Apple skin patterning is associated with differential expression of MYb10

Background Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown. Results Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes. Conclusions Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.

Identification and characterization of flowering genes in kiwifruit : sequence conservation and role in kiwifruit flower development

Background Flower development in kiwifruit (Actinidia spp.) is initiated in the first growing season, when undifferentiated primordia are established in latent shoot buds. These primordia can differentiate into flowers in the second growing season, after the winter dormancy period and upon accumulation of adequate winter chilling. Kiwifruit is an important horticultural crop, yet little is known about the molecular regulation of flower development. Results To study kiwifruit flower development, nine MADS-box genes were identified and functionally characterized. Protein sequence alignment, phenotypes obtained upon overexpression in Arabidopsis and expression patterns suggest that the identified genes are required for floral meristem and floral organ specification. Their role during budbreak and flower development was studied. A spontaneous kiwifruit mutant was utilized to correlate the extended expression domains of these flowering genes with abnormal floral development. Conclusions This study provides a description of flower development in kiwifruit at the molecular level. It has identified markers for flower development, and candidates for manipulation of kiwifruit growth, phase change and time of flowering. The expression in normal and aberrant flowers provided a model for kiwifruit flower development.

Model-driven service engineering in home telecare

Health care systems are highly dynamic not just due to developments and innovations in diagnosis and treatments, but also by virtue of emerging management techniques supported by modern information and communication technology. A multitude of stakeholders such as patients, nurses, general practitioners or social carers can be integrated by modeling complex interactions necessary for managing the provision and consumption of health care services. Furthermore, it is the availability of Service-oriented Architecture (SOA) that supports those integration efforts by enabling the flexible and reusable composition of autonomous, loosely-coupled and web-enabled software components. However, there is still the gap between SOA and predominantly business-oriented perspectives (e.g. business process models). The alignment of both views is crucial not just for the guided development of SOA but also for the sustainable evolution of holistic enterprise architectures. In this paper, we combine the Semantic Object Model (SOM) and the Business Process Modelling Notation (BPMN) towards a model-driven approach to service engineering. By addressing a business system in Home Telecare and deriving a business process model, which can eventually be controlled and executed by machines; in particular by composed web services, the full potential of a process-centric SOA is exploited.

Protocol : A highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 l of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

Background Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to characterise cis-regulatory sequences that are necessary for transcription activation in a complex list of co-ordinately regulated genes.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

Background Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. Methods We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). Results 15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Conclusion Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

Staurosporine augments EGF-mediated EMT in PMC42-LA cells through actin depolymerisation, focal contact size reduction and Snail1 induction : a model for cross-modulation

Background A feature of epithelial to mesenchymal transition (EMT) relevant to tumour dissemination is the reorganization of actin cytoskeleton/focal contacts, influencing cellular ECM adherence and motility. This is coupled with the transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and Zeb1/δEF1. These genes, overexpressed in breast carcinomas, are known targets of growth factor-initiated pathways, however it is less clear how alterations in ECM attachment cross-modulate to regulate these pathways. EGF induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor staurosporine (ST) induces EMT in embryonic neural epithelial cells, with F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of aPKC. Methods PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2, Zeb1/δEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin) were visualized by confocal microscopy. A public database of human breast cancers was assessed for expression of Snail1 and Snail2 in relation to outcome. Results When PMC42-LA were treated with EGF, Snail2 was the principal E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with more extreme EMT and Zeb1/δEF1 induction seen with ST+EGF. ST reduced stress fibres and focal contact size rapidly and independently of gene transcription. Gene expression analysis by MT-PCR indicated that ST repressed many genes which were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the public database of breast cancers revealed tumours exhibiting higher Snail1 expression have an increased risk of disease-recurrence. This was not seen for Snail2, and Zeb1/δEF1 showed a reverse correlation with lower expression values being predictive of increased risk. Conclusion ST in combination with EGF directed a greater EMT via actin depolymerisation and focal contact size reduction, resulting in a loosening of cell-ECM attachment along with Snail1-Zeb1/δEF1 induction. This appeared fundamentally different to the EGF-induced EMT, highlighting the multiple pathways which can regulate EMT. Our findings add support for a functional role for Snail1 in invasive breast cancer.

Regulation of ROCK1 via Notch1 during breast cancer cell migration into dense matrices

Background The behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated. Results We generated high-density (HD) matrices that mimicked tumour collagen content of 20 mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion Increased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

The ENHANCES study--Enhancing Head and Neck Cancer patients' Experiences of Survivorship: study protocol for a randomized controlled trial

Background Few cancers pose greater challenges than head and neck (H&N) cancer. Residual effects following treatment include body image changes, pain, fatigue and difficulties with appetite, swallowing and speech. Depression is a common comorbidity. There is limited evidence about ways to assist patients to achieve optimal adjustment after completion of treatment. In this study, we aim to examine the effectiveness and feasibility of a model of survivorship care to improve the quality of life of patients who have completed treatment for H&N cancer. Methods This is a preliminary study in which 120 patients will be recruited. A prospective randomised controlled trial of the H&N Cancer Survivor Self-management Care Plan (HNCP) involving pre- and post-intervention assessments will be used. Consecutive patients who have completed a defined treatment protocol for H&N cancer will be recruited from two large cancer services and randomly allocated to one of three study arms: (1) usual care, (2) information in the form of a written resource or (3) the HNCP delivered by an oncology nurse who has participated in manual-based training and skill development in patient self-management support. The trained nurses will meet patients in a face-to-face interview lasting up to 60 minutes to develop an individualised HNCP, based on principles of chronic disease self-management. Participants will be assessed at baseline, 3 and 6 months. The primary outcome measure is quality of life. The secondary outcome measures include mood, self-efficacy and health-care utilisation. The feasibility of implementing this intervention in routine clinical care will be assessed through semistructured interviews with participating nurses, managers and administrators. Interviews with patients who received the HNCP will explore their perceptions of the HNCP, including factors that assisted them in achieving behavioural change. Discussion In this study, we aim to improve the quality of life of a patient population with unique needs by means of a tailored self-management care plan developed upon completion of treatment. Delivery of the intervention by trained oncology nurses is likely to be acceptable to patients and, if successful, will be a model of care that can be implemented for diverse patient populations.

Correlates of sedentary behaviours in preschool children : a review

Background Sedentary behaviour has been linked with a number of health outcomes. Preschool-aged children spend significant proportions of their day engaged in sedentary behaviours. Research into the correlates of sedentary behaviours in the preschool population is an emerging field, with most research being published since 2002. Reviews on correlates of sedentary behaviours which include preschool children have previously been published; however, none have reported results specific to the preschool population. This paper reviews articles reporting on correlates of sedentary behaviour in preschool children published between 1993 and 2009. Methods A literature search was undertaken to identify articles which examined correlates of sedentary behaviours in preschool children. Articles were retrieved and evaluated in 2008 and 2009. Results Twenty-nine studies were identified which met the inclusion criteria. From those studies, 63 potential correlates were identified. Television viewing was the most commonly examined sedentary behaviour. Findings from the review suggest that child's sex was not associated with television viewing and had an indeterminate association with sedentary behaviour as measured by accelerometry. Age, body mass index, parental education and race had an indeterminate association with television viewing, and outdoor playtime had no association with television viewing. The remaining 57 potential correlates had been investigated too infrequently to be able to draw robust conclusions about associations. Conclusions The correlates of preschool children's sedentary behaviours are multi-dimensional and not well established. Further research is required to provide a more comprehensive understanding of the influences on preschool children's sedentary behaviours to better inform the development of interventions.

Calculate excess mortality during heatwaves using Hilbert-Huang transform algorithm

Background Heatwaves could cause the population excess death numbers to be ranged from tens to thousands within a couple of weeks in a local area. An excess mortality due to a special event (e.g., a heatwave or an epidemic outbreak) is estimated by subtracting the mortality figure under ‘normal’ conditions from the historical daily mortality records. The calculation of the excess mortality is a scientific challenge because of the stochastic temporal pattern of the daily mortality data which is characterised by (a) the long-term changing mean levels (i.e., non-stationarity); (b) the non-linear temperature-mortality association. The Hilbert-Huang Transform (HHT) algorithm is a novel method originally developed for analysing the non-linear and non-stationary time series data in the field of signal processing, however, it has not been applied in public health research. This paper aimed to demonstrate the applicability and strength of the HHT algorithm in analysing health data. Methods Special R functions were developed to implement the HHT algorithm to decompose the daily mortality time series into trend and non-trend components in terms of the underlying physical mechanism. The excess mortality is calculated directly from the resulting non-trend component series. Results The Brisbane (Queensland, Australia) and the Chicago (United States) daily mortality time series data were utilized for calculating the excess mortality associated with heatwaves. The HHT algorithm estimated 62 excess deaths related to the February 2004 Brisbane heatwave. To calculate the excess mortality associated with the July 1995 Chicago heatwave, the HHT algorithm needed to handle the mode mixing issue. The HHT algorithm estimated 510 excess deaths for the 1995 Chicago heatwave event. To exemplify potential applications, the HHT decomposition results were used as the input data for a subsequent regression analysis, using the Brisbane data, to investigate the association between excess mortality and different risk factors. Conclusions The HHT algorithm is a novel and powerful analytical tool in time series data analysis. It has a real potential to have a wide range of applications in public health research because of its ability to decompose a nonlinear and non-stationary time series into trend and non-trend components consistently and efficiently.

A qualitative investigation of adolescents’ perceived mechanisms of change from a universal school-based depression prevention program

A recent meta-analysis provides evidence supporting the universal application of school-based prevention programs for adolescent depression. The mechanisms underlying such successful interventions, however, are largely unknown. We report on a qualitative analysis of 109 Grade 9 students’ beliefs about what they gained from an evidence-based depression prevention intervention, the Resourceful Adolescent Program (RAP-A). Fifty-four percent of interviewees articulated at least one specific example of program benefit. A thematic analysis of responses revealed two major themes, improved interpersonal relationships and improved self-regulation, both stronger than originally assumed. A more minor theme also emerged—more helpful cognitions. It is postulated that both improved interpersonal relationships and improved self-regulation are likely to enhance one another, and more helpful cognitions may express its contribution through enhanced self-regulation. These findings broaden our understanding of the impact of depression prevention programs, beginning to illuminate how such programs benefit participants.

STI-GMaS : an open-source environment for simulation of sexually-transmitted infections

Background Sexually-transmitted pathogens often have severe reproductive health implications if treatment is delayed or absent, especially in females. The complex processes of disease progression, namely replication and ascension of the infection through the genital tract, span both extracellular and intracellular physiological scales, and in females can vary over the distinct phases of the menstrual cycle. The complexity of these processes, coupled with the common impossibility of obtaining comprehensive and sequential clinical data from individual human patients, makes mathematical and computational modelling valuable tools in developing our understanding of the infection, with a view to identifying new interventions. While many within-host models of sexually-transmitted infections (STIs) are available in existing literature, these models are difficult to deploy in clinical/experimental settings since simulations often require complex computational approaches. Results We present STI-GMaS (Sexually-Transmitted Infections – Graphical Modelling and Simulation), an environment for simulation of STI models, with a view to stimulating the uptake of these models within the laboratory or clinic. The software currently focuses upon the representative case-study of Chlamydia trachomatis, the most common sexually-transmitted bacterial pathogen of humans. Here, we demonstrate the use of a hybrid PDE–cellular automata model for simulation of a hypothetical Chlamydia vaccination, demonstrating the effect of a vaccine-induced antibody in preventing the infection from ascending to above the cervix. This example illustrates the ease with which existing models can be adapted to describe new studies, and its careful parameterisation within STI-GMaS facilitates future tuning to experimental data as they arise. Conclusions STI-GMaS represents the first software designed explicitly for in-silico simulation of STI models by non-theoreticians, thus presenting a novel route to bridging the gap between computational and clinical/experimental disciplines. With the propensity for model reuse and extension, there is much scope within STI-GMaS to allow clinical and experimental studies to inform model inputs and drive future model development. Many of the modelling paradigms and software design principles deployed to date transfer readily to other STIs, both bacterial and viral; forthcoming releases of STI-GMaS will extend the software to incorporate a more diverse range of infections.