The 'Healthy Dads, Healthy Kids' program was designed to help overweight fathers lose weight and positively influence the health behaviors of their children. The aim of the current study was to evaluate the previously established program in a community setting, in a large effectiveness trial.Stage 1 - program refinement and resource development (ii) Stage 2 - community randomized controlled trial (iii) Stage 3 - community effectiveness trial. The program will be evaluated in five Local Government Areas in the Hunter Valley Region of NSW, Australia. For the community randomized controlled trial, 50 overweight/obese men (aged 18-65 years) from one Local Government Area with a child aged between 5-12 years of age will be recruited. Families will be randomized to either the program or a 6-month wait-list control group. Fathers and their children will be assessed at baseline, post-intervention (3-months) and 6-months. Inclusion criteria are: body mass index 25-40 kg/m2; no participation in other weight loss programs during the study; pass a health-screening questionnaire; and access to a computer with Internet facilities. In the community trial, the program will be evaluated using a non-randomized, prospective design in five Local Government Areas. The exclusion criteria is body mass index < 25 kg/m2 or lack of doctor's approval. Measures will be collected at baseline, 3-, 6- and 12-months. The program involves fathers attending seven face-to-face group sessions (three with children) over 3-months. Measures: The primary outcome is fathers' weight. Secondary outcomes for both fathers and children include: waist circumference, blood pressure, resting heart rate, physical activity, sedentary behaviors and dietary intake. Father-only measures include portion size, alcohol consumption, parenting for physical activity and nutrition and parental engagement. Process evaluation will determine the fidelity, dose (delivered and received), reach, recruitment and context of the program.The Healthy Dads, Healthy Kids community trial consists of three stages: (i) As a unique approach to reducing obesity prevalence in men and improving lifestyle behaviours in children, our findings will provide important evidence relating to the translation of Healthy Dads, Healthy Kids, which will enable it to be delivered on a larger scale.ACTRN12610000608066Australia and New Zealand Clinical Trials Register (ANZCTR):  Obesity is a serious public health concern associated with numerous negative health consequences . Its preHealthy Dads, Healthy Kids (HDHK) pilot, which examined the impact of a lifestyle program targeting fathers on children's physical activity and dietary habits [As parent behaviors, attitudes, parenting styles and practices have a profound influence on children's health behaviors ,11, thery habits .The HDHK program was designed to help overweight fathers lose weight and role model positive health behaviors in order to positively influence the physical activity and dietary behaviors of their children. Preliminary studies have shown the program to be highly feasible and efficacious , with apDespite the results of the HDHK pilot study, efficacy was established in a university-based research project that was delivered by highly qualified staff in a closely monitored trial. While efficacy is an essential first step and evaluates outcomes under ideal conditions, effectiveness measures the impact of an intervention when implemented in a real-world setting and represents a more realistic evaluation of the likely intervention effect . While eTherefore, the generalizability and effectiveness of the HDHK program requires testing in an appropriate trial to determine the impact of the program when delivered by trained community-based facilitators and with longer-term follow-up. This type of translational research will maximize the public health impact and reach of the HDHK program. There is an urgent need to translate obesity prevention and treatment programs with demonstrated efficacy into real-world settings  to reducThe HDHK community trial consists of 3 major stages:Stage 1 - Program refinement and resource developmentStage 2 - Community randomized controlled trial (RCT)Stage 3 - Community effectiveness trialThe HDHK community program will be evaluated in five Local Government Areas  in the Hunter Valley Region of New South Wales, Australia. In these areas, there are high levels of employment within the mining and associated manufacturing industries. In Australia, 52% of men who work in the mining industry are engaged in shift work , which iStage 1 - Program refinement and resource development (2010)One local government area (Singleton) was targeted in order to develop and test strategies for engaging communities, promoting the program among residents, and recruiting participants. Stage 1 was also used to further refine and develop key resources for families and for facilitators. For example, the session content was extensively reviewed and updated and more participant-centered learning experiences were embedded throughout the program. These developments were informed by the process evaluation of the pilot study  and fromStage 2 - Community RCT designWith the facilitator training and resources developed, the impact of the program in a community setting will be evaluated with trained local facilitators, employing a RCT design.2) fathers (aged 18-65 years) with a child aged between 5 and 12 years of age (i.e. primary school age) will be recruited from the local communities using the following strategies: local media releases , school newsletters items, school-based presentations, fliers distributed through community notice boards and local businesses, paid advertisements in local newspapers, local networks of sports clubs, service clubs, local government, and community health facilities. Fathers will be screened for eligibility via telephone. Ethics approval has been obtained from the University of Newcastle Human Research Ethics Committee. Written informed consent will be obtained from the fathers prior to their participation as well as child assent.Overweight or obese  and randomized using a computer-based random number-producing algorithm, to intervention or control group. To ensure concealment, the sequence will be generated by a statistician and given to the project manager. Randomization will be completed by a research assistant not involved in assessments and the allocation sequence will be concealed when enrolling participants. Fathers and their children will be assessed at baseline, post-intervention (3-months) and 6-months (3-months post-intervention).Fifty fathers will be recruited from one LGA (Maitland) and randomized to either the HDHK program or a 6-month wait-list control group. Fathers (and their children) will be stratified by BMI category  a sample size of 20 fathers for each group will be needed at 6 months. Assuming a 20% attrition rate [For the RCT, based on 80% power to detect a significant weight loss difference between groups of 3 kg, assuming SD = 5  will be collected at baseline, post-intervention, and 6 and 12 months post-baseline. We aim to recruit 15-20 fathers from each area. Results from each program will be pooled and it is expected at least 200 families will participate in the community trial of the HDHK program.The aims of the program are to help fathers achieve their weight loss goals, become healthy role models, and promote healthy behaviors for their children. Table The HDHK program is based on Social Cognitive Theory (SCT)  and FamiSessions on healthy eating for families focus on various aspects of parental influence on children's dietary intake incorporating Satter's 'trust' paradigm , which sThe HDHK program targets male facilitators with relevant discipline knowledge and experience. Although experienced physical education (PE) teachers are not specifically targeted, they are ideally suited to deliver the program. PE teachers have experience in teaching children, qualifications in education, expertise in health-related areas, and access to and understanding of schools. Importantly, they have the distinct skills and expertise to safely and effectively run the practical sessions. The training workshops are 6-8 hours of face-to-face contact and are designed to cover the background information to the program, session-by-session content knowledge, and the mechanics of the program .A network of trained facilitators will be established to deliver the program in their local areas. Two facilitators will be paid to deliver the program. The lead facilitator's role is to present all materials and lead the delivery of the sessions. The co-facilitator provides a supporting role for the lead during all sessions , administrative support prior to  and post (participant feedback questionnaires) all sessions. Both facilitators are responsible for completing the facilitator session feedback (independently) and for liaising with each other regarding equipment and resource requirements for each session.A variety of data collection methods will be used to assess program impact, implementation, participation and fidelity. For the community RCT, participants will be blinded to group allocation at baseline assessment. Baseline assessments will be made 1-2 weeks before program commencement. Assessors will be trained by the same experienced researcher, and for anthropometric measurements, the protocols used are those prescribed by the International Society for the Advancement of Kinanthropometry . A summaBackground details and socio-demographic variables will be collected by questionnaire including age, marital status, occupation, gross annual family income, educational level, ethnic origin, language spoke at home, socioeconomic status (SES) and postcode. SES is based on postal code of residence using the Index of Relative Socioeconomic Advantage and Disadvantage from the Australian Bureau of Statistics census-based Socio-Economic Indexes for Areas (SEIFA) .The primary outcome measure is body weight of the fathers at 6-month follow-up. Weight will be measured with fathers wearing light clothing, without shoes on a digital scale to 0.1 kg . Weight will be recorded twice and the average of the two measures reported. The same protocol will be followed to record children's weight.2). Height will be measured to 0.3 cm using the stretch stature method and a stadiometer (VR High Speed Counter) . Height will be recorded twice and the average of the two measures reported. For children, height and weight will be used to calculate BMI (kg/m2) and age- and sex-adjusted standardized scores (z-scores) based upon the UK reference data [will be calculated using the standard equation  level with the umbilicus and (ii) at the greatest circumference between the lower costal border and the iliac crest; and in children: (i) level with the umbilicus and (ii) at the narrowest point. Each measurement will be recorded with a non-extensible steel tape . Two measures will be taken and if the measures differ by more than one centimeter, a third will be recorded. The average of the measures will be reported and a waist children .Systolic and diastolic blood pressures will be measured using a NISSEI/DS-105E digital electronic blood pressure monitor  under standardized procedures. Participants will be seated for at least five minutes before blood pressure and RHR is recorded. Blood pressure and RHR will be measured three times and the average of the three measures will be reported.Yamax SW700 pedometers  will be used to objectively measure physical activity; they are considered to be more reliable than other brands of pedometers . They haSelf-reported physical activity will be collected using a modified version  of the vAssessed in the RCT by the mother using a modified Children's Leisure Activities Study Survey (CLASS), which has been validated in children 5 - 6 and 10 - 12 year of age . In the For the eldest child in the RCT, the mother will complete a modified version of the CLASS, which has been validated in children 5 - 6 and 10 - 12 year of age . In the will be assessed in the RCT using the Australian Eating Survey (AES). AES is a 120-item semi-quantitative Food Frequency Questionnaire (FFQ), used previously in Australian youth up to 16 years  and currDietary intake for fathers in the community trials will be assessed using a modified version of the AES. Eight questions related to the key nutritional messages of the program will be drawn from the full AES survey .For the eldest participating child in the RCT, their mothers will complete the Australian Child and Adolescent Eating Survey (ACAES), developed and validated for use with Australian children, aged 10 to 16 years  and prevwill be assessed using portion size photographs from the Dietary Questionnaire for Epidemiological Studies Version 2 (DQES v2), FFQ from the Cancer Council Victoria . These pAlcohol consumption will be measured using an adaptation of the Australian Government Department of Veteran Affairs, Alcohol Use Disorders Identification Test (AUDIT) 2009 . This inThe Child Feeding Questionnaire (CFQ) is a 31-item validated questionnaire, designed for completion by parents of children aged 2 to 11 years . The CFQwill be assessed using the Parenting Strategies for Eating and Activity Scale (PEAS), which has been shown to be a valid measure of parenting strategies related to children's obesity-related behaviors . During This questionnaire uses a combination of previously validated parenting measures focusing on: (i) Parenting for physical activity , attendance (at program sessions), retention (retention rates at 6-month follow-up) and participant perceptions collected from fathers about program satisfaction via a questionnaire  that focuses on: open ended feedback of positives and negatives of the program; multiple choice questions regarding prior weight-loss experiences and reasons for joining the program; and Likert scale (5-point) questions anchored from (1) strongly disagree to (5) strongly agree regarding perceptions of program content, program structure, quality of facilitators, quality of program resources, and perceived changes made during the program. Additional information regarding the long term feasibility of the program will be obtained using a semi-structured post program phone interview and a long term process evaluation questionnaire .The project is based on the principles of community consultation and will actively seek the ideas and opinions of the communities in which the project will run, including via community forums and leadership from a community advisory panel. Community advisory panel members will represent a broad range of areas including education, local government, health, sports groups and service clubs. The communities will be consulted on the best approaches to engage and recruit participants and run the program in a sustainable manner. The community consultations will also help to identify key local individuals and organizations that can potentially assist in embedding the HDHK program in the local community. The consultations will also assist in identifying suitable local venues for the HDHK program and gaining information about local conditions which may impact on the delivery of the HDHK program (e.g. timing of delivery). Additionally, in order to draw on the strengths and resources of each community and to promote capacity building for sustainability of the program, appropriately qualified community members will be invited to train as facilitators who will conduct the program in their communities. Community consultation meetings will be held in each LGA prior to commencing the program. This will facilitate collaborative partnerships between the researchers and community, and contribute to the sustainability of the program within each community. Any interested community member will be able to attend.http://www.healthydadshealthykids.com.au which provides information for potential participants and other interested parties.Stage 1 focuses on developing training and implementation manuals that will provide step-by-step instructions on how to run the program. These resources will allow the community to continue to run the program indefinitely after the three year program has been completed. This, along with the training and implementation resources, will provide the basis for gaining support for investment in these strategies by other interested communities, and more importantly governments and health care policy makers. We have also developed a HDHK website t tests for continuous variables and chi-squared (χ2) tests for categorical variables. The significance level will be set at 0.05 for all analyses. Analyses will be performed separately for fathers and children and included all randomized participants. Generalized linear mixed models (GLM) will be fitted with an unstructured covariance structure for all primary and secondary outcomes. Differences between means and 95% confidence intervals (CIs) will be determined using GLM.Analyses will be performed using PASW Statistics 19 . Data will be presented as mean (SD) for continuous variables and counts (percentages) for categorical variables. Characteristics of completers versus dropouts will be tested using independent d [d = M1 - M2/σpooled). We will also examine a range of secondary outcomes to support the primary outcome . Additional exploratory models will be fitted to examine if the fathers who have the greatest reduction in weight are also those who have the greatest improvements in the secondary outcomes.GLM will be used to assess all outcomes for the impact of group (Intervention and control), time  and the group-by-time interaction, these three terms forming the base model. Baseline scores for subjects who dropped out at 3-months and/or 6-months will be retained consistent with an intention-to-treat (ITT) analysis. Mixed models are more robust to the biases of missing data, and provide better control of Type 1 and Type 2 errors than last observation carried forward (LOCF) in Analysis of Variance . Similard  and calcFor the community trial, the mean change between pre and post-intervention outcomes will be analyzed using paired Student's t-test when change data are normally distributed. Wilcoxon matched pairs signed rank tests will be used for non-parametric tests. Per protocol analyses for both the RCT and the community trial will be performed for those who attended at least 70% of HDHK sessions and the follow-up assessment visits.The primary aim of this study is to evaluate the impact of the HDHK program in a community setting. This study will test the previously established efficacious HDHK program  in a larTranslating research results into evidence-based community programs that are widely disseminated is a major public health challenge . The proOur community-based participatory research approach focuses on local relevance of health issues and collaborative partnerships between the researchers and community; builds on the strengths and resources of a community; and promotes capacity building. Translating efficacious interventions into community settings has the potential to make a significant public health impact. The potential public health benefits relate to our key research outcome, which is the improvement in the overall health and quality of life of participants (both fathers and children) through (a) positive changes in behaviors relating to physical activity and food intake (b) positive role modeling and engagement of fathers in their children's health and life and (c) community benefits through reductions in the burden of disease and direct/indirect health costs that are associated with poor lifestyle behaviors.The authors declare that they have no competing interests.The study chief investigators PJM, DRL, CEC, RC, RF, ADO, RCP, TB and VC were responsible for identifying the research question, design of the study, obtaining ethics approval, and the acquisition of funding and overseeing study implementation. Research assistants MDY, AM, and PhD student AL have contributed to the development of intervention materials, recruiting participants and study implementation. All authors were responsible for the drafting of this manuscript and have read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/876/prepub


The interconnections between the two pathways are still poorly understood, but they may be connected in specialized membrane domains, and specific knockouts strongly suggest that both routes have different layers of mutual control and are co-affected by drugs. With the goal of shedding light on the functional integration of the yeast sphingolipid-ergosterol (SL-E) pathway, we constructed a dynamic model of the ergosterol pathway using the guidelines of Biochemical Systems Theory (BST) . The resulting model was merged with a previous mathematical model of sphingolipid metabolism in yeast . The S-system format within BST was used for analyses of consistency, stability, and sensitivity of the SL-E model, while the GMA format was used for dynamic simulations and predictions. Model validation was accomplished by comparing predictions from the model with published results on sterol and sterol-ester dynamics in yeast. The validated model was used to predict the metabolomic dynamics of the SL-E pathway after drug treatment. Specifically, we simulated the action of drugs affecting sphingolipids in the endoplasmic reticulum and studied changes in ergosterol associated with microdomains of the plasma membrane (PM).The  Sphingolipids and sterols constitute two of the major classes of eukaryotic lipids. Although, for the most part, these two classes can be studied metabolically and functionally in isolation, there is a growing body of evidence to suggest more intimate connections between the two. In particular, the two pathways are subject to cross- and co-regulation under many conditions Because the ergosterol and sphingolipid pathways constitute complex systems, it is difficult to predict with intuition alone the effects of changes in one pathway on the operation of the other; moreover, it is difficult to predict which specific steps could provide most efficacious drug targets and what the sequelae of alterations in particular enzyme activities could be. To achieve sufficient understanding of the dynamic interactions between the two biosynthetic pathways and their regulatory effects on the formation and destruction of putative microdomains, it seems necessary to work toward reliable mathematical and computational systems models. Such models may eventually help us appreciate differences in the structure and function of membrane microdomains of normal or cancer cells, and other pathological conditions +-ATPase in vitro, perhaps these domains are also enriched in complex sphingolipids, and microdomain-associated proteins, such as solute transporters, Pma1p, and probably several glycosylphosphatidylinositols (GPI's)-linked proteins It should be noted that the existence of biological microdomains in cells has not been firmly established, in contrast to the physical demonstration of microdomains in experimental models of membranes. In yeast, the plasma membrane has at least three different domains, one containing a variety of solute transporters, another with the plasma membrane He.g., Any metabolic model of microdomain dynamics requires better insights into the biosynthesis of sphingolipids and sterols. The biosynthetic sterol route in fungi and vertebrates has been described in the literature with different names, including the mevalonate pathway The ergosterol pathway is readily divided into two functional parts, namely the heme-dependent and the heme-independent sections . The hemERG11 knockouts and strains with a non-functional Erg11p enzyme are non-viable unless exogenous ergosterol is supplied in the medium Cholesterol and ergosterol are the final products of the sterol biosynthetic routes for mammalian and yeast cells, respectively. These sterol end products are essential components of lipid membranes, and their production is tightly regulated at multiple cellular levels Saccharomyces cerevisiae and integrate it with an existing sphingolipid-glycerolipid model that was formulated with methods of Biochemical Systems Theory (BST) In this work, we construct and validate a mathematical model of the biosynthetic ergosterol pathway in A mathematical representation of the heme-dependent section of the ergosterol pathway had been established previously with methods of Metabolic Flux Analysis S6.Sterol biosynthesis is localized in the ER 14C]-acetate bolus  at the stage of steryl-esters , which we achieved by alteration of the steryl-ester synthase enzymes  (see S6 for details of this specific implementation). When the labeled palmitoyl-CoA (L12) is included as co-substrate for the formation of labeled steryl-esters, the model indicates a moderate increase in the maximal amplitude (MA) of the steryl-ester pools (simulation results not shown).As a first analysis, the ability of the model to predict dynamic changes in sterol metabolism was evaluated. Taylor and Parks conducted -inositol pulse-chase experiment X39) shows an early increase which is probably due to the contribution of the flux through Yeh2p (X182). The CS ergosterol pool (X37) increases mostly as a consequence of the external acetate label, given as a pulse-chase as in X35) is the steryl-ester with the early sustained increase, while the steryl-ester in close contact with the PM (X40) shown a latter sustained increase. The outer PM ergosterol (X36) is not represented because of its low value. The Steryl Lanosterol (X33) and Steryl Zymosterol (X34) present an initial rise and posterior plateau starting in 700 min. At the end of the simulation the Steryl Lanosterol (X33) and Steryl Zymosterol (X34) present higher values than the Steryl Ergosterols pools (X35 and X40) (dynamics not shown).S4 and S6 respectively. Table S11 presents the PLAS The SL-E model is available in PLAS X133) activity, emulating a pathway block exerted by drugs such as aureobasidin, galbonolide, or khafrefungin X133) for a 30-min external labeled acetate pulse-chase perturbation. The knockout conditions and posterior perturbations where done with one minute of difference assuming the drug have reached their enzyme site of action after that time. The comparison of In order to predict the sterol and steryl-ester dynamics when key sphingolipid biosynthetic enzymes are impaired, the effects of specific drugs affecting enzymes of the sphingolipid biosynthetic route were decreased by 99% in their specific activities. In the first analysis, we decreased IPC synthase (X37). This suggests that the vesicular co-transport of ergosterol and CS is principally responsible for the decrease in CS ergosterol (X37). The inner PM ergosterol (X39) presents an MA increase of 14% which indicates a redistribution of the ergosterol from the outer to the inner PM leaflet. Pools not predominantly affected by a decreased IPC synthase activity are: ergosterol-ester-1 (X35), ergosterol-ester-2 (X40), the endoplasmic reticulum ergosterol (X32), and the outer PM ergosterol not associated with DIMs (X36) (not shown).The comparison of ergosterol sub-populations in X157) X37) decreases by 63%. As in the case of IPC synthase inhibition, much of this decrease in the CS-associated ergosterol pool (X37) can be explained by the impaired contribution of the vesicular transport of ergosterol and CS from this pool. Fourth, there is a 43% increase in MA of ergosterol at the inner side of PM (X39), which reflects the ergosterol redistribution dynamics inside the PM when the ergosterol associated with the CS is impaired. This increase is qualitatively consistent with the increase in free ergosterol in PM under lcb1-100 conditions in Bauman's model  increased slightly  from lcb1-100 strains at restrictive temperatures by fluorescent filipin-stained sterols lcb1-100 strains at a restrictive temperature, an increase in the intracellular ergosterol as detected by the fluorescent ergosterol-binding protein filipin, but they were unable to visualize a change in the endoplasmic reticulum filipin staining pattern. In addition, they observed a slower equilibration rate for the ER-PM [3H]-ergosterol in lcb1-100 strains at both permissive and restrictive temperatures. This slower ER ergosterol (X32) recovery dynamics can also be observed in our simulation  shows a rise in the PM inner ergosterol (X39) and ER ergosterol (X32), a decrease in the PM outer ergosterol associated with the complex sphingolipids (X37), and a general slower recovery dynamics, which can be explained as a consequence of the decrease in the vesicular ergosterol flux and the increase of the non-vesicular ergosterol moving through the fluxes v32,39 and v39,32  on the total mass in the SL-E model shows a 26% and 55% decrease in the CS-associated ergosterol (X37) after 60 min and 120 min of the computational experiment, respectively (The simulation of the effects of a greatly reduced activity (1%) of SPT; (ectively .X37) after 60 and 120 minutes, respectively (not shown). In the literature a similar 23% decrease in the DIM associated ergosterol  reduction occurs in spite of the reduced vesicular flux, which does not represent more than 2% of the total ergosterol forward-backward flux .X37) and increases in the non-CS associated ergosterol sub-populations.Simulations with 1% IPC synthase activity  show resX134) was decreased to 12.5% of its basal value  present a slight but consistent increase under the reduced SPT and IPC synthase conditions .The first column from lcb-100 strain after 60 minutes at the restrictive temperature, using immunoblotting Gas1p antibodies  are the metabolites most affected in all computationally tested conditions . de novo sphingolipid synthesis that are needed for the formation of complex sphingolipids. The finding is indirectly consistent with results of labeling the lip1 mutant (ceramide synthase) 2C rather than the IPC.In general, a decrease in complex sphingolipids was observed for all simulated conditions . The comFor the first time, the sphingolipid and the ergosterol biosynthetic routes have been integrated into a single mathematical model.lcb1-100 strain, a temperature conditional mutant for the SPT, the first enzyme of the sphingolipid route.Model validation was accomplished by comparing the dynamics of sterols and sterol esters with published time courses. Under different conditions and strains, the sphingolipid-ergosterol model (SL-E) was able to simulate the experimental levels of specific ergosterol sub-populations obtained with the The validated SL-E model was used to predict the metabolomic dynamics of the sphingolipid-ergosterol pathways after drug treatment. Specifically, we simulated the effect of drugs affecting sphingolipid and sterol enzymes and studied subsequent changes in sterols and sterol esters sub-populations.Sensitivity analysis indicates that the SL-E model is robust, showing generally low sensitivities for the parameters with respect to concentrations and fluxes see , and S9.X171). Because the thiolase/synthase is a common enzyme shared with mammalian cells, only the mechanisms associated with ergosterol transport are interesting as antifungal drug targets. These ergosterol processes are in fact the target of polyenes antifungals such as Nystatin and Amphotericin B de novo synthesis of sterols and steryl-esters after perturbations with external radioactive acetate  by decreasing the SPT (X157) to 1% their basal value. This suggests that the vesicular co-transport of ergosterol and CS is principally responsible for the decrease in CS-associated ergosterol (X37). Interestingly, a similar 34% decrease for the newly synthesized cholesterol in rafts was reported in mammalian kidney cells when the vesicular transport from the Golgi compartment to the cell surface was impaired by decreasing the incubation temperature from 37°C to 15°C  acetate . Second,lcb1-100 mutant strain X37)  . This relcb1-100 DIM-associated ergosterol X37)   are bothlcb1-100 results lcb1-100 strain exhibits lower complex sphingolipid levels even at a permissive temperature It is also important to remember that the comparison of simulated SPT inhibition  with lcba) a reduction in the plasma membrane CS-ergosterol level, b) a moderate increase of the free ergosterol sub-populations, and c) slightly longer sterol recovery times after [14C]-acetate perturbations. The steryl-ester dynamics are affected only when the specific activity of SPT was decreased.As a whole, from the predictions from in silico conditions tested, such as in the lcb1-100 temperature conditional mutant.Predictions in e.g. The SL-E model is able to reproduce the very efficient homeostatic sterol balance in yeast that has been observed under different experimental conditions e.g. . The SL-14C]-acetate chase experiments for the sterol and steryl-ester pools, especially for the steryl-esters  can catalyze the incorporation of fatty-acyl chains of different lengths U-14C] potassium acetate after 30 minutes of labeling. This process raises the possibility that the chase contributes to the sharp decrease in the radioactivity incorporation rates observed at the third and fourth time points for the sterols and steryl-esters 14C]acetate pulse and the pulse-chase experiments in the studies of Taylor and Parks are not clear, and the first documented time points correspond to 250 minutes   minutes  and 566  minutes   and the Plasma Membrane Associated structures described by the Daum group was realized During the development and implementation of the SL-E model, two aspects of the sterol and steryl-esters biology were highlighted. First, in spite of the fact that the esterified pools and associated enzymes are not essential for yeast viability  for details) facilitates the identification of processes that influence the pathway more than others.  shows that there are three principal groups of sterol related metabolites with different degrees of sensitivity, namely the sterol precursors, the ergosterol sub-populations, and the steryl-ester sub-populations, of which the last group exhibits the highest sensitivities. These three blocks of sterol-related metabolites are expected to be more influenced by perturbations from the rest of the SL-E model.The scope of the SL-E model is quite broad. Foremost, it can complement experimental research as a tool aiding our understanding of yeast lipidomics under different conditions. For instance, the comprehensive sensitivity analysis of the model . An example of this type of analysis was recently executed with the sphingolipid pathway alone The model is also a valuable tool for testing hypotheses regarding the dynamics of sterols and their acylated forms after specific changes in sphingolipid and/or sterol associated enzymes from the SL-E model , which could have different properties and functions e.g., . The DIMERG10, the gene coding for acetoacetyl-CoA thiolase HMG1 and HMG2 occurs during anaerobic growth and in cells moving from log-growth to stationary phase. Specifically, HMG1 is preferentially expressed during logarithmic aerobic growth, and HMG2 is translated during the stationary phase and/or under anaerobic growth S. cerevisiae preferentially synthesizes its own ergosterol even in the presence of external sterols Preliminary model analysis implied the need to include the end product feedback regulation of ergosterol. Furthermore, it is reported that ergosterol promotes transcriptional feedback regulation upon de novo and salvage production of ceramides, as well as the formation and transport of complex sphingolipids between the Golgi and the plasma membrane. It also includes glycerolipids directly related with the sphingolipid pathway and the formation of CS from phytoceramide (X7) and dihydroceramide (X3), with phosphatidylinositol  as a cosubstrate, through the inositol phosphorylceramide synthase (X133) reactions  for the wild type and the sphingosine-phosphate lyase (X150) knockout The sphingolipid component of the SL-E model includes the eactions . Predictv32,37 and v37,32Due to the lack of specific data for the proteins associated with the vesicular transport, the SL-E model does not include a specific protein  involved in the transport of ergosterol and CS from vesicular ER to PM through the fluxes transferases and flippases), which facilitate the movement of lipids between membranes and the trans-bilayer forward-backward ER-PM ergosterol flip-flop. These flippases play an important role in maintaining ergosterol homeostasis at both sides of the membrane  because it seems that the contribution of this ergosterol pool is low due to its strong association with complex sphingolipids The SL-E model does not include a trans-bilayer inner-outer leaflet flip-flop movement for the ergosterol DIM lipid-ordered pool   see .In addition to ergosterol, the inner leaflet of the yeast plasma membrane contains other abundant lipids, such as the anionic phosphatidylinositol (PI) and phosphatidylserine (PS) and the neutral phosphatidylethanolamine (PE) To summarize these findings and assumptions, the current SL-E model represents a number of processes at the membrane, but does not account for specific structures at the inner leaflet of PM or for transmembrane proteins that could contribute to the formation of inner leaflet microdomains.X182) contributes to the ergosterol pool (X39) at the PM inner leaflet . Moreover, because the yeast population at steady-state contains the same numbers of cells growing and decaying, and because yeast cell size For the total mass simulations , and 3, v40,39, which is catalyzed by the plasma membrane hydrolase Yep2p (X182), was set to zero because Yep1p is the sole active steryl-ester hydrolase under heme-deficient conditions, while Yeh2p and Tgl1p are inactivated Specifically, the vesicular co-transport of ergosterol and CS and the fluxes of lipid particles were balanced. The flux a priori guidelines for setting up mathematical models of complex systems. In this situation, Biochemical System Theory (BST) There are no N is the stoichiometric matrix, and iν in the metabolic system. In BST, these fluxes are represented as power-law functions of the formWhere Each term contains exclusively those variables that have a direct effect on this flux. Each variable is raised to a powere.g., n equations, one for each dependent metabolite variable, and the products may also include up to m further independent variables whose concentrations remain constant over time. Typical examples are constant substrates and enzyme activities.Thus, each BST equation in the generalized mass action (GMA) format reads(3)e.g., . There aiX , is represented by a differential equation that contains the difference between all influxes and effluxes, and these are formulated as specific power-law functions, as discussed above.It is straightforward to design a GMA model from a metabolic diagram. In fact, this step can be accomplished with computer software Materials S1 for details).We therefore used our earlier GMA model of the sphingolipid-glycerolipid system For stability and sensitivity analysis we linearized this system in a logarithmic coordinate system cf. .Zymomonas mobilisMaterials S1.In order to compare our simulation results with data on sterol and steryl-ester dynamics reported in the literature X32), ergosterol associated with the inner and outer plasma membrane (PM) not in microdomains , and the CS associated ergosterol pool (X37). In S. cerevisiae, the highest ergosterol concentration is found in the PM S2 for details). One ER ergosterol sub-pool is in close contact with the PM and associated with the PAM (PM associated membrane) structures described by Pichler and collaborators X32).The ergosterol pool was subdivided into four sub-populations: ergosterol in the ER , thus leaving a smaller proportion of non-complex sphingolipid associated ergosterol (X36) at this side of the PM.Finally, Baumann and collaborators established a 9∶1 relationship between free sterol and ergosterol associated with DIMs The steryl-esters are confined to structures known as lipid particles (LP), which are composed principally of triacylglycerols, sterol-esters, and moderate quantities of triacylglycerol and ergosterol biosynthetic enzymes S2 for details).Similar to the treatment of the different ergosterol sub-populations, and assuring the correct relative concentrations between the different organelles, the steryl-esters levels where calculated from an observed linear relationship between the endoplasmic reticulum sterols X35 and X40) in a ratio of 10∶1, with the smaller pool (X40) in direct contact with the PM. The existence of LP sub-populations had been reported by different laboratories and by different approaches. First, Daum and collaborators observed two sub-populations of differently sized LPs X40) is a consequence of the high STE hydrolase steryl ester activity (X182) detected in the PM Δ/tgl1Δ double mutant strains does not efficiently mobilize steryl-esters in vivo is consistent with limited access of Yeh2p to the full intracellular LP steryl-ester pool, as is predicted from the localization and topology of Yeh2p The ergosterol ester in LP was split into two sub-populations .The major ergosterol flux moves material from the ER to other membranes through close contact of large sections of the ER with the plasma membrane and with other organelles such as the plasma membrane and the mitochondria X28) through the farnesyltransferase (X179) reaction to the formation of multiple isoprenoid metabolites, while the second flux channels ergosterol from the ER to the mitochondria and other organelles (X186). The latter flux is assumed to occur through direct ergosterol translocation, because reports Table S3 for details).Fluxes, such as the bidirectional flux between ER and LP, were estimated from the assumption of a classic Michaelis-Menten process. The calculated fluxes between ER and PM indicate a net flux toward the steryl-ester formation, which is consistent with data addressing the exponential growth phase S. cerevisiae represses expression of acetyl energy dependent transporters a of acetate). Thus, the uptake rate constant of undissociated acetate was set to 0.0022 Fig. S1 for details).In the presence of glucose, Table S4 for details). The presence of some non-zero imaginary eigenvalues permits oscillations in response to specific perturbations to the system; some of these oscillations were observed in the dynamics of some simulation results . The high percentage of sensitivities smaller than one in magnitude indicates that the vast majority of perturbations in parameters are attenuated by the model.Based on previously published models for the sphingolipid-glycerolipid pathway ts e.g., , S2. GloFigure S1Experimentally observed exponential decay trend line for external radioactive acetate. Experimental points (triangles) data adapted from Wang et al. (see X125) was replaced with radioactive acetate (L125). al. see . For all(TIF)Click here for additional data file.Figure S2SL-E model dynamics after external radioactive acetate pulse bolus perturbation. Perturbation similar to the one in X33+X34+X35+X40), Sterol (X30+X31+X32+X36+X37+X39). Ergosterol sub-populations: ergosterol in endoplasmic reticulum (X32), ergosterol steryl-ester-1 (X35), plasma membrane ergosterol in outer leaflet (X36), ergosterol associated with the complex sphingolipids (X37), plasma membrane ergosterol in inner leaflet (X39), ergosterol steryl-ester-2 (X40). (A) Wild-type condition. Dynamic simulation for steryl-esters pool and total sterols after pulse-chase bolus with labeled acetate. (B) Wild-type ergosterol and steryl-ester sub-populations. (C) Decrease to 1% in IPC synthase activity (X133). Dynamic simulation for the steryl-esters pool and total sterols after pulse-chase bolus with labeled acetate. (D) Decrease to 1% in IPC synthase activity (X133). Ergosterol and steryl-ester sub-populations. (E) Decrease to 1% in serine palmitoyl transferase activity (X157). Dynamic simulation for steryl-ester pool and total sterols after pulse-chase bolus with labeled acetate. (F) Decrease to 1% in serine palmitoyl transferase activity (X157). Ergosterol and steryl-ester sub-populations. (P) Pulse-chase bolus with labeled external acetate. Transported labeled acetate (X125) and cytoplasmic acetate (X38).(TIF)Click here for additional data file.Table S1Condensed literature information for the SL-E metabolites. Included time dependent metabolites and time independent non-enzymatic parameters.(PDF)Click here for additional data file.Table S2Condensed literature information for the SL-E enzymes.(PDF)Click here for additional data file.Table S3Incoming and outgoing fluxes at model nodes.(PDF)Click here for additional data file.Table S4Eigenvalues for the flux balanced SL-E S-system model.(PDF)Click here for additional data file.Table S5Global sensitivity statistics for the SL-E model.(PDF)Click here for additional data file.Table S6SL-E logarithmic gains (of metabolites) with magnitudes greater than one. First row and column correspond to dependent and time independent variables, respectively. The key for the variable names are given in S2. Rows not presented have only values smaller than one.(PDF)Click here for additional data file.Table S7SL-E rate constant sensitivities (of metabolites) with magnitudes greater than one. First row and column correspond to dependent variables and rate constants respectively. The key for the variable names is given in . Rows not present have only values smaller than one.(PDF)Click here for additional data file.Table S8SL-E logarithmic gains (of fluxes) with magnitudes greater than one. First row and column correspond to the fluxes and the independent variables respectively. The key for the variable names is given in S2. Rows not presented have only values smaller than one.(PDF)Click here for additional data file.Table S9SL-E rate constant sensitivities (of fluxes) with magnitudes greater than one. First row and column correspond to dependent variables and rate constants, respectively. The key for the variable names is given in S2. Rows not presented have only values smaller than one.(PDF)Click here for additional data file.Table S10Simulation of the effect of statins in the SL-E model. The total mass of ergosterol (Erg.) and complex sphingolipid (CS) sub-populations in the flux balanced SL-E model is presented under different experimental conditions, after 120 minutes. Wild type condition is represented by the basal values; the two experimental conditions correspond to: A) fold changes with respect to their corresponding basal values after decreasing the specific activity of Thiolase/Synthase and HMG Reductase to 10%, 1%, and 0.01%. B) Same conditions as in A but with an simultaneous decrease to 1% the activity of IPC Synthase (X133). Complex sphingolipids: IPC, MIPC, and M(IP)2C in Golgi compartment (Golgi CS), or plasma membrane (PM). Last row correspond at the result of divide the DIM ergosterol, and the PM CS's.(PDF)Click here for additional data file.Table S11Variables equivalence from Plas  into Matlab® .(PDF)Click here for additional data file.Equations S1GMA mass equations for pools of total masses. In these equations, a superscript indicates fluxes for the complex sphingolipids that were not represented in (PDF)Click here for additional data file.Equations S2Power-Law terms from Equations S1. In these equations, a superscript indicates fluxes for the complex sphingolipids that were not represented in (PDF)Click here for additional data file.Equations S3GMA mass balance equations for labeled pools. The structures of the mass balance equations are similar to the Equations in S8, but for clarity we use (PDF)Click here for additional data file.Equations S4Terms from Equations S3. A superscript indicates deviations from the total pool; the method automatically computes equations for the labeled fractions.(PDF)Click here for additional data file.Equations S5GMA mass balance equations for unlabeled pools. The structures of the mass balance equations are similar to the Equations in S8, but for clarity we use (PDF)Click here for additional data file.Equations S6Terms from Equations S5.(PDF)Click here for additional data file.Material S1Mathematical Framework.(DOC)Click here for additional data file.Materials S2Modifications of our former sphingolipid (SL) model.(DOC)Click here for additional data file.Material S3SL-E GMA model in Plas  format. File used for the dynamic simulations.(DOC)Click here for additional data file.Material S4SL-E flux balanced GMA and Flux aggregated model in Plas  format. Files used for total mass experiments and sensitivity analysis.(DOC)Click here for additional data file.Material S5SL-E GMA model in Matlab®  format. Files used for the dynamic simulations.(DOC)Click here for additional data file.Materials S6SL-E flux balanced GMA model in Matlab®  format. Files used for total mass experiments.(DOC)Click here for additional data file.


CLIMATE Schools Combined intervention, a universal approach to preventing substance use and mental health problems among adolescents.Anxiety, depressive and substance use disorders account for three quarters of the disability attributed to mental disorders and frequently co-occur. While programs for the prevention and reduction of symptoms associated with (i) substance use and (ii) mental health disorders exist, research is yet to determine if a combined approach is more effective. This paper describes the study protocol of a cluster randomised controlled trial to evaluate the effectiveness of the CLIMATE Schools Combined intervention; (ii) CLIMATE Schools - Substance Use; (iii) CLIMATE Schools - Mental Health, or (iv) Control .Participants will consist of approximately 8400 students aged 13 to 14-years-old from 84 secondary schools in New South Wales, Western Australia and Queensland, Australia. The schools will be cluster randomised to one of four groups; (i) The primary outcomes of the trial will be the uptake and harmful use of alcohol and other drugs, mental health symptomatology and anxiety, depression and substance use knowledge. Secondary outcomes include substance use related harms, self-efficacy to resist peer pressure, general disability, and truancy. The link between personality and substance use will also be examined.CLIMATE Schools - Substance Use, or CLIMATE Schools - Mental Health or the Control condition , we expect students who receive the CLIMATE Schools Combined intervention to show greater delays to the initiation of substance use, reductions in substance use and mental health symptoms, and increased substance use and mental health knowledge.Compared to students who receive the universal ACTRN12613000723785.This trial is registered with the Australian and New Zealand Clinical Trials registry,  Substance use, anxiety and depressive disorders account for three quarters of the disability attributed to mental disorders . The peaAn array of substance use prevention programs have existed for some time -11. HoweProblems with the implementation and dissemination of drug prevention programs are well-recognised in the drug prevention field ,21-26. FThe effectiveness of prevention programs for anxiety and depressive symptoms is also contentious. For example, ‘targeted’ prevention programs for young people at high risk of a depressive disorder, have been shown to be effective over the long term, while universal school-based programs for the prevention of depression in young people have not consistently demonstrated positive effects -38. HerePrograms to prevent and reduce anxiety symptoms have also yielded mixed results. A targeted intervention, comparing a 10 session cognitive-behavioural therapy (CBT) intervention with a waitlist control among 128  highly anxious 14 year olds, found the CBT group had half the risk of meeting criteria for an anxiety disorder compared to the control group 20% vs. 39%) at two-years follow up % at two-. HoweverThe results of a randomised controlled trial (RCT) of a ‘targeted’ version of the FRIENDS program, delivered by trained school staff, as opposed to clinicians, were disappointing in that the intervention group showed no gains . The majTaken together, the aforementioned programs show promising results and highlight the importance of implementation fidelity for the development of more effective programs to prevent and reduce substance use and symptoms of anxiety and depression. Another key point is that current programs are designed to target either substance use or mental health disorders, despite research indicating that these disorders co-occur . As suchCLIMATE Schools Combined (CSC) program, will combine the universal CLIMATE Schools - Substance Use program and the universal CLIMATE Schools - Mental Health program. This program will utilise Internet-based delivery in schools, which has been identified as an effective method to address implementation issues [The current study seeks to evaluate an innovative combined approach to prevent and decrease substance use and mental health symptoms in Australian adolescents. The proposed universal program, known as the n issues ,44CLIMATE Schools Combined program consists of both the CLIMATE Schools - Substance Use program and the CLIMATE Schools - Mental Health program. All CLIMATE Schools programs include both computer-based and manualised classroom activities. The computer-based component is completed individually via the Internet, where students are engaged through contemporary cartoon storylines that impart information about substance use, and anxiety and depressive symptoms. Students are provided with confidential login details to access the CLIMATE Schools website. The classroom activities are delivered by the teacher and aim to reinforce the learning outcomes outlined in the cartoons and allow interactive communication between students. Teachers are provided with access to all program materials including an outline of activities, implementation guidelines, links to the education syllabus and summaries for each lesson. Implementation issues are also addressed, as the program is designed to link with the Australian National Curriculum and Australian state-based Health and Physical Education syllabuses. The program is also facilitated by the Internet to maximise efficient, complete and consistent delivery, as well as ensure high implementation fidelity.The CLIMATE Schools - Substance Use program aims to reduce the use of alcohol and cannabis, Australia’s most commonly used licit (excluding caffeine) and illicit drugs [The universal it drugs . The proit drugs ,46.CLIMATE Schools - Substance Use program consists of twelve 40-minute lessons. The first six lessons focus specifically on alcohol and are delivered approximately six months prior to the remaining six lessons, which focus on cannabis.The CLIMATE Schools - Substance Use program has been established using a cluster RCT in 10 schools in Sydney, Australia (n = 764) [CLIMATE Schools program, exhibited high follow-up rates, thereby suggesting that the Internet is an effective method of delivery. Importantly, students and teachers rated the program as acceptable and enjoyable. Specifically, 100% of teachers who implemented this program in their classroom rated it as superior to other substance use prevention programs, and over 90% of students reported information delivered in this format was easy to learn and would like more school subjects to be taught using this method.The efficacy of the n = 764) ,48. ResuCLIMATE Schools - Mental Health program aims to prevent and reduce anxiety and depressive symptoms in young people. The program is designed for adolescents aged 14-15 years old, as research demonstrates that programs are effective in reducing these symptoms when delivered at this time [The universal his time ,38. ThisCLIMATE Schools- Mental Health program is currently being updated and is based on two modules from the online school education program provided by the ‘This Way Up’ clinic, titled ‘Overcoming Anxiety’ and ‘Combating Depression’. Originally six lessons each, the two modules have been condensed into the six lesson CLIMATE Schools - Mental Health program, in order to reduce content overlap and decrease time demands for participating schools. In revising the program, an experienced clinical psychologist amended the script of the online cartoon component, and illustrations were updated to reflect current youth trends and to maintain consistency with the CLIMATE Schools - Substance Use program. Focus groups were conducted with approximately 30 students from two independent schools in Sydney, Australia to ensure that the modifications accurately reflected realistic scenarios and language of Year 9 students. Once the CLIMATE Schools - Mental Health program has been modified, a number of health and educational professionals will be approached to comment on the program’s clinical and educational validity, and content will be revised accordingly.The CLIMATE Schools - Substance Use program and the CLIMATE Schools - Mental Health program.The primary objective of the CSC Study is to evaluate an innovative Internet-based prevention program for the prevention and reduction of substance use, anxiety and depressive symptoms, by combining the universal CLIMATE Schools - Substance Use program, and (3) the CLIMATE Schools - Mental Health program in relation to the following outcomes:It is hypothesised that the CSC program will be more effective than; (1) school-based Health and Physical Education as usual, (2) the 1) reducing the use and harmful use of alcohol and cannabis2) reducing overall levels of anxiety and depression3) increasing knowledge related to alcohol, cannabis anxiety and depression.Secondary aims include examining the effects of the interventions on alcohol use related harms, self-efficacy to resist peer pressure, general disability, and truancy. The link between personality and substance use, as well as mental health will also be examined.1 Ethical approval has been granted from the UNSW Human Research Ethics Committee (HC13073) and from each school.In 2012 the CSC project was funded as part of a National competitive funding round.CLIMATE Schools - Combined condition (CSC), (2) the CLIMATE Schools - Substance Use condition (CS-SU), (3) the CLIMATE Schools - Mental Health condition (CS-MH), or (4) the Control condition (CO). See Table To establish the effectiveness of the integrated CSC intervention, a cluster RCT will be conducted in 84 Australian secondary schools between 2014 and 2016. Cluster randomisation will be employed to avoid contamination of the control group with the intervention group through student communication. Participating schools will be randomly allocated to one of four groups; (1) the CLIMATE Schools programs, school participation rates for similar trials were approximately 30 per cent [To account for cluster randomisation, sample size calculations were based on recent sample size requirements developed by Heo & Leon . to deteper cent ,51. TherRalloc function in Stata, and schools will be allocated to one of the four intervention groups shown in Figure The recruitment, inclusion, and randomisation of the participants (schools and students) will commence in September 2013. A total of 270 schools will be selected from a list of all public, independent and catholic schools in NSW, QLD and WA. The Principals of these schools will be sent a letter outlining the aims of the research, school requirements, time-frames, details of the randomisation procedure and an invitation for their school to participate. School Principals will then be followed up with a further email and phone call to invite their school to participate and explain the study requirements. Following school consent, randomisation will occur using the Information and consent forms will be sent home to parents/guardians of all Year 8 students (Year 9 for QLD) aged between 13-14 years, at participating schools. Only students who receive parental consent and give consent themselves will be eligible to be involved in the study.CLIMATE Schools - Substance Use program during Year 8 (Year 9 in QLD), while aged 13-14 years old. Those students allocated to the CS-MH group will receive the CLIMATE Schools - Mental Health program during Year 9 (Year 10 in QLD), when aged 14-15 years old. Students in the CSC group will receive both the CLIMATE Schools - Substance Use and CLIMATE Schools - Mental Health programs over Year 8 and 9 (Year 9 and 10 in QLD).Following the baseline assessment, students in the CS-SU group will receive the Control group will receive their usual Health and Physical Education classes  over the three years of the intervention period. Control schools will be asked to record any substance use and/or mental health education they deliver during the year, including how many lessons and the format of such lessons. Control schools will be offered the use of the CSC program following completion of the study. See Figure Students randomised to the CLIMATE Schools program  and 18, 24 and 30 months after baseline. Schools will have the option to complete the assessments either online or by paper and pencil. Each student will be assigned a unique username and password to login to the study website and all survey data obtained is strictly confidential. Table Regardless of the condition to which schools are assigned, all students will be assessed via a self-report questionnaire at; baseline, immediately pre and post each Demographic data including gender, age, country of birth, academic performance, and truancy rates will be obtained to determine baseline equivalence of the groups.Preventure’ program [CLIMATE Schools trials [very likely’ to ‘very unlikely’, how likely it is that they will try alcohol, cannabis and any other drugs in the future.Students will be asked to rate the frequency and quantity of their alcohol consumption in standard drinks, frequency of drinking to excess (defined as having more than five standard drinks on a single occasion), age of first alcohol consumption for both a sip, full drink and drinking to excess, the maximum number of standard drinks they have consumed on one occasion, the proportion of their friends who drink and their intention to try alcohol. These questions were originally adapted from the School Health and Alcohol Harm Reduction Project (SHAHRP) ‘Patterns of Alcohol’ index  and tria program , and refs trials ,48,51,54s trials  and the s trials . This alneglected my responsibilities”, as a consequence of drinking alcohol, on a five point Likert scale ranging from ‘never’ to ‘more than 6 times’. Alcohol related knowledge will be assessed using a 16-item questionnaire originally adapted from the SHAHRP ‘Knowledge of Alcohol’ index [CLIMATE Schools programs [CLIMATE Schools trials [Alcohol related harms will be assessed using the 23-item Rutgers Alcohol Problem Index (RAPI) . Studentl’ index , and refprograms ,48,51,54s trials ,48,51,547-item scale’ (GAD-7) [The ‘Kessler 6’ scale (K6)  will be  (GAD-7) . The 3-i (GAD-7) . Quality (GAD-7) ,62. The  (GAD-7)  will be The 23-item ‘Substance Use Risk Profile Scale’ (SURPS)  will be ‘Resistive Self-Regulatory Efficacy Scale’ [The 10-item y Scale’ ,66 will CLIMATE Schools programs, students and teachers will be asked to evaluate the program. Students will be asked to indicate how acceptable, appropriate and enjoyable they found the program and to indicate how likely it is they will use the information in their own lives. Teachers will be asked to rate the program; a) overall, b) in comparison to other substance use education programs, and c) in relation to the educational quality of the program. Teachers will also be asked to indicate how easy the program was to implement, how well it held students’ attention, and how likely it is that they would use the program in the future.Upon completion of the CLIMATE Schools programs will be asked to complete a logbook, indicating which lessons and activities they completed with their class and to write down any adaptations they made to the program. To ensure completion and fidelity of the online part of the program, students are required to view the lessons in full and in order. Students are not able to access the next lesson until they have completed the previous online lesson in full.All teachers delivering the CLIMATE Schools programs. Specifically teachers will be asked about the length, content and delivery method of this education.Health and Physical Education teachers across all of the four study groups will be asked to record any substance use and/or mental health education that was delivered to students and/or their parents that was in addition to the Single-level analyses; one-way analyses of variance , Chi-square , and Mann-Whitney U-test  will be used to examine baseline equivalence and attrition between groups. Due to the multi-level and hierarchical nature of the data, mixed effects regression will be used to examine intervention by time interaction effects. To account for intracluster variance within States or schools, intervention effects will primarily be examined using hierarchical linear modelling (HLM) for normally distributed data and hierarchical generalized linear modelling using a Poisson distribution for count data. Outcome variables will be centred at post-test to allow for comparisons between groups immediately after the intervention, and growth terms will be analysed to determine the magnitude of the follow-up effects. Analyses will be conducted using the program Stata. If unconditional models reveal that less than 10% of systematic variance exists at the between State or between school level for any outcome variable, HLM will be abandoned and single-level analyses will be used . For theThe present study protocol presents the design of a cluster RCT to evaluate the effectiveness of the CSC intervention; an integrated universal program to prevent alcohol and cannabis use and symptoms of anxiety and depression among adolescents. The primary aims of the CSC study are to evaluate the effectiveness of the CSC intervention compared to universal prevention programs for substance use and anxiety/depression when delivered alone and compared to school-based Health and Physical Education as usual in relation to; a) reducing the uptake and harmful use of alcohol and other drugs, b) reducing rates of anxiety and depression, and c) increasing mental health and substance use related knowledge.The preventative focus of the CSC study on comorbid substance use and mental health symptoms in young people is a key strength of the study. To date, school prevention programs for anxiety, depression and substance use have mostly been designed to target one disorder at a time, rather than targeting all these common disorders. The development and evaluation of a combined prevention approach to mental health and substance use problems is a critical step in acknowledging the comorbidity of these problems, including common antecedents, as well as influencing policy to improve mental health and wellbeing.The CSC intervention has been designed to overcome many obstacles and limitations of current school-based preventative programs. First, the program has been specifically designed to link with the curriculum, and is thereby potentially practical, acceptable, and scalable to all schools in Australia. Furthermore, implementation issues that have hindered previous school-based programs have been addressed through the use of innovative online delivery methods, which are designed to increase program fidelity and increase student engagement.Finally, the study has been powered to allow for comparisons between all four of the intervention conditions, both nationally and within each of the three States for effect sizes as low as .15. Such a large sample will allow for the detection of even small differences between groups. This will enable the investigators to establish the efficacy of the combined program over and above the stand-alone programs, as well as normative substance use and mental health education in Physical Health and Education classes as usual.One limitation of the study is the inclusion of participant self-report measurement for the primary outcomes of substance use and mental health symptoms. The use of self-report measures introduces the possibility of over or under reporting by participants. However, self-report measures have demonstrated excellent discriminant  and predResearch has highlighted the comorbidity of anxiety, depressive and substance use disorders, yet there are no existing prevention programs which adopt a combined approach to prevent and reduce symptoms associated with these common disorders. Furthermore, while a number of preventative programs for substance use and mental health symptoms are available, many yield minimal effect sizes, are not universally feasible and do not embody effective implementation strategies. The CSC study will be the first trial, internationally, to test a combined prevention model, whilst addressing the aforementioned limitations identified in current preventive programs.The CSC intervention has the potential to reduce substance use, as well as reduce rates of anxiety and depression at higher levels than either; a) separate substance use and mental health programs and, b) traditional classroom Health and Physical Education. Importantly, this study offers the potential for a paradigm shift, such that future preventive programs and research adopts a combined approach into understanding comorbid substance use and mental health disorders.Ethics approval has been granted by the UNSW Human Research Ethics Committee (HC13073).1National Health and Medical Research Council of Australia (NHMRC) Targeted Mental Health Grant.CLIMATE Schools program in Australia. These programs are distributed not for profit. The other authors declare that they have no competing interests.MT, NN and GA are three of the developers on the CLIMATE Schools - Mental Health module. All authors were involved in study design and will be involved in data analysis and reporting of the study results. All authors read and approved the final manuscript.MT, NN, GA, TS and CC are the Chief Investigators on the CSC study NHMRC grant in Australia. LBirrell, ZT, NN and MT are responsible for ethics and clinical trial submission, recruitment of schools, and data collection. SA and NM are responsible for trail co-ordination, school recruitment and data collection in Western Australia. LH is responsible for trail co-ordination, school recruitment and data collection in Queensland. GA, LM and LBrownhill are responsible to modification of the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-244X/14/32/prepub