Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75(NTR) in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75NTR was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75NTR, CD24 antigens and ALDH activity (ALDEFLUOR® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

Speaker attribution of multiple telephone conversations using a complete-linkage clustering approach

In this paper we propose and evaluate a speaker attribution system using a complete-linkage clustering method. Speaker attribution refers to the annotation of a collection of spoken audio based on speaker identities. This can be achieved using diarization and speaker linking. The main challenge associated with attribution is achieving computational efficiency when dealing with large audio archives. Traditional agglomerative clustering methods with model merging and retraining are not feasible for this purpose. This has motivated the use of linkage clustering methods without retraining. We first propose a diarization system using complete-linkage clustering and show that it outperforms traditional agglomerative and single-linkage clustering based diarization systems with a relative improvement of 40% and 68%, respectively. We then propose a complete-linkage speaker linking system to achieve attribution and demonstrate a 26% relative improvement in attribution error rate (AER) over the single-linkage speaker linking approach.

Quality Assessment Matrix : A Collaborative Evaluation Using Multiple Lines of Evidence

Welcome to the Quality assessment matrix. This matrix is designed for highly qualified discipline experts to evaluate their course, major or unit in a systematic manner. The primary purpose of the Quality assessment matrix is to provide a tool that a group of academic staff at universities can collaboratively review the assessment within a course, major or unit annually. The annual review will result in you being read for an external curricula review at any point in time. This tool is designed for use in a workshop format with one, two or more academic staff, and will lead to an action plan for implementation.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

An assessment of MMP and TIMP gene expression in cell lines and stroma : tumour differences in microdissected breast cancer biopsies

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans’ ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

Phenotypic changes in artemisinin-resistant plasmodium falciparum lines in vitro: evidence for decreased sensitivity to dormancy and growth inhibition

The appearance of Plasmodium falciparum parasites with decreased in vivo sensitivity but no measurable in vitro resistance to artemisinin has raised the urgent need to characterize the artemisinin resistance phenotype. Changes in the temporary growth arrest (dormancy) profile of parasites may be one aspect of this phenotype. In this study, we investigated the link between dormancy and resistance, using artelinic acid (AL)-resistant parasites. Our results demonstrate that the AL resistance phenotype has (i) decreased sensitivity of mature-stage parasites, (ii) decreased sensitivity of the ring stage to the induction of dormancy, and (iii) a faster recovery from dormancy.

Cost-effectiveness of a telephone-delivered education programme to prevent early childhood caries in a disadvantaged area : a cohort study

Objectives Early childhood caries is a highly destructive dental disease which is compounded by the need for young children to be treated under general anaesthesia. In Australia, there are long waiting periods for treatment at public hospitals. In this paper, we examined the costs and patient outcomes of a prevention programme for early childhood caries to assess its value for government services. Design Cost-effectiveness analysis using a Markov model. Setting Public dental patients in a low socioeconomic, socially disadvantaged area in the State of Queensland, Australia. Participants Children aged 6 months to 6 years received either a telephone prevention programme or usual care. Primary and secondary outcome measures A mathematical model was used to assess caries incidence and public dental treatment costs for a cohort of children. Healthcare costs, treatment probabilities and caries incidence were modelled from 6 months to 6 years of age based on trial data from mothers and their children who received either a telephone prevention programme or usual care. Sensitivity analyses were used to assess the robustness of the findings to uncertainty in the model estimates. Results By age 6 years, the telephone intervention programme had prevented an estimated 43 carious teeth and saved £69 984 in healthcare costs per 100 children. The results were sensitive to the cost of general anaesthesia (cost-savings range £36 043–£97 298) and the incidence of caries in the prevention group (cost-savings range £59 496–£83 368) and usual care (cost-savings range £46 833–£93 328), but there were cost savings in all scenarios. Conclusions A telephone intervention that aims to prevent early childhood caries is likely to generate considerable and immediate patient benefits and cost savings to the public dental health service in disadvantaged communities.

Protocol for a randomised blocked design study using telephone and text-messaging to support cardiac patients with diabetes : a cross cultural international collaborative project

Background The prevalence of type 2 diabetes is rising internationally. Patients with diabetes have a higher risk of cardiovascular events accounting for substantial premature morbidity and mortality, and health care expenditure. Given healthcare workforce limitations, there is a need to improve interventions that promote positive self-management behaviours that enable patients to manage their chronic conditions effectively, across different cultural contexts. Previous studies have evaluated the feasibility of including telephone and Short Message Service (SMS) follow up in chronic disease self-management programs, but only for single diseases or in one specific population. Therefore, the aim of this study is to evaluate the feasibility and short-term efficacy of incorporating telephone and text messaging to support the care of patients with diabetes and cardiac disease, in Australia and in Taiwan. Methods/design A randomised controlled trial design will be used to evaluate a self-management program for people with diabetes and cardiac disease that incorporates the use of simple remote-access communication technologies. A sample size of 180 participants from Australia and Taiwan will be recruited and randomised in a one-to-one ratio to receive either the intervention in addition to usual care (intervention) or usual care alone (control). The intervention will consist of in-hospital education as well as follow up utilising personal telephone calls and SMS reminders. Primary short term outcomes of interest include self-care behaviours and self-efficacy assessed at baseline and four weeks. Discussion If the results of this investigation substantiate the feasibility and efficacy of the telephone and SMS intervention for promoting self management among patients with diabetes and cardiac disease in Australia and Taiwan, it will support the external validity of the intervention. It is anticipated that empirical data from this investigation will provide valuable information to inform future international collaborations, while providing a platform for further enhancements of the program, which has potential to benefit patients internationally.

Behavioral evaluation of eight rat lines selected for high and low anxiety-related responses

Anxiety traits can be stable and permanent characteristics of an individual across time that is less susceptible of influences by a particular situation. One way to study trait anxiety in an experimental context is through the use of rat lines, selected according to contrasting phenotypes of fear and anxiety. It is not clear whether the behavioral differences between two contrasting rat lines in one given anxiety test are also present in others paradigms of state anxiety. Here, we examine the extent to which multiple anxiety traits generalize across selected animal lines originally selected for a single anxiety trait. We review the behavioral results available in the literature of eight rat genetic models of trait anxiety - namely Maudsley Reactive and Non-reactive rats, Floripa H and L rats, Tsukuba High and Low Emotional rats, High and Low Anxiety-related rats, High and Low Ultrasonic Vocalization rats, Roman High and Low Avoidance rats, Syracuse High and Low Avoidance rats, and Carioca High and Low Conditioned Freezing rats - across 11 behavioral paradigms of innate anxiety or aversive learning frequently used in the experimental setting. We observed both convergence and divergence of behavioral responses in these selected lines across the 11 paradigms. We find that predisposition for specific anxiety traits will usually be generalized to other anxiety provoking stimuli. However this generalization is not observed across all genetic models indicating some unique trait and state interactions. Genetic models of enhanced-anxiety related responses are beginning to help define how anxiety can manifest differently depending on the underlying traits and the current environmentally induced state.

Dynamic load sharing in heavy vehicle suspensions - further analysis of the equalisation effects of larger air lines

This paper provides details on comparative testing of axle-to-chassis forces of two heavy vehicles (HVs) based on an experimental programme carried out in 2007. Dynamic forces at the air springs were measured against speed and roughness values for the test roads used. One goal of that programme was to determine whether dynamic axle-to-chassis forces could be reduced by using larger-than-standard diameter longitudinal air lines. This paper presents a portion of the methodology, analysis and results from that programme. Two analytical techniques and their results are presented. The first uses correlation coefficients of the forces between air springs and the second is a student’s t-test. These were used to determine the causality surrounding improved dynamic load sharing between heavy vehicle air springs with larger air lines installed longitudinally compared with the standard sized air lines installed on the majority of air-sprung heavy vehicles.

Pathogenicity of three RPV isolates of Barley Yellow Dwarf Virus on barley, wheat and wheat alien addition lines

Barleys with and without the Yd2 resistance factor, wheat alien addition stocks with other barley yellow dwarf virus (BYDV) resistance factors and true wheats were challenged with three Australian isolates of BYDV-RPV. Yd2 resistance was effective against two of the BYDV-RPV isolates and inoculated barleys which carry Yd2 did not develop BYD symptoms and shoot growth was not affected. However, barleys with Yd2 were susceptible to the third BYDV-RPV isolate. All barley lines inoculated with the third virus isolate developed typical BYD symptoms (yellowing), shoot growth was reduced compared to uninfected controls and virus titres determined by ELISA were high and similar in barleys with and without Yd2. In contrast, resistances from Thinopyrum intermedium and Agropyron pulcherrimum in wheat backgrounds were effective against all three BYDV-RPV isolates. Shoot growth of inoculated plants with either of these resistance factors did not differ from uninfected controls and virus titres determined by ELISA were very low.