Ewing's Sarcoma EWS protein regulates skeletogenesis by modulation of SOX9

Ewing sarcoma is the second most common form of bone cancer in adolescents, characterized by the presence of an aberrant chimeric fusion gene EWS/FLI1. Wildtype EWS has been proposed to play a role in splicing and transcription. Currently, how these functions affect early developmental stages is unknown. To elucidate the function of EWS in early development, we analyzed an ewsa zebrafish mutant line originally isolated from an insertional mutagenesis method. We generated a Maternal Zygotic (MZ) ewsa/ewsa line because ewsa/wt and ewsa/ewsa zebrafish appear to be normal and are fertile. Alizarin Red staining revealed that there are skeletal formation defects in the lower jaw with an aberrant angle and position of the dentary and basihyal bones in adult MZ ewsa/ewsa mutants. Alcian blue staining revealed that the MZ ewsa/ewsa mutation leads to craniofacial defects with higher numbers of smaller cells with disorganized polarization compared to wt/wt zebrafish at four days post fertilization (dpf). In addition, there were reduced intervertebral discs and asymmetrical vertebrae leading to curved spines in MZ ewsa/ewsa mutants. MZ ewsa/ewsa mutants display disorganized alignment of Sox9 expressing neural crest cells at 27hpf. Because both craniofacial skeletons and vertebrae arise from Sox9 expressing cells, we hypothesized that EWS interacts with Sox9 and modulates the transcriptional regulation activity of Sox9. Co-immunoprecipitation (IP) experiment revealed that EWS interacts with SOX9. Furthermore, qPCR analysis identified that known SOX9 target genes are either upregulated (ctgfa, ctgfb, col2a1a, col2a1b) or downregulated (sox5, nog1, nog2, bmp4) in MZ ewsa/ewsa mutants compared to wt/wt zebrafish embryos. This is the first evidence for a tissue specific role of EWS in skeletogenesis and suggests a novel mechanism by which Sox9 transcriptional regulation is modulated as it directs endochondral bone and cartilage development

Ewing Sarcoma Eswa Protein Regulates Chondrogenesis of Meckel's Cartilage through Modulation of Sox9 in Zebrafish

Ewing sarcoma is the second most common skeletal (bone and cartilage) cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ) ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel’s cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf). Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b) or downregulated (sox5, nog1, nog2, and bmp4) in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP) experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel’s cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel’s cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.This manuscript was supported by the Massman Family Ewing Sarcoma Research Fund, the Sarcoma Foundation of America, P20RR016475 / P20GM103418 and P20 RR032682-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Genetic dissection of a model complex trait using the Drosophila Synthetic Population Resource

Genetic dissection of complex, polygenic trait variation is a key goal of medical and evolutionary genetics. Attempts to identify genetic variants underlying complex traits have been plagued by low mapping resolution in traditional linkage studies, and an inability to identify variants that cumulatively explain the bulk of standing genetic variation in genome-wide association studies (GWAS). Thus, much of the heritability remains unexplained for most complex traits. Here we describe a novel, freely available resource for the Drosophila community consisting of two sets of recombinant inbred lines (RILs), each derived from an advanced generation cross between a different set of eight highly inbred, completely resequenced founders. The Drosophila Synthetic Population Resource (DSPR) has been designed to combine the high mapping resolution offered by multiple generations of recombination, with the high statistical power afforded by a linkage-based design. Here, we detail the properties of the mapping panel of >1600 genotyped RILs, and provide an empirical demonstration of the utility of the approach by genetically dissecting alcohol dehydrogenase (ADH) enzyme activity. We confirm that a large fraction of the variation in this classic quantitative trait is due to allelic variation at the Adh locus, and additionally identify several previously unknown modest-effect trans-acting QTL (quantitative trait loci). Using a unique property of multiparental linkage mapping designs, for each QTL we highlight a relatively small set of candidate causative variants for follow-up work. The DSPR represents an important step toward the ultimate goal of a complete understanding of the genetics of complex traits in the Drosophila model system.This work was supported by the following NIH R01 grants: RR024862 to S.J.M. and A.D.L., GM085260 to S.J.M., GM085251 to A.D.L., GM078338 to S.S., and GM074244 to K.W.B

A pilot randomised controlled trial investigating a mindfulness-based stress reduction (MBSR) intervention in individuals with pulmonary arterial hypertension (PAH): the PATHWAYS study

Background: Pulmonary arterial hypertension (PAH) is an uncommon condition with progressive heart failure and premature death. Treatment costs up to £120,000 per patient per year, and the psychological burden of PAH is substantial. Mindfulness-based stress reduction (MBSR) is an intervention with the potential to reduce this burden, but to date, it has not been applied to people with pulmonary hypertension. We wished to determine whether a trial of MBSR for people with PAH would be feasible. Methods: A customised gentle MBSR programme of eight sessions was developed for people with physical disability due to PAH, and they were randomised to group-based MBSR or treatment as usual. The completeness of outcome measures including Beck Anxiety Index, Beck Depression Inventory and standard physical assessment at 3 months after randomisation were recorded. Health care utilisation was measured. Attendance at the sessions and the costs involved in delivering the intervention were assessed. Semi-structured interviews were conducted to explore the acceptability of the MBSR intervention and when appropriate the reasons for trial non-participation. Results: Fifty-two patients were recruited, but only 34 were randomised due to patients finding it difficult to travel to sessions. Twenty-two completed all questionnaires and attended all clinics, both routine and additional in order to collect outcomes measures. The MSBR sessions were delivered in Bristol, Cardiff and London, costing, on average, between £2234 (Cardiff) and £4128 (London) per patient to deliver. Attendance at each session averaged between two patients in Bristol and Cardiff and three in London. For those receiving treatment as usual, clinician blinding was achievable. Interviews revealed that people who attended MBSR found it interesting and helpful in managing their symptoms and minimising the psychological component of their disease. Conclusions: We found that attendance at group MBSR was poor in people with chronic PAH within the context of a trial. Achieving better MBSR intervention attendance or use of an Internet-based programme might maximise the benefit of MBSR

Supporting good practice in the provision of services to people with comorbid mental health and alcohol and other drug problems in Australia: describing key elements of good service models

<p>Abstract</p> <p>Background</p> <p>The co-occurrence of mental illness and substance use problems (referred to as "comorbidity" in this paper) is common, and is often reported by service providers as the expectation rather than the exception. Despite this, many different treatment service models are being used in the alcohol and other drugs (AOD) and mental health (MH) sectors to treat this complex client group. While there is abundant literature in the area of comorbidity treatment, no agreed overarching framework to describe the range of service delivery models is apparent internationally or at the national level. The aims of the current research were to identify and describe elements of good practice in current service models of treatment of comorbidity in Australia. The focus of the research was on models of service delivery. The research did not aim to measure the client outcomes achieved by individual treatment services, but sought to identify elements of good practice in services.</p> <p>Methods</p> <p>Australian treatment services were identified to take part in the study through a process of expert consultation. The intent was to look for similarities in the delivery models being implemented across a diverse set of services that were perceived to be providing good quality treatment for people with comorbidity problems.</p> <p>Results</p> <p>A survey was designed based on a concept map of service delivery devised from a literature review. Seventeen Australian treatment services participated in the survey, which explored the context in which services operate, inputs such as organisational philosophy and service structure, policies and procedures that guide the way in which treatment is delivered by the service, practices that reflect the way treatment is provided to clients, and client impacts.</p> <p>Conclusions</p> <p>The treatment of people with comorbidity of mental health and substance use disorders presents complex problems that require strong but flexible service models. While the treatment services included in this study reflected the diversity of settings and approaches described in the literature, the research found that they shared a range of common characteristics. These referred to: service linkages; workforce; policies, procedures and practices; and treatment.</p

Physical and Psychosocial Effects of Wii Video Game Use among Older Women

This study investigated the physical and psychosocial effect of exergaming in community dwelling older adult women. In a pilot study consisting of a six-week baseline period and a six-week intervention period, participants (N = 11, mean age = 73.5 years, SD = 9.0) played Nintendo Wii Sports twice weekly. We measured full body movements using accelerometers, and assessed psychosocial effects through end-of-study focus group meetings. There were large self-reported psychological effects related to positive changes in self perception. The game-play deepened social connections within the group and provided a basis for shared experiences with younger aged family members. Physically, the game-play showed significantly higher maximum energy expenditure (t = -4.52, p &lt; 0.05) than baseline, but no significant difference in overall energy expenditure. Findings from the quantitative data showed that Wii-play did not have substantial physical effects; nevertheless, qualitative data revealed that the participants perceived an improved sense of physical, social and psychological wellbeing

Validation of a Portable eDNA Detection Kit for Invasive Carps

Loop-mediated isothermal amplification (LAMP) is a rapid molecular detection technique that has been used as a diagnostic tool for detecting human and animal pathogens for over 20 years and is promising for detecting environmental DNA shed by invasive species. We designed a LAMP assay to detect the invasive carps, silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), black carp (Mylopharyngodon piceus), and grass carp (Ctenopharyngodon idella). To determine the sensitivity of the LAMP assay, we determined limit of detection (LOD) for each invasive carp species and compared with the performance of a grass carp quantitative PCR (qPCR) assay in LOD and in a mesocosm study. We used two grass carp densities, 3 juvenile grass carp in one mesocosm and 33 juvenile grass carp in the other. Prior to adding grass carp to the mesocosms, we added 68 kg of fathead minnows (Pimephales promelas) to each mesocosm to simulate farm ponds used for raising bait fish. We filtered 500 mL of water per sample to compare LAMP and qPCR analysis, and we collected 50 mL grab samples that were only analyzed using qPCR to gain additional data using a higher-throughput method to monitor environmental DNA (eDNA) levels throughout the study period. No eDNA for any of the four invasive carp species was detected in water collected from the mesocosms during the three days prior to adding grass carp. Forty-eight hours after grass carp addition to mesocosms, we detected grass carp eDNA in the mesocosm containing 33 grass carp using the LAMP assay. However, we failed to detect any grass carp DNA in the mesocosm containing 3 grass carp with the LAMP assay throughout the study. We analyzed the data using an occupancy model and found that the 500 mL filter samples yielded a higher eDNA capture probability than 50 mL grab samples in the mesocosm containing three grass carp but had similar eDNA capture probability in the mesocosm containing 33 grass carp. Both LAMP and qPCR reliably detected grass carp eDNA 2 days after grass carp addition, but detections were more consistent with qPCR. The LAMP assay may have utility for certain niche uses because it can be used to rapidly analyze eDNA samples and is robust to inhibition, despite having some limitations

Reporting the limits of detection and quantification for environmental DNA assays

Background: Environmental DNA (eDNA) analysis is increasingly being used to detect the presence and relative abundance of rare species, especially invasive or imperiled aquatic species. The rapid progress in the eDNA field has resulted in numerous studies impacting conservation and management actions. However, standardization of eDNA methods and reporting across the field is yet to be fully established, with one area being the calculation and interpretation of assay limit of detection (LOD) and limit of quantification (LOQ). Aims: Here, we propose establishing consistent methods for determining and reporting of LOD and LOQ for single‐species quantitative PCR (qPCR) eDNA studies. Materials & Methods/ Results: We utilize datasets from multiple cooperating laboratories to demonstrate both a discrete threshold approach and a curve‐fitting modeling approach for determining LODs and LOQs for eDNA qPCR assays. We also provide details of an R script developed and applied for the modeling method. Discussion/Conclusions: Ultimately, standardization of how LOD and LOQ are determined, interpreted, and reported for eDNA assays will allow for more informed interpretation of assay results, more meaningful interlaboratory comparisons of experiments, and enhanced capacity for assessing the relative technical quality and performance of different eDNA qPCR assays