Extracellular matrix composition affects outgrowth of dendrites and dendritic spines on cortical neurons.

The composition of the extracellular matrix (ECM) in nervous tissue plays an important role in controlling neuronal outgrowth and synapse development. Changes in both protein and glycosaminoglycan components of the ECM occur with tissue injury and may affect neuron growth. To investigate neuron responses to alterations in fibronectin (FN), a major component of the wound ECM, we grew cortical neurons on cell-derived decellularized matrices composed of wild type FN (FN+/+) or of a mutant form of FN (FNΔ/+) from which the III13 heparin-binding site had been deleted by CRISPR-Cas 9 gene editing. The most significant effect of the mutant FN was a reduction in dendrite outgrowth. Not only were dendrites shorter on mutant FNΔ/+-collagen (COL) matrix than on wild type (FN+/+-COL) matrix, but the number of dendrites and dendritic spines per neuron and the spine densities were also dramatically reduced on FNΔ/+-COL matrices. Mass spectrometry and immunostaining identified a reduction in tenascin-C (TN-C) levels in the mutant matrix. TN-C is an ECM protein that binds to the III13 site of FN and modulates cell-matrix interactions and has been linked to dendrite development. We propose that TN-C binding to FN in the wound matrix supports dendrite and spine development during repair of damaged neural tissue. Overall, these results show that changes in ECM composition can dramatically affect elaboration of neurites and support the idea that the ECM microenvironment controls neuron morphology and connectivity.

Nucleation of fibronectin fibril assembly requires binding between heparin and the 13th type III module of fibronectin.

Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III13 module of FN, and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III13 to control FN assembly, we deleted both III13 alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII13 cells assembled fewer FN matrix fibrils and less DOC-insoluble FN matrix than wildtype cells. Little if any mutant FN matrix was assembled when purified ΔIII13 FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III13 caused the deficiency in assembly by ΔIII13 cells. Addition of heparin promoted the assembly of wildtype FN by CHO cells, but it had no effect on the assembly of ΔIII13 FN. Furthermore, heparin binding stabilized the folded conformation of III13 and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III13 and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII13 cells require both exogenous wildtype FN and heparin in the culture medium to maximize assembly site formation. Our results show that heparin-promoted growth of fibril nucleation sites is dependent on III13. We conclude that HS/heparin binds to III13 to promote and control the nucleation and development of FN fibrils.

Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites.

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.

Confident Body, Confident Child: Outcomes for Children of Parents Receiving a Universal Parenting Program to Promote Healthful Eating Patterns and Positive Body Image in Their Pre-Schoolers-An Exploratory RCT Extension.

Objective: A four-arm randomized controlled trial (RCT) conducted in Victoria, Australia, previously evaluated parent-report outcomes following Confident Body, Confident Child: a program for parents to promote healthful eating patterns and positive body image in pre-schoolers. This exploratory study evaluated data from children of parents in the trial at 18 months follow-up. Method: Participants were 89 children (58 girls, 31 boys) of parents across all RCT arms (group A: Confident Body, Confident Child (CBCC) resource + workshop, n = 27; group B: CBCC resource only, n = 26; group C: nutrition booklet, n = 18; group D: wait-list control, n = 18). Children's eating patterns, body image and weight bias were assessed via play-based interview. Results: Children of CBCC parents reported higher body esteem. Children of nutrition booklet parents reported stronger weight bias. Children of CBCC workshop parents reported lower External Eating. Discussion: This exploratory study suggests that CBCC may promote healthy eating patterns and child body image 18 months after parents receive the intervention.

An imprinted non-coding genomic cluster at 14q32 defines clinically relevant molecular subtypes in osteosarcoma across multiple independent datasets.

BACKGROUND: A microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We assessed the clinical utility of this miRNA set and their association with methylation status. METHODS: We integrated coding and non-coding RNA data from three independent annotated clinical osteosarcoma cohorts (n = 65, n = 27, and n = 25) and miRNA and methylation data from one in vitro (19 cell lines) and one clinical (NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset, n = 80) dataset. We used time-dependent receiver operating characteristic (tdROC) analysis to evaluate the clinical value of candidate miRNA profiles and machine learning approaches to compare the coding and non-coding transcriptional programs of high- and low-risk osteosarcoma tumors and high- versus low-aggressiveness cell lines. In the cell line and TARGET datasets, we also studied the methylation patterns of the MEG3 imprinting control region on 14q32 and their association with miRNA expression and tumor aggressiveness. RESULTS: In the tdROC analysis, miRNA sets on 14q32 showed strong discriminatory power for recurrence and survival in the three clinical datasets. High- or low-risk tumor classification was robust to using different microRNA sets or classification methods. Machine learning approaches showed that genome-wide miRNA profiles and miRNA regulatory networks were quite different between the two outcome groups and mRNA profiles categorized the samples in a manner concordant with the miRNAs, suggesting potential molecular subtypes. Further, miRNA expression patterns were reproducible in comparing high-aggressiveness versus low-aggressiveness cell lines. Methylation patterns in the MEG3 differentially methylated region (DMR) also distinguished high-aggressiveness from low-aggressiveness cell lines and were associated with expression of several 14q32 miRNAs in both the cell lines and the large TARGET clinical dataset. Within the limits of available CpG array coverage, we observed a potential methylation-sensitive regulation of the non-coding RNA cluster by CTCF, a known enhancer-blocking factor. CONCLUSIONS: Loss of imprinting/methylation changes in the 14q32 non-coding region defines reproducible previously unrecognized osteosarcoma subtypes with distinct transcriptional programs and biologic and clinical behavior. Future studies will define the precise relationship between 14q32 imprinting, non-coding RNA expression, genomic enhancer binding, and tumor aggressiveness, with possible therapeutic implications for both early- and advanced-stage patients.

Energy drinks, weight loss, and disordered eating behaviors.

OBJECTIVE: The present study examined energy drink consumption and relations with weight loss attempts and behaviors, body image, and eating disorders. PARTICIPANTS/METHODS: This is a secondary analysis using data from 856 undergraduate students who completed the American College Health Association-National College Health Assessment II confidentially online during February 2012. RESULTS: This study revealed that the majority reported lifetime consumption of energy drinks (68.4%) and a substantial minority (30.2%) reported past-30-day consumption. Chi-square and t test results suggest that consumption is associated with concerns about personal appearance, weight loss attempts, and disordered eating behaviors (eg, vomiting). Hierarchical logistic regressions revealed that after controlling for demographics, the relations between energy drink consumption and the act of trying to lose weight, the use of diet pills, and the use of vomiting/laxatives remained significant. CONCLUSIONS: The current findings suggest that energy drink consumption is associated with weight loss attempts, poor body image, and unhealthy weight loss behaviors. Future research should examine the prevalence of energy drink consumption for the purpose of weight loss.

Ethnic and gender differences in normative perceptions of substance use and actual use among college students.

The primary objective of this study was to examine whether perceived norms about cigarette, alcohol, and marijuana use differed from actual use by ethnicity using data from the 2011 College Health Survey (N = 974). Analyses of covariance controlling for year in college and sorority or fraternity membership indicated a higher mean discrepancy scores for all substances for minority students compared with White students. Results suggest that minorities may be at an increased risk for substance use due to their perception that the typical student is using more than they actually are. An implication is that social marketing messages may need to be tailored to recognize these discrepancies.

Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.

Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514-0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research.

MicroRNA paraffin-based studies in osteosarcoma reveal reproducible independent prognostic profiles at 14q32.

BACKGROUND: Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly because prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability. METHODS: We performed miRNA and mRNA microarray formalin-fixed, paraffin-embedded assays in 65 osteosarcoma biopsy and 26 paired post-chemotherapy resection specimens and used the only publicly available miRNA dataset, generated independently by another group, to externally validate our strongest findings (n = 29). We used supervised principal components analysis and logistic regression for survival and chemoresponse, and miRNA activity and target gene set analysis to study miRNA regulatory activity. RESULTS: Several miRNA-based models with as few as five miRNAs were prognostic independently of pathologically assessed chemoresponse (median recurrence-free survival: 59 months versus not-yet-reached; adjusted hazards ratio = 2.90; P = 0.036). The independent dataset supported the reproducibility of recurrence and survival findings. The prognostic value of the profile was independent of confounding by known prognostic variables, including chemoresponse, tumor location and metastasis at diagnosis. Model performance improved when chemoresponse was added as a covariate (median recurrence-free survival: 59 months versus not-yet-reached; hazard ratio = 3.91; P = 0.002). Most prognostic miRNAs were located at 14q32 - a locus already linked to osteosarcoma - and their gene targets display deregulation patterns associated with outcome. We also identified miRNA profiles predictive of chemoresponse (75% to 80% accuracy), which did not overlap with prognostic profiles. CONCLUSIONS: Formalin-fixed, paraffin-embedded tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA and mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for formalin-fixed, paraffin-embedded-based miRNA biomarker studies in cancer.

Substance use in a population-based clinic sample of people with first-episode psychosis.

BACKGROUND: Substance use is implicated in the cause and course of psychosis. AIMS: To characterise substance and alcohol use in an epidemiologically representative treatment sample of people experiencing a first psychotic episode in south Cambridgeshire. METHOD: Current and lifetime substance use was recorded for 123 consecutive referrals to a specialist early intervention service. Substance use was compared with general population prevalence estimates from the British Crime Survey. RESULTS: Substance use among people with first-episode psychosis was twice that of the general population and was more common in men than women. Cannabis abuse was reported in 51% of patients (n=62) and alcohol abuse in 43% (n=53). More than half (n=68, 55%) had used Class A drugs, and 38% (n=43) reported polysubstance abuse. Age at first use of cannabis, cocaine, ecstasy and amphetamine was significantly associated with age at first psychotic symptom. CONCLUSIONS: Substance misuse is present in the majority of people with first-episode psychosis and has major implications for management. The association between age at first substance use and first psychotic symptoms has public health implications.

Visuospatial learning and executive function are independently impaired in first-episode psychosis.

BACKGROUND: Demonstrating specific cognitive impairments in psychotic disorders is difficult. However, specific deficits in memory and executive functions have often been claimed. The Cambridge Neuropsychological Test Automated Battery (CANTAB) tasks of IDED attention-shifting (an executive task) and visuospatial paired associates learning (PAL, a memory task) require intact frontal and temporo-hippocampal functioning, respectively; both have been suggested as markers of disease progress in psychosis. METHOD: Seventy-one subjects with a first-episode psychosis or at-risk mental state were assessed on these two tasks during referral to a specialist service, the Cambridge-based CAMEO early intervention team. RESULTS: Performance on the two tasks was dissociated. Poor performance on the PAL test was associated with increased symptom levels and poorer global function, while failure on the IDED executive test was not found to have significant clinical associations. Duration of illness was not associated with performance on either task. CONCLUSIONS: Visuospatial PAL failure may be a marker of clinical severity at the onset of psychosis while IDED performance may reflect a more stable, trait-like impairment. Dissociated performance on the executive and associative learning tasks may reflect independent, neurally dissociated impairments that do not arise in a fixed order. This may explain some of the heterogeneity of cognitive function seen in early psychosis.