positive life change or posttraumatic growth that is commonly reported after a diagnosis of cancer. Adopting a salutogenic framework acknowledges that the cancer experience is a dynamic psychosocial process with both negative and positive repercussions. Next, we describe the situational and individual factors that are associated with posttraumatic growth and the types of positive life change that are prevalent in this context. Finally, we discuss the implications of this research in a therapeutic context and the directions of future posttraumatic growth research with cancer survivors. This chapter will present both quantitative and qualitative research that indicates the potential for personal growth from adversity rather than just mere survival and return to pre-diagnosis functioning. It is important to emphasise however, that the presence of growth and prevalence of resilience does not negate the extremely distressing nature of a cancer diagnosis for the patient and their families and the suffering that can accompany treatment regimes. Indeed, it will be explained that for growth to occur, the experience must be one that quite literally shatters previously held schemas in order to act as a catalyst for change.

The cancer stem-cell hypothesis : its emerging role in lung cancer biology and its relevance for future therapy

The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review.