Developing a three-dimensional in-vitro cell culture model for studying breast and prostate cancer using starPEG-heparin hydrogels

Introduction Hydrogels prepared from poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs) (1). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSC). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyse the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via Alamar Blue assays, light microscopy, and immunofluorescence staining for cytokeratin 8/18, Ki67 and E-Cadherin. Cancer cell lines were then pre-grown in hydrogels for 5-7 days and then re-seeded into starPEG-heparin hydrogels functionalised with RGD, SDF-1, bFGF and VEGF as spheroids with HUVECs and MSC and grown for 14 days as a tri-culture in Endothelial Cell Growth Medium (ECGM; Promocell). Cell lines were also seeded as a single cell suspension into the functionalised tri-culture system. Cultures were fixed in 4% paraformaldehyde and analysed via immunostaining for Von Willebrand Factor and CD31, as well as the above mentioned markers, and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualised in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. HUVEC tube formation and cancer line spheroid formation occured after 3-4 days. Interaction was visualised between tumours and HUVECs via confocal microscopy. Slightly increased interaction was seen between cancer tumours and micro-vascular tubes when seeded as single cells compared with the pre-formed spheroid approach. Further studies intend to utilise cytokine gradients to further optimise the ECM environment of in situ tumour angiogenesis. Discussion and Conclusions Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVECs and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer. References 1. Tsurkan MV, Chwalek K, Prokoph S, Zieris A, Levental KR, Freudenberg U, Werner C. Advanced Materials. 25, 2606-10, 2013. Disclosures The authors declare no conflicts of interest

The Fleet Store : devising a public retail exhibition to promote creative, innovative and sustainable business models for emerging fashion designers

1.Description of the Work The Fleet Store was devised as a creative output to establish an exhibition linked to a fashion business model where emerging designers were encouraged to research new and innovative strategies for creating design-driven and commercial collections for a public consumer. This was a project that was devised to break down the perceptions of emerging fashion designers that designing commercial collections linked to a sustainable business model is a boring and unnecessary process. The focus was to demystify the business of fashion and to link its importance to a design-driven and public outcome that is more familiar to fashion designers. The criterion for participation was that all designers had to be registered as a business with the Australian Taxation Office. Designers were chosen from the Creative Enterprise Australia Fashion Business Incubator, the QUT fashion graduate alumni and current QUT fashion design and double degree (fashion and business) students with existing businesses. The project evolved from a series of collaborative workshops where designers were introduced to new and innovative creative industries’ business models and the processes, costings and timings involved to create a niche, sustainable business for a public exhibition of design-driven commercial collections. All designers initiated their own business infra-structure but were then introduced to the concept of collaboration for successful and profitable exhibition and business outcomes. Collaborative strategies such as crowd funding, crowd sourcing, peer to peer mentoring and manufacturing were all researched, and strategies for the establishment of the retail exhibition were all devised in a collaborative environment. All participants also took on roles outside their ‘designer’ background to create a retail exhibition that was creative but also had critical mass and aesthetic for the consumer. The Fleet Store ‘popped up’ for 2 weeks (10 days), in a heritage-listed building in an inner city location. Passers-by were important, but the main consumer was enlisted by the use of interest and investment from crowd sourcing, crowd funding, ethical marketing, corporate social responsibility projects and collaborative public relations and social media strategies. The research has furthered discussion on innovative strategies for emerging fashion designers to initiate and maintain sustainable businesses and suggests that collaboration combined with a design-driven and business focus can create a sustainable and economically viable retail exhibition. 2. Research Statement Research Background The research field involved developing a new ethical, design-driven, collaborative and sustainable model for fashion design practice and management. The research asked can a public, design-driven, collaborative retail exhibition create a platform for promoting creative, innovative and sustainable business models for emerging fashion designers. The methodology was primarily practice-led as all participants were designers in their own right and the project manager acted as a mentor and curator to guide the process and analyse the potential of the research question. The Fleet Store offers new knowledge in design practice and management; with the creation of a model where design outcomes and business models are inextricably linked to the success of the creative output. Key innovations include extending the commercialisation of emerging fashion businesses by creating a curated retail gallery for collaborative and sustainable strategies to support niche fashion designer labels. This has contributed to a broader conversation on how to nurture and sustain competitive Australian fashion designers/labels. Research Contribution and Significance The Fleet Store has contributed to a growing body of research into innovative and sustainable business models for niche fashion and creative industries’ practitioners. All participants have maintained their business infra-structure and many are currently growing their businesses, using the strategies tested for the Fleet Store. The exhibition space was visited by over 1,000 people and sales of $27,000 were made in 10 days of opening. (Follow up sales of $3,000 has also been reported.) Three of the designers were ‘discovered’ from the exhibition and have received substantial orders from high profile national buyers and retailers for next season delivery. Several participants have since collaborated to create other pop up retail environments and are now mentoring other emerging designers on the significance of a collaborative retail exhibition to consolidate niche business models for emerging fashion designers.

Psychological health and the risk of diabetes mellitus in Australian women: A 21-year prospective study

Background Symptoms of depression can be recurrent or limited to one episode. This study discusses the prospective association between psychological health, measured as change in depression symptoms, and the risk of diabetes mellitus in Australian women. Methods Data obtained from the Mater-University of Queensland Study of Pregnancy. Depression was measured using the Delusions-Symptoms: States Inventory. To examine possible transitions over time, depression was grouped into four categories and assessed at different phases over the 21-year period. Multiple logistic regression models and sensitivity analysis to assess the robustness of our analytical strategy were performed. Results Three hundred and one women reported diabetes 21 years after the index pregnancy. Almost one-third of the women who reported depression symptoms continued to report these at a subsequent follow-up (FU) phase. About 1 in 20 women who had not reported depression symptoms at the 5-year FU did so at the subsequent 14-year FU. In prospective analyses, we did not find a significant association between diabetes and negative change (not depressed to depressed, at subsequent phase); however, for women with positive history of symptoms of depression and women with persistent symptoms, there was a 1.97-fold (95% confidence interval [CI]: 1.14–3.40) to 2.23-fold (95% CI: 1.09–4.57) greater risk of diabetes. Conclusions Our study suggests that an increased risk of diabetes is significantly associated with persistent depression symptoms. It highlights the importance of recognizing depression symptoms in terms of women's psychological wellbeing and thus provides a basis for targeting those most at risk.

Projecting future air pollution-related mortality under a changing climate: Progress, uncertainties and research needs

Background Climate change may affect mortality associated with air pollutants, especially for fine particulate matter (PM2.5) and ozone (O3). Projection studies of such kind involve complicated modelling approaches with uncertainties. Objectives We conducted a systematic review of researches and methods for projecting future PM2.5-/O3-related mortality to identify the uncertainties and optimal approaches for handling uncertainty. Methods A literature search was conducted in October 2013, using the electronic databases: PubMed, Scopus, ScienceDirect, ProQuest, and Web of Science. The search was limited to peer-reviewed journal articles published in English from January 1980 to September 2013. Discussion Fifteen studies fulfilled the inclusion criteria. Most studies reported that an increase of climate change-induced PM2.5 and O3 may result in an increase in mortality. However, little research has been conducted in developing countries with high emissions and dense populations. Additionally, health effects induced by PM2.5 may dominate compared to those caused by O3, but projection studies of PM2.5-related mortality are fewer than those of O3-related mortality. There is a considerable variation in approaches of scenario-based projection researches, which makes it difficult to compare results. Multiple scenarios, models and downscaling methods have been used to reduce uncertainties. However, few studies have discussed what the main source of uncertainties is and which uncertainty could be most effectively reduced. Conclusions Projecting air pollution-related mortality requires a systematic consideration of assumptions and uncertainties, which will significantly aid policymakers in efforts to manage potential impacts of PM2.5 and O3 on mortality in the context of climate change.

Psychological services for asylum seekers in the community: Challenges and solutions

Objective: Given the Australian government’s current policy of placing asylum seekers into the community while their applications for asylum are being considered, it is important that mental health practitioners are able to offer culturally safe, sensitive, and competent services in this context. Method: A qualitative approach was used to interview seven practitioners, who provided services to asylum seekers at a specialist health clinic in the community. These professionals used their experience to identify the psychosocial issues faced by these asylum seekers, the challenges of providing culturally effective services to this group, and how these services can be improved. Result: Data were thematically analysed using NVivo. The findings indicated that participants perceived that clients experienced psychological, health, and cultural difficulties. The stress and uncertainty around visa applications emerged as the most severe factor impacting asylum seekers’ mental health. Working effectively with interpreters and culturally adapting assessment and treatment for these clients emerged as effective strategies. Gathering information in a conversational way and using clients’ different cultural explanatory models were methods used to identify and address mental health issues, rather than using formal measures. Interventions were eclectic and holistic, and reflected treatments that were appropriate for the clients’ cultural backgrounds. Conclusion: The study has implications for how psychological services are offered to asylum seekers in the community. Further, it identifies areas that can be incorporated in the future training of practitioners.

Genetic variants underlying risk of endometriosis: Insights from meta-analysis of eight genome-wide association and replication datasets

BACKGROUND Endometriosis is a heritable common gynaecological condition influenced by multiple genetic and environmental factors. Genome-wide association studies (GWASs) have proved successful in identifying common genetic variants of moderate effects for various complex diseases. To date, eight GWAS and replication studies from multiple populations have been published on endometriosis. In this review, we investigate the consistency and heterogeneity of the results across all the studies and their implications for an improved understanding of the aetiology of the condition. METHODS Meta-analyses were conducted on four GWASs and four replication studies including a total of 11 506 cases and 32 678 controls, and on the subset of studies that investigated associations for revised American Fertility Society (rAFS) Stage III/IV including 2859 cases. The datasets included 9039 cases and 27 343 controls of European (Australia, Belgium, Italy, UK, USA) and 2467 cases and 5335 controls of Japanese ancestry. Fixed and Han and Elkin random-effects models, and heterogeneity statistics (Cochran's Q test), were used to investigate the evidence of the nine reported genome-wide significant loci across datasets and populations. RESULTS Meta-analysis showed that seven out of nine loci had consistent directions of effect across studies and populations, and six out of nine remained genome-wide significant (P < 5 × 10(-8)), including rs12700667 on 7p15.2 (P = 1.6 × 10(-9)), rs7521902 near WNT4 (P = 1.8 × 10(-15)), rs10859871 near VEZT (P = 4.7 × 10(-15)), rs1537377 near CDKN2B-AS1 (P = 1.5 × 10(-8)), rs7739264 near ID4 (P = 6.2 × 10(-10)) and rs13394619 in GREB1 (P = 4.5 × 10(-8)). In addition to the six loci, two showed borderline genome-wide significant associations with Stage III/IV endometriosis, including rs1250248 in FN1 (P = 8 × 10(-8)) and rs4141819 on 2p14 (P = 9.2 × 10(-8)). Two independent inter-genic loci, rs4141819 and rs6734792 on chromosome 2, showed significant evidence of heterogeneity across datasets (P < 0.005). Eight of the nine loci had stronger effect sizes among Stage III/IV cases, implying that they are likely to be implicated in the development of moderate to severe, or ovarian, disease. While three out of nine loci were inter-genic, the remaining were in or near genes with known functions of biological relevance to endometriosis, varying from roles in developmental pathways to cellular growth/carcinogenesis. CONCLUSIONS Our meta-analysis shows remarkable consistency in endometriosis GWAS results across studies, with little evidence of population-based heterogeneity. They also show that the phenotypic classifications used in GWAS to date have been limited. Stronger associations with Stage III/IV disease observed for most loci emphasize the importance for future studies to include detailed sub-phenotype information. Functional studies in relevant tissues are needed to understand the effect of the variants on downstream biological pathways.

Predicting the need for adaptive radiotherapy in head and neck cancer

Background and purpose Adaptive radiotherapy (ART) can account for the dosimetric impact of anatomical change in head and neck cancer patients; however it can be resource intensive. Consequently, it is imperative that patients likely to require ART are identified. The purpose of this study was to find predictive factors that identify oropharyngeal squamous cell carcinoma (OPC) and nasopharyngeal carcinoma (NPC) patients more likely to need ART. Materials and methods One hundred and ten patients with OPC or NPC were analysed. Patient demographics and tumour characteristics were compared between patients who were replanned and those that were not. Factors found to be significant were included in logistic regression models. Risk profiles were developed from these models. A dosimetric analysis was performed. Results Nodal disease stage, pre-treatment largest involved node size, diagnosis and initial weight (categorised in 2 groups) were identified as significant for inclusion in the model. Two models were found to be significant (p = 0.001), correctly classifying 98.2% and 96.1% of patients respectively. Three ART risk profiles were developed. Conclusion Predictive factors identifying OPC or NPC patients more likely to require ART were reported. A risk profile approach could facilitate the effective implementation of ART into radiotherapy departments through forward planning and appropriate resource allocation.

Populations of models, experimental designs and coverage of parameter space by Latin Hypercube and Orthogonal Sampling

In this paper we have used simulations to make a conjecture about the coverage of a t-dimensional subspace of a d-dimensional parameter space of size n when performing k trials of Latin Hypercube sampling. This takes the form P(k,n,d,t) = 1 - e^(-k/n^(t-1)). We suggest that this coverage formula is independent of d and this allows us to make connections between building Populations of Models and Experimental Designs. We also show that Orthogonal sampling is superior to Latin Hypercube sampling in terms of allowing a more uniform coverage of the t-dimensional subspace at the sub-block size level. These ideas have particular relevance when attempting to perform uncertainty quantification and sensitivity analyses.

Living well with diabetes: 24-month outcomes from a randomized trial of telephone-delivered weight loss and physical activity intervention to improve glycemic control

OBJECTIVE: To evaluate the effectiveness of a telephone-delivered behavioral weight loss and physical activity intervention targeting Australian primary care patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Pragmatic randomized controlled trial of telephone counseling (n = 151) versus usual care (n = 151). Reported here are 18-month (end-of-intervention) and 24-month (maintenance) primary outcomes of weight, moderate-to-vigorous-intensity physical activity (MVPA; via accelerometer), and HbA1c level. Secondary outcomes include dietary energy intake and diet quality, waist circumference, lipid levels, and blood pressure. Data were analyzed via adjusted linear mixed models with multiple imputation of missing data. RESULTS: Relative to usual-care participants, telephone counseling participants achieved modest, but significant, improvements in weight loss (relative rate [RR] -1.42% of baseline body weight [95% CI -2.54 to -0.30% of baseline body weight]), MVPA (RR 1.42 [95% CI 1.06-1.90]), diet quality (2.72 [95% CI 0.55-4.89]), and waist circumference (-1.84 cm [95% CI -3.16 to -0.51 cm]), but not in HbA1c level (RR 0.99 [95% CI 0.96-1.02]), or other cardio-metabolic markers. None of the outcomes showed a significant change/deterioration over the maintenance period. However, only the intervention effect for MVPA remained statistically significant at 24 months. CONCLUSIONS: The modest improvements in weight loss and behavior change, but the lack of changes in cardio-metabolic markers, may limit the utility, scalability, and sustainability of such an approach.

Study of reproducibility of human arterial plaque reconstruction and its effects on stress analysis based on multispectral in vivo magnetic resonance imaging

Purpose: To quantify the uncertainties of carotid plaque morphology reconstruction based on patient-specific multispectral in vivo magnetic resonance imaging (MRI) and their impacts on the plaque stress analysis. Materials and Methods: In this study, three independent investigators were invited to reconstruct the carotid bifurcation with plaque based on MR images from two subjects to study the geometry reconstruction reproducibility. Finite element stress analyses were performed on the carotid bifurcations, as well as the models with artificially modified plaque geometries to mimic the image segmentation uncertainties, to study the impacts of the uncertainties to the stress prediction. Results: Plaque reconstruction reproducibility was generally high in the study. The uncertainties among interobservers are around one or the subpixel level. It also shows that the predicted stress is relatively less sensitive to the arterial wall segmentation uncertainties, and more affected by the accuracy of lipid region definition. For a model with lipid core region artificially increased by adding one pixel on the lipid region boundary, it will significantly increase the maximum Von Mises Stress in fibrous cap (>100%) compared with the baseline model for all subjects. Conclusion: The current in vivo MRI in the carotid plaque could provide useful and reliable information for plaque morphology. The accuracy of stress analysis based on plaque geometry is subject to MRI quality. The improved resolution/quality in plaque imaging with newly developed MRI protocols would generate more realistic stress predictions.

Impact of calcification and intraluminal thrombus on the computed wall stresses of abdominal aortic aneurysm

Background: Increased biomechanical stresses within the abdominal aortic aneurysm (AAA) wall contribute to its rupture. Calcification and intraluminal thrombus can be commonly found in AAAs, but the relationship between calcification/intraluminal thrombus and AAA wall stress is not completely described. Methods: Patient-specific three-dimensional AAA geometries were reconstructed from computed tomographic images of 20 patients. Structural analysis was performed to calculate the wall stresses of the 20 AAA models and their altered models when calcification or intraluminal thrombus was not considered. A nonlinear large-strain finite element method was used to compute the wall stress distribution. The relationships between wall stresses and volumes of calcification and intraluminal thrombus were sought. Results: Maximum stress was not correlated with the percentage of calcification, and was negatively correlated with the percentage of intraluminal thrombus (r = -0.56; P = .011). Exclusion of calcification from analysis led to a significant decrease in maximum stress by a median of 14% (range, 2%-27%; P < .01). When intraluminal thrombus was eliminated, maximum stress increased significantly by a median of 24% (range, 5%-43%; P < .01). Conclusion: The presence of calcification increases AAA peak wall stress, suggesting that calcification decrease the biomechanical stability of AAA. In contrast, intraluminal thrombus reduces the maximum stress in AAA. Calcification and intraluminal thrombus should both be considered in the evaluation of wall stress for risk assessment of AAA rupture.

Overfitting Bayesian mixture models with an unknown number of components

This paper proposes solutions to three issues pertaining to the estimation of finite mixture models with an unknown number of components: the non-identifiability induced by overfitting the number of components, the mixing limitations of standard Markov Chain Monte Carlo (MCMC) sampling techniques, and the related label switching problem. An overfitting approach is used to estimate the number of components in a finite mixture model via a Zmix algorithm. Zmix provides a bridge between multidimensional samplers and test based estimation methods, whereby priors are chosen to encourage extra groups to have weights approaching zero. MCMC sampling is made possible by the implementation of prior parallel tempering, an extension of parallel tempering. Zmix can accurately estimate the number of components, posterior parameter estimates and allocation probabilities given a sufficiently large sample size. The results will reflect uncertainty in the final model and will report the range of possible candidate models and their respective estimated probabilities from a single run. Label switching is resolved with a computationally light-weight method, Zswitch, developed for overfitted mixtures by exploiting the intuitiveness of allocation-based relabelling algorithms and the precision of label-invariant loss functions. Four simulation studies are included to illustrate Zmix and Zswitch, as well as three case studies from the literature. All methods are available as part of the R package Zmix, which can currently be applied to univariate Gaussian mixture models.

Epithelial-mesenchymal axis in head and neck cancer cell lines

Objectives: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumour type which necessitates multiple invitro models to attain an appreciation of its multiple subtypes. The phenomenon of epithelial-mesenchymal transition (EMT) isimportant to the development of a metastatic cancer cell phenotype being relevant to the ability of cancer cells to intravasate intovasculature and to invade tissues. The role of EMT in human papilloma virus (HPV) positive HNSCC is not well understood. Thispaper aims to characterize seven HNSCC cell lines (FaDu, SCC-25, SCC-15, CAL27, RPMI2650) including two new HPV-16positive HNSCC cell lines (UD-SCC2, 93-VU-147T) for their epithelial and mesenchymal properties. Materials and methods: A panel of HNSCC cell lines from multiple head and neck anatomical sites were profiled for basalexpression of epithelial and mesenchymal characteristics at mRNA, protein and functional levels (proliferative, migratory andinvasive properties). Furthermore, 3D spheroid forming capabilities were investigated. Results: We found that the HPV-16 positive cell line, in particular UD-SCC2 demonstrated a more invasive and mesenchymalphenotype at the molecular and functional levels suggesting HPV infection may mediate some of these cellular properties.Moreover, HPV-negative cell lines were not strictly epithelial presenting with a dynamic range of expression. Conclusions: This study presents the molecular and phenotypic diversity of HNSCC cell lines. It highlights the need formore studies in this field and a scoring system where HNSCC cell lines are ranked according to their respective epithelial andmesenchymal nature. This data will be useful to anyone modelling HNSCC behaviour, providing a molecular context which willenable them to decipher cell phenotypes and to develop therapies which block EMT progression.

Complete and interpretable conformance checking of business processes

This article presents a method for checking the conformance between an event log capturing the actual execution of a business process, and a model capturing its expected or normative execution. Given a business process model and an event log, the method returns a set of statements in natural language describing the behavior allowed by the process model but not observed in the log and vice versa. The method relies on a unified representation of process models and event logs based on a well-known model of concurrency, namely event structures. Specifically, the problem of conformance checking is approached by folding the input event log into an event structure, unfolding the process model into another event structure, and comparing the two event structures via an error-correcting synchronized product. Each behavioral difference detected in the synchronized product is then verbalized as a natural language statement. An empirical evaluation shows that the proposed method scales up to real-life datasets while producing more concise and higher-level difference descriptions than state-of-the-art conformance checking methods.