An operationally simple, convenient, and mild strategy for the synthesis of triazole-substituted titanocenes via strain-driven 1,3-dipolar cycloadditions between azide-functionalized titanocenes and cyclooctyne has been developed. It features the first synthesis of titanocenes containing azide groups. These compounds constitute ‘second-generation’ functionalized titanocene building blocks for further synthetic elaboration. Our synthesis is modular and large numbers of the complexes can in principle be prepared in short periods of time. Some of the triazole-substituted titanocenes display high cyctotoxic activity against BJAB cells. Comparison of the most active complexes allows the identification of structural features essential for biological activity. Typical examples of the reaction are summarized in With the new titanocene building blocks in hand we turned our attention to their further functionalization through the strain-driven 1,3-dipolar cycloaddition with cyclooctyne. The original conditions of Wittig , the rea3513 and 17 are obtained in high yield. This opens further interesting perspectives for the immobilization of titanocene complexes.The results demonstrate that the strain-driven 1,3-dipolar cycloaddition is a convenient and very mild route to triazole-functionalized cationic titanocenes. The yields between 75% and 92% are satisfying. It should be noted that the polyether-substituted complexes 50 values, instead of nonspecific cytotoxicity, which is usually reported as LC50 values, because cytotoxic drugs operate by specific induction of apoptosis. So we determined the AC50 values of our titanocenes, i.e., the concentrations causing specific apoptosis in 50% of lymphoma cells, counting all cells with membrane damage.One of the pertinent features of titanocenes is their cytotoxicity –5. There50 > 100 µM). Introduction of the triazole ring through 1,3-dipolar cycloaddition markedly changes the activity of our titanocenes as a function of the substitution pattern. The most active complexes are highlighted in The azide-substituted complexes showed no significant apoptosis induction (AC18, together with a ketone-substituted titanocene, displays the highest activity against the BJAB cell line of our cationic carbonyl-substituted titanocenes. Comparison of the three most active complexes also allows the identification of structural features essential for cytotoxic activity. First, a bulky substitution of the cyclopentadienyl ligand is favorable. Second, positioning of the triazol in close proximity – ortho-substitution in 16 leads to a lower AC50 value than para-substitution in 15 – of the metal center enhances the biological activity.Gratifyingly, In summary, we have devised an operationally simple, convenient, and mild strategy for the synthesis of triazole substituted titanocenes via strain-driven 1,3-dipolar cycloadditions between azide-functionalized titanocenes and cyclooctyne. It features the first synthesis of titanocenes containing azide groups. These compounds constitute functionalized ‘second-generation’ titanocene building blocks for further synthetic elaboration. Our synthesis is modular and large numbers of the complexes can in principle be prepared in short periods of time. Some of the triazole-substituted titanocenes display high cyctotoxic activity against BJAB cells. Comparison of the most active complexes allows the identification of structural features essential for biological activity.Future studies will concentrate on the preparation of neutral and of enantiomerically pure triazole-substituted titanocenes, also via metal catalyzed cycloadditions, for further increasing the biological activity of the complexes and for applications in enantioselective –55 cycliFile 1Experimental procedures and compound characterization, cytotoxicity studies.


Pernaute et al. identify miRNA-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These miRNA families are needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death. Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify microRNA (miRNA)-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we found that three miRNA families, miR-20, miR-92, and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the proapoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are primed for not only differentiation but also cell death. In the early mammalian embryo, two different states of pluripotency can be distinguished: a naïve state present in the epiblast of the preimplantation embryo and in mouse embryonic stem cells (mESCs) and a state primed for differentiation present in the epiblast of the post-implantation embryo and in epiblast stem cells (EpiSCs) . These tMicroRNAs (miRNAs) are ∼22-nucleotide (nt)-long RNAs that specifically bind to target mRNAs and impair their translation, often leading to the mRNAs’ subsequent degradation. miRNAs have an essential role in multiple processes, including development, maintenance of tissue homeostasis, and disease. Most miRNA target recognition occurs through perfect matching between the second-eighth nucleotide of the miRNA, named the miRNA seed sequence, and the 3′ untranslated region (UTR) of the target mRNA . AccordiDicer, Dgcr8, and Ago2; in all cases, gene loss results in embryonic lethality by ∼7.5 d post-coitum (dpc) (Dicer) results in a massive increase in cell death in the epiblast  . Interesum (dpc) , but at epiblast . TogetheDicer deletion in EpiSCs and embryos indicates that three of these families exert the highest impact on the transcriptome of primed pluripotent stem cells and control the apoptotic threshold in these cells by regulating the levels of expression of the proapoptotic protein BIM. Furthermore, we found that the extensive cell death in the epiblast induced by loss of miRNAs is precluded by concurrent loss of Bim, confirming that this protein is the main target of miRNAs regulating cell survival during early embryogenesis.Here, by miRNA expression profiling of mouse embryos from 5.5 to 8.5 dpc, we identify four miRNA seed families that account for the majority of miRNA expression during early development. Analysis of the effects of A number of studies have characterized the miRNA expression profile of hESCs and mESCs as well as preimplantation mouse embryos . HoweverThese studies found that the 30 most abundant miRNAs at each developmental stage accounted for 77%–85% of the total detected miRNA expression . Most of these miRNAs are expressed from just five miRNA clusters . The miRFour miRNA seed families are encoded by the clusters highly expressed in the post-implantation epiblast and therefore account for the majority of miRNA expression observed there: the miR-19, miR-20, miR-92, and miR-302 families . The miRTogether, these data show that four miRNA families found within five miRNA clusters account for most miRNA expression in the primed pluripotent stage in the mouse embryo in vivo. Interestingly, orthologous miRNA families are expressed early in development in other vertebrates, suggesting that they play a conserved role at these stages .Dicer in the control of cell survival in the epiblast at post-implantation stages  by direct differentiation from Dicerfx/fx ESCs (Dicer deletion and miRNA depletion within a short window of time (Many studies have addressed miRNA function in the naïve state of pluripotency (mostly in mESCs); however, little is known regarding the role of these regulators in the primed pluripotent state. We previously described an essential role for n stages . To stud/fx ESCs . This mo of time , the mai of time  or in th of time , indicatDicer-deleted with undeleted EpiSCs by microarray analysis. A total of 509 genes were misregulated upon miRNA loss, 261 of which were up-regulated by at least 1.2-fold in Dicer-deleted cells. The degree to which these up-regulated genes are regulated by miRNAs was analyzed using the data analysis platform Babelomics , pointing to a caspase-dependent type of cell death. Caspase-dependent cell death is known as apoptosis and can be induced by two different pathways. The mitochondrial pathway results in mitochondrial membrane depolarization and cytochrome c release to the cytosol, which triggers the activation of effector caspases (Caspase 3 and Caspase 7). In contrast, the nonmitochondrial pathway relies primarily on direct activation of effector caspases by Caspase 8 independently of the mitochondria . This was confirmed by qPCR and Western blot analysis of proapoptotic factors in Dicerfx/fx and Dicer-deleted cells . This points to increased BIM expression as the primary cause for the apoptosis induced by miRNA depletion in the primed pluripotent state.Activation of the mitochondrial apoptotic machinery mostly depends on the balance between pro- and anti-apoptotic members of the Bcl2 protein family. Various signaling pathways have a prosurvival or proapoptotic effect depending on the regulation that they exert over the balance of Bcl2 family members. In miRNA-depleted EpiSCs, we observed no difference in the activation of proapoptotic  or anti-apoptotic (STAT3 and AKT) signaling pathways or in the expression of the AKT inhibitor PTEN . Similarhttp://www.targetscan.org). We observed that the increase in BIM expression occurred as early as day 4 after the induction of Dicer deletion , which is when the levels of miRNAs decrease and the cells start dying by apoptosis . Furthermore, deletion of only one Bim allele rescued the cell survival in three out of six Dicer−/− embryos  DIG-labeled on the 3′ end following previously described methods, and TUNEL staining in embryos was performed as described elsewhere  following the manufacturer's instructions. Pools of ∼200 embryos at 5.5 dpc, 100 embryos at 6.5 dpc, 20 embryos at 7.5 dpc, and 10 embryos at 8.5 dpc from different litters were used for total RNA extraction. From 5.5 to 7.5 dpc, the whole embryo was analyzed, including the extraembryonic regions. TaqMan miRNA probes (Applied Biosystems) were used to perform miRNA qPCRs in stem cells. cDNA was synthetized with the TaqMan miRNA reverse transcription kit (Applied Biosystems), and qPCR for individual miRNAs was performed using TaqMan universal PCR master mix (Applied Biosystems). sno135 and sno202 were used to normalize miRNA expression in ESCs and EpiSCs, respectively. For the qPCR-based microarrays in embryos, total RNA samples were processed and analyzed by Geneservice using the TaqMan mouse miRNA assay set version 1.0 (Applied Biosystems). miRNA expression was normalized against sno202. It remains unclear whether miR-709 and miR-720 are indeed miRNAs  was used for protein extraction. Western blot was performed following standard procedures. Membrane and cytosolic extracts were separated following previously published methods  with minTotal RNA was isolated using RNeasy (Qiagen). SuperScript III reverse transcriptase (Invitrogen) was used to synthetize cDNA, and SYBR PCR master mix (Qiagen) was used to perform the qPCR reaction. mRNA expression levels were calculated using the comparative Ct value method, and b-Actin was used for normalization. Primer sequences and microarray analysis are detailed in the Supplemental Material.


Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response.Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway.The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity.The online version of this article (doi:10.1186/s12964-014-0080-8) contains supplementary material, which is available to authorized users. It is commonplace for a single receptor to recognize a number of different ligands and a single ligand to bind to a number of different receptors . In manyA prominent example of this type of promiscuity is the prolactin receptor (PRL-R), which uses the same set of amino acids on its extracellular domain (ECD) to bind to two different hormones: prolactin (PRL) ,3 and grWe present, here, a methodology that utilizes a novel class of affinity reagents generated by phage display selections that induce allosteric changes in a target protein that can effectively differentiate between ligands that bind to the same receptor epitope. These affinity reagents are based on the antigen-antibody fragment (Fab) antibody frameworks and are called “synthetic antibodies” or sABs . The ext10 member reduced genetic code phage display library [To generate sABs against hPRL-R, the immobilized ECD was incubated with a 10 library . The sABTo gain insight into the structural basis of the interactions between the sABs and hPRL-R, we determined the crystal structure of the receptor in complex with sAB A8. In the structure, the sAB binds to the opposite face of the ECD away from the hormone-binding site Figure a. Intera50 values for each hormone-sAB combination were determined  [Kd ~6nM) [50 values for hGH were ~5 fold higher than those for PRL   than PRLKd ~6nM) , the IC5We note that biological signaling requires the hormones to bind the ECD at both Site1 and the lower affinity Site2. Thus, it is possible that the sABs have an even more profound effect on ECD binding at Site2. Unfortunately, because of its more transient nature, it is not possible to quantitatively measure the influence of sABs on binding of the hormone at Site2. Thus, we instead quantified the effects using relevant biological readouts of the intracellular signaling mechanism mediated by the hPRL-R using T47D, a breast cancer cell-line. First, we examined the effects of sABs on hormone-induced receptor internalization, which is a ubiquitous feature of cytokine receptor activation. We tested the ability of the four sABs to block hPRL-R-mediated internalization of cy5-labeled hPRL or hGH. Fluorescence microscopy indicated that sABs A8, A9 and A10 significantly decreased or completely abolished the internalization of hPRL, whereas the control sAB A4 had no effect on hormone internalization (Figure We next examined the concentration and time-dependent effects of the sABs on hPRL-R signaling. A luciferase reporter assay  revealedi.e. hGH-bound vs. hPRL-bound, that ultimately define the function of the receptor. Therefore, the ability to utilize phage display to rationally engineer sophisticated reagents as highly selective inhibitors with defined behavior can serve as a powerful approach to investigate the diversity of structural states of cell-surface receptors and to design new drugs.The structural information gained from the A8-hPRL-R complex reveals the mechanism by which the sAB functions as an allosteric antagonist of PRL activity. The ability of a number of cytokine receptors, including hPRL-R, to bind to multiple ligands, which manifest in different biological responses, brings about important questions relating subtle changes in receptor conformation on the cell-surface to downstream effects inside the cell. Our work indicates that sABs can distinguish between such subtle differences in receptor structure,


Following the approval of lisdexamfetamine dimesylate (LDX) in several European countries for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children and adolescents with an inadequate response to methylphenidate (MPH) treatment, the aim of the present analysis was to establish the response to LDX in subgroups of patients with different ADHD medication histories.This was a post hoc subgroup analysis of data from a 7-week, European, double-blind, dose-optimized, Phase III study. Patients aged 6–17 years were randomized 1:1:1 to LDX, placebo, or osmotic-release oral system methylphenidate (OROS-MPH). OROS-MPH was included as a reference arm rather than as a direct comparator. Efficacy was assessed in patients categorized according to their ADHD medication history using the ADHD Rating Scale IV and Clinical Global Impressions-Improvement (CGI-I) scores.The difference between active drug and placebo in least-squares mean change from baseline to endpoint in ADHD Rating Scale IV total score  was similar between the overall study population  and treatment-naïve individuals  or patients previously treated with any ADHD medication . In addition, similar proportions of patients receiving active treatment were categorized as improved based on CGI-I score (CGI-I of 1 or 2) in the overall study population and among treatment-naïve individuals or patients previously treated with any ADHD medication.In these post hoc analyses, the response to LDX treatment, and to the reference treatment OROS-MPH, was similar to that observed for the overall study population in subgroups of patients categorized according to whether or not they had previously received ADHD medication. Attention-deficit/hyperactivity disorder (ADHD) is characterized by persistent symptoms of hyperactivity/impulsivity and/or inattention, and is estimated to affect approximately 5.9%–7.1% of children and adolescents worldwide.5P<0.001) and that a significantly (P<0.05) greater proportion of patients receiving LDX were classified as treatment responders, based on two of the three response criteria examined, than those receiving OROS-MPH.12Psychostimulants, including methylphenidate (MPH) and amphetamine, are recognized as effective pharmacological treatments for ADHD.The present post hoc analyses examined the impact of previous ADHD medication on the efficacy of LDX treatment in SPD489-325. Because of the recent second-line approval of LDX in several European countries, European patients being considered for LDX treatment should have received other ADHD medications, and for most this is likely to be an MPH-based formulation. Therefore, for clinicians in Europe, it is particularly important to establish the efficacy of LDX in patients who have been previously treated with ADHD medication. The aim of this analysis was to establish whether the LDX treatment response for subgroups of patients with different ADHD treatment histories was similar to that of the overall study population. This analysis did not attempt to compare the relative effects of LDX and the reference treatment OROS-MPH within these patient subgroups because the small number of patients involved would limit the robustness of such analyses to support inferential statistics.The design of study SPD489-325 has been described in detail previously.This study enrolled male and female children and adolescents (aged 6–17 years) who satisfied the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) criteria for a primary diagnosis of ADHD, and who had a baseline ADHD-RS-IV total score of 28 or higher. Medication history was obtained at baseline. Patients were excluded from the study if they had previously failed to respond, based on the investigators’ judgment, to an adequate course (dose and duration) of OROS-MPH therapy or if their current ADHD medication provided effective control of symptoms with acceptable tolerability. In addition, individuals with documented allergy, hypersensitivity, or intolerance to amphetamines or MPH were excluded. All patients attended an initial screening visit which included documentation of their lifetime history of therapies for ADHD; use of any investigational compound, clonidine, antipsychotic, anxiolytic, sedative-hypnotic or antidepressant medication within 30 days prior to screening excluded a patient from the study. The use of herbal preparations or melatonin was prohibited during the study. Continued participation in behavioral therapy was permitted, provided that the patient had been receiving the therapy for at least one month at the time of the baseline visit. With the exception of oppositional defiant disorder, this study also excluded patients having a comorbid psychiatric diagnosis with significant symptoms and individuals with a conduct disorder.Following the initial screening visit (visit 1), participants completed a washout period (3–42 days); for all previous medications, the washout period was a minimum of five times the half-life of the medication. Eligible patients were then randomized (1:1:1) at baseline (visit 0) to receive once-daily LDX , placebo, or OROS-MPH  for a 7-week, double-blind, dose-optimized evaluation period (visits 1–7). Investigators were not blinded to patients’ treatment history. The evaluation period was immediately followed by a one-week washout and a follow-up visit (visit 8). Endpoint was defined as the last on-therapy, post-randomization visit at which a valid efficacy score was obtained.The primary efficacy outcome in study SPD489-325 was the change in ADHD-RS-IV total score from baseline to endpoint.The key secondary efficacy outcome utilized the CGI scale,In these post hoc analyses, changes in ADHD-RS-IV total scores and in the percentage of patients categorized as “improved” on the basis of CGI-I scores were assessed for patients dichotomized according to previous treatment with ADHD medication (treatment-naïve or previously treated). In addition, changes in ADHD-RS-IV total scores were examined specifically in patients who had reported that they had previously received MPH treatment at any time, and in those who had reported receiving MPH treatment in the period immediately prior to randomization in SPD489-325 (defined as having received MPH treatment at any point during the 30 days prior to randomization).The safety population comprised all patients who were randomized and received at least one dose of investigational product. The full analysis set was defined as all patients who were randomized and received at least one dose of investigational product but excluded 15 patients from one site owing to violations of good clinical practice. Dosing information and efficacy outcomes were assessed in the full analysis set. Statistical analyses included the calculation of least-squares means, which were based on type III sum of squares from an analysis of covariance model for the change from baseline in ADHD-RS-IV total score, including treatment, country, and age groups as fixed effects and baseline value as a covariate. The precision of results and the similarities between subgroups were estimated based on 95% confidence intervals. Effect sizes were calculated as the difference between the least-squares mean scores for the active treatment and placebo groups, divided by the root mean square error obtained from the analysis of covariance model. Effect sizes of 0.2, 0.5, and 0.8 correspond to small, medium, and large magnitudes of effect, respectively.Patient disposition and baseline characteristics for the overall study population have been fully described previously and were similar across the three treatment groups .10 A totMean ADHD-RS-IV total scores at baseline were similar in treatment-naïve patients and those who had previously received any ADHD medication. In all subgroups of patients, mean ADHD-RS-IV total scores decreased from baseline to endpoint . Based oCompared with placebo, there were also a greater proportion of patients with an improved CGI-I score (CGI-I score of 1 or 2) at endpoint in the LDX treatment group and in the OROS-MPH reference arm, in both treatment-naïve and previously treated patients . The difMean (standard deviation) ADHD-RS-IV total scores at baseline were similar across treatment groups in treatment-naïve patients and in those who had previously received MPH, either at any time or immediately prior to randomization . The difThe different previously treated subgroups each received similar mean (standard deviation) doses of LDX  or OROS-MPH .Establishing the efficacy of LDX among previously treated patients is of clinical relevance in Europe, where LDX is approved for use for patients with ADHD who have responded inadequately to MPH. In this randomized, controlled trial, approximately half (54.8%) of patients in the study had received previous ADHD medication and, of these, the majority  had received MPH. The present post hoc analyses indicated that the response to LDX treatment was similar in subgroups of patients categorized according to whether or not they had previously received ADHD medication, and was similar to that observed for the overall study population.Several lines of evidence indicate that the outcome of treatment with one ADHD medication does not predict the success of treatment with a second.22In the present study, the response to LDX was robust in all subgroups, as indicated by the large effect sizes for the reduction in ADHD-RS-IV total score. However, effect sizes were larger in previously treated patients than among treatment-naïve individuals. Analysis of ten randomized, placebo-controlled trials assessing the efficacy of the nonstimulant ADHD medication atomoxetine in children indicated that a placebo response was more likely in treatment-naïve children than in patients who had previously been treated with ADHD medication.The OROS-MPH active reference arm was included in SPD489-325 to provide internal validation of the study design and to contextualize results. A previous post hoc analysis compared the relative benefits of LDX and OROS-MPH treatment in the overall SPD489-325 study population, finding that LDX was significantly more effective than OROS-MPH on several treatment outcomes.Because the study excluded patients who were well controlled on their current medication with acceptable tolerability, it is noteworthy that OROS-MPH was similarly effective in the overall study population and in patients who had previously received MPH therapy. One possible explanation for this is that the dosage of previous treatment was suboptimal, whereas in the present study both active treatments were dose-optimized. A second possibility is that the formulation of MPH may have differed between the study drug, OROS-MPH, and previous MPH treatment. For example, patients may previously have received short-acting MPH which has been associated with lower levels of treatment adherence than once-daily, long-acting formulations such as OROS-MPH.Full safety outcomes from this study have been published previouslyWhen interpreting the LDX treatment response in these analyses, it should be noted that study SPD489-325 was not powered to detect significant effects between subgroups and, although the subgroups examined here were based on prerandomization patient characteristics, they were not prespecified. In addition, it is unclear whether the specific inclusion and exclusion criteria relating to previous medication response/exposure would have impacted on the LDX treatment response. The short-term nature of this study and the exclusion of patients with comorbidities may limit the conclusions to be drawn from the present findings. Finally, the fact that safety outcomes were not assessed in the previously treated subgroups is another limitation of the present study.In conclusion, these post hoc analyses of a randomized, double-blind, placebo-controlled trial, which enrolled a large number of patients at multiple centers across Europe, support the use of LDX as an effective therapy for treatment-naïve children and adolescents with ADHD as well as those who have received previous ADHD medication.


There is a demand to make first-line treatments, including cognitive behavioural therapy (CBT) for adolescent anxiety disorders, more widely available. Internet-based CBT is proposed to circumvent access and availability barriers and reduce health care system costs. Recent reviews suggest more evidence is needed to establish the treatment effects of Internet-based CBT in children and adolescents and to determine related economic impacts.This pilot trial aims to collect the necessary data to inform the planning of a full-scale RCT to test the effectiveness of the Internet-based CBT program Breathe .We are conducting a 27-month, 2-arm parallel-group, pilot randomized controlled trial (RCT). Outcomes will inform the planning of a full-scale RCT aimed to test the effectiveness of Internet-based CBT with a population of adolescents with moderate to mild anxiety problems. In the pilot RCT we will: (1) define a minimal clinically important difference (MCID) for the primary outcome measure ; (2) determine a sample size for the full-scale RCT; (3) estimate recruitment and retention rates; (4) measure intervention acceptability to inform critical intervention changes; (5) determine the use of co-interventions; and (6) conduct a cost-consequence analysis to inform a cost-effectiveness analysis in the full-scale RCT. Adolescents aged 13-17 years seeking care for an anxiety complaint from a participating emergency department, mobile or school-based crisis team, or primary care clinic are being screened for interest and eligibility. Enrolled adolescents are being randomly allocated to either 8 weeks of Internet-based CBT with limited telephone and e-mail support, or a control group with access to a static webpage listing anxiety resources. Adolescents are randomly assigned using a computer generated allocation sequence. Data are being collected at baseline, treatment completion, and at a 3-month follow-up.Currently, adolescents are being enrolled in the study. Enrolment is taking place between March 2014 and February 2016; data collection will conclude May 2016. We expect that analysis and results will be available by August 2016.In many communities, the resources available for front-line anxiety treatment are outweighed by the need for care. This pilot RCT is an essential step to designing a robust RCT to evaluate the effectiveness of an Internet-based CBT program for adolescents with moderate to mild anxiety problems.Clinicaltrials.gov NCT02059226; http://clinicaltrials.gov/ct2/show/NCT02059226 (Archived by WebCite at http://www.webcitation.org/6epF8v7k4) Anxiety disorders are the most common mental disorders diagnosed in adolescence, with prevalence before age 18 reported as greater than 30% . The medCognitive behavioral therapy (CBT) as a first-line treatment for anxiety disorders in children and adolescents is well established ,21,22 anTrained mental health professionals have traditionally delivered CBT, but the structured and sequential nature of CBT translates well to computer-based delivery. Within this approach, the use of the Internet as a delivery mode provides researchers with unobtrusive ways to capture data and examine access patterns, compliance, and usability issues . InterneThree recent systematic reviews of the literature have shown that self-led, computer-based and computer-assisted CBT, in combination with in-person or therapist-guided care, are viable and effective treatment approaches for children and adolescents ,31,32. Slow to very low [While the reported treatment effects of Internet-based CBT for treating adolescent anxiety are promising ,34, publvery low ,32. Key very low ,31,32. Mvery low ,34. Revivery low ,31,32.Breathe . In the pilot RCT we will (1) define a minimal clinically important difference (MCID), as defined by adolescents, for the primary outcome measure, (2) determine a sample size for a full-scale RCT, (3) estimate recruitment and retention rates to determine the number of study sites needed, (4) measure intervention acceptability to inform critical intervention changes, (5) determine the use of co-interventions, and (6) conduct a cost-consequence analysis to inform a cost-effectiveness analysis for a full-scale RCT.We designed a pilot randomized controlled trial (RCT) that addresses several of the limitations highlighted in the literature to date. This pilot trial aims to collect the necessary data to inform the planning of a full-scale RCT to test the effectiveness of the Internet-based CBT program Breathe program), or a control  group with 8 weeks of access to a static webpage with general anxiety resources and information. Study enrolment is taking place from March 2014 to February 2016; data collection will conclude May 2016. Details of the study design are illustrated in We are conducting a 27-month, 2-arm parallel-group, pilot RCT. Eighty adolescents will be randomly assigned to either 8 weeks of Internet-based CBT with telephone and e-mail support  seeking care from a pediatric emergency department, mobile or school-based mental health team, or primary care setting that is participating as a study recruitment site. Study inclusion criteria are: (1) ages 13 to 17 years, (2) self-reported mild to moderate anxiety, (3) ability to read and write English, (4) regular access to a telephone and a computer system with high speed Internet service, and (5) ability to use a computer to interact with web material. Adolescents aged 13 to 14 years must also have a consenting parent.Adolescents are screened for study inclusion criteria via a secure online recruitment process or telephone-based recruitment phone call. Demography is collected during the screening process to characterize adolescents who meet and do not meet clinical eligibility. Screening criteria (1), (3), (4), and (5) are assessed through yes/no answers. We are using the Screen for Child Anxiety Related Emotional Disorders (SCARED)  to screeWe are also screening for risk of suicide using the Ask Suicide-Screening Questions (ASQ) . The ASQPotentially eligible participants (and their parents) can receive oral, written, and/or online information about the study. In the Canadian provinces involved in this study, there is no specified age of consent. A minor’s ability to consent or assent is decided after assessing capacity to consent. In this study, 15 to 17 year-olds will be allowed to consent to the study on their own behalf. We will require parental consent for adolescents aged 13 to 14 years. This decision was not based on any age of consent law; we want to acknowledge the role that parents have in their younger adolescent’s activities and their ability to provide perspective on their child’s potential participation in the study. Requiring parental consent will provide us with a formal mechanism to engage with parents before study enrolment. We will seek assent from adolescents aged 13 to 14 years even if they were assessed as being able to consent.Randomization takes place after informed consent/assent is obtained. Adolescents are randomly assigned using a computer-generated allocation sequence. Participants are randomly allocated to one of two treatment conditions and receive this allocation information via e-mail from a graduate student trainee affiliated with the project. The e-mail also provides login/website information to the allocated intervention. Given the study objectives, the pilot was designed as open-label.thBreathe module (post-treatment) and 3 months post-treatment (follow-up). Adolescents in the control group complete the MASC2 at baseline and 8 weeks after study enrolment via IRIS. Change in anxiety symptoms in adolescents will be measured using the total score from the Multidimensional Anxiety Scale for Children . The MASC2 is one of the most widely used self-report measures in clinical trials in adolescents with anxiety disorders. It assesses physical symptoms, social anxiety, harm avoidance, separation/panic, and total anxiety, and has excellent 3-month test-retest reliability  and exceAll secondary outcome data are collected via self-report using IRIS. The MCID will be determined for the primary outcome measure following module 8 completion (post-intervention). Adolescents allocated to the treatment condition are asked to indicate the minimum change in anxiety for which they would consider treatment participation. We will use the MCID to guide interpretation and application of outcome change in a full-scale RCT. To estimate the MCID, we will use adolescents’ global ratings of change on a 10-point Likert scale (-5 to +5), a commonly used anchor ,45.Satisfaction will be measured in adolescents allocated to the treatment condition at the end of module 8 (post-intervention) to infer intervention acceptability. A 12-item instrument developed by the research team will measure satisfaction based on the adolescent’s report of the program’s engagement and sense of safety/privacy, expectations and usefulness, communication, and technical (intervention) management. For 10 statements, a 5-point Likert response format ranging from strongly agree (score 5) to strongly disagree (score 1) will be used. Scores range from 10 to 50 with scores of 40 or greater indicating higher acceptability. Two open-ended questions to inform the larger trial will allow adolescents to identify challenges or barriers that were faced in taking part in the trial, and the extent to which adolescents used the skills learned.Treatment adherence will be measured at 8 weeks to further evaluate intervention acceptability. Adherence is measured by documenting the number of treatment modules and homework tasks completed. We will also record the number of tailored modules completed by each participant (treatment arm) and site visits (control arm). These data are being collected through IRIS.Health care resource use will be detailed for a preliminary cost-consequence analysis ,47. The We have been conservative in estimating our ability to enroll and retain participants, by assuming that 50% will refuse to participate and 50% of enrolled adolescents will drop out. A total of 160 adolescents are being invited to participate with the expectation that we will enroll 80 adolescents (40 assigned to each study arm) and retain 40 adolescents (20 per arm) by module 8 completion (post-intervention). In keeping with the standard goals of a pilot RCT, a sample size of 40 adolescents at post-intervention (20 per arm) will provide sufficient data on the primary outcome to calculate a sample size based on 90% power for a full-scale RCT ,49.Breathe program consists of 8 core CBT treatment modules , (6) contingency management (examining the function of anxiety from a reinforcement perspective) and modeling (viewing videos of others confronting feared situations), and (7) skills for maintenance and relapse prevention. Animations, embedded video, audio playback, graphic novel style vignettes, image maps, timed prompts and on screen pop-ups provide an interactive and multimodal experience to promote sustained engagement with the treatment content across modules.Consistent with published CBT treatment recommendations ,21,22, tAsk the Expert feature where they can e-mail a trained research team member with questions regarding the program and/or treatment. Breathe successfully underwent iterative usability testing cycles with five clinicians and five adolescents prior to the pilot study to ensure adherence to CBT therapeutic principles, developmental appropriateness for the target age group, satisfaction, and engagement with the multi-media content.Adolescents are instructed at the beginning of the trial to use the program weekly. Adolescents who do not log in for one week receive an e-mail encouraging them to log in to complete their weekly module. During the program, adolescents have access to an Breathe is delivered via the Internet-based platform IRIS, which supports the integration of persuasive design mechanisms [Breathe personalizes the program for adolescents via three primary mechanisms: tailoring content ; self-monitoring ; and automated suggestions/reminders . For example, an adolescent who indicates issues with alcohol or other drug use would receive material on these topics, while an adolescent who indicates alcohol or other drug use is not an issue would not. Breathe also engages the adolescent’s parent(s) by sending e-mails with educational materials about the nature of adolescent anxiety and highlights of key topics their child is working on in the program. Adolescents are given the choice as to whether they want parents to receive these e-mail updates.chanisms  for Intecheck-in and check-out whereby they answer questions related to anxiety symptoms and indicate whether they are having thoughts of self-harm or harming others. This check-in/check-out is a typical clinical feature of CBT programs. The research team monitors the adolescents’ answers through automated indicators built into the IRIS program. If IRIS flags a safety issue , a research team member contacts the adolescent and their parent(s) by phone follow-up within 36 hours and directs them to emergency services if the adolescent requires more immediate care. Additionally, the adolescent is automatically directed onscreen to a safety video on self-harm and a pop-up box appears in the program encouraging the adolescent to notify a parent/guardian of their thoughts and to seek immediate help. Access to the Ask the Expert feature in Breathe and e-mail contact also allow adolescents to identify distressing issues that may be activated during treatment. Such adolescents are re-assessed to determine if continuing in the study is advisable. Those adolescents not able to use the program independently or who are struggling with additional mental health issues may be referred to a higher level of care. Any serious adverse event will be reported to the institutional ethics board according to the standard operating procedures. Adolescents randomized to the control group are provided with contact information for local emergency resources .The risks for adolescents participating in this pilot trial are considered minimal, but those in the treatment group are monitored each time they start and finish a module in the intervention program, to assess clinical functioning. Adolescents complete a Breathe program at the end of their 8-week control group participation. At that time, the study will follow the same processes to monitor safety and support adolescents who may require additional help, as detailed above.Adolescents assigned to the control group are provided with access to a static webpage that houses suggested anxiety-related trade publications, print-based workbooks for adolescents, and the names of organizations and websites where the adolescent might find support. There is no interactivity or personalization included in the webpage. Adolescents assigned to the control group are provided with the option to use the Adolescent reports of anxiety, intervention acceptability and adherence, use of co-interventions, recruitment and retention rates, and demography will be described by numerical summaries . The mean difference in primary outcome change scores (and SDs) from baseline to the completion of module 8 (post-intervention) will be calculated with 95% CIs. SD values from the primary outcome change scores will be used to guide the full-scale RCT sample size calculations . AdolescCurrently, adolescents are being enrolled in the study. Enrolment is taking place between March 2014 and February 2016; data collection will conclude May 2016. We expect that analysis and results will be available by August 2016.As current systems-based models of health care look to enhance local community resource capacity, stepped care options and improved access to mental health services for adolescents are needed. Recent systematic reviews have shown that self-led, computer-based and computer-assisted CBT are viable treatment approaches for adolescents, but suggest that maturation of the evidence base is needed ,31,32. TBreathe program compared to a web-based, minimal control intervention providing psychoeducation (static webpage) in reducing anxiety symptoms in adolescents with moderate to mild anxiety problems. The MCID, sample calculation, and co-intervention use determined from the pilot RCT will inform full-scale trial methodology. Our sample calculation will ensure the full-scale trial is adequately powered, and will address sample size limitations noted in the literature to date [We will use our pilot RCT results to develop a protocol for a full-scale trial. This trial will be designed to test the effectiveness of the  to date . The use to date . Our cos


MicroRNA-21 (miR-21) has been suggested to play a significant role in the prognosis of carcinoma. The recognition of novel biomarkers for the prediction of cancer outcomes is urgently required. However, the potential prognostic value of miR-21 in various types of human malignancy remains controversial. The present meta-analysis summarises and analyses the associations between miR-21 status and overall survival (OS) in a variety of tumours.Eligible published studies were identified by searching the PubMed and Chinese Biomedicine databases. The patients’ clinical characteristics and survival results were pooled, and a pooled hazard ratio (HR) with 95% confidence intervals (95% CI) was used to calculate the strength of this association. A random-effects model was adopted, and then, meta-regression and subgroup analyses were performed. In addition, an analysis of publication bias was also conducted.2 = 76.0%, p = 0.001, showing that miR-21 might be a considerable prognostic factor for poor survival in cancer patients.Twenty-seven eligible articles (including 31 studies) were identified that included survival data for 3273 patients. The pooled HR suggested that high miR-21 was clearly related to worse overall survival , with a heterogeneity measure index of IMiR-21 might be a potentially useful biomarker for predicting cancer prognosis in future clinical applications.The online version of this article (doi:10.1186/1471-2407-14-819) contains supplementary material, which is available to authorized users. MicroRNAs (miRNAs) are a class of endogenous, small (approximately 22 nucleotides), non-coding, highly conserved and single-stranded RNAs that negatively regulate mRNA and protein expression by forming base-pairs with target mRNAs and sequentially induce translational repression and mRNA cleavage , 2. MoreMiR-21 stands out as the most commonly dramatically up-regulated miRNA in both solid and haematological malignancies , and it Prognostic factors may identify subsets of patients with a worse prognosis and facilitate the selection of a more aggressive treatment strategy. The discovery of molecular biological prognostic factors would be helpful in a more accurate prediction of clinical outcome and may also reveal novel predictive factors and therapeutic targets . HoweverWe performed this meta-analysis according to the guidelines of the Meta-analysis of Observational Studies in Epidemiology group (MOOSE)  and PRISth, 2014, and no lower date limit was used. The search was performed without language restriction. We also contacted some of the authors of the identified studies to obtain some unavailable data. Reference lists from relevant primary studies and review articles were also scanned for additional relevant publications. To ensure the quality of the meta-analysis, two authors (Li Jinhui & Wang Wenjia) independently performed the search and identification according to the standardised approach, and the final selection of a study for inclusion in the meta-analysis was reached by consensus.Studies were identified via an electronic search of PubMed and Chinese Biomedicine databases using the following keywords: (microRNA-21 OR miR-21 OR miR-21 OR mir21) AND . The search ended on June 19To be eligible for inclusion, studies met the following criteria: (I) they reported research on patients with any type of carcinoma; (II) they measured the expression of miR-21 and reported the corresponding cut-off value; (III) they investigated the association between miR-21 expression and overall survival (OS); (IV) the hazard ratio (HR) for overall survival according to miR-21 status either had to be reported or could be calculated from the information presented; (V) the study sample size was higher than twenty individuals; (VI) when the same author or group reported results obtained from the same patient population in more than one article, the most recent report or the most informative one was included in this analysis to avoid overlapping between cohorts; and (VII) they used tissue samples (without any neoadjuvant therapy) obtained from surgically resected tumours and corresponding noncancerous or normal tissues for comparison.Overall survival was defined as the interval between the medical treatment and the death of patients or the last observation.All eligible publications were reviewed by two reviewers (Li Jinhui & Wang Wenjia), and they then extracted the study data based on a predefined standardised form including the characteristics of eligible studies, the baseline information of patients and the survival analysis data  according to REMARK guidelines . Disagre2 statistics, which describe the proportion of total variation in meta-analysis estimates due to between-study heterogeneity. The variation is measured from 0-100%, with increasing I2 values indicating a larger impact of between-study heterogeneity in the meta-analysis [p value was less than 0.05 and was quantified using the I2 metric  [p value less than 0.05 was considered to be statistically significant. All analyses were performed using STATA version 12.0 software .To quantitatively combine the survival data, we extracted the HRs and their 95% CIs to assess the impact of the miR-21 status on tumour prognosis. A combined HR > 1 implied a worse survival for the group with miR-21 overexpression. This negative impact of miR-21 on survival was considered statistically significant if the 95% CI for the combined HR did not overlap 1. To assess heterogeneity among the studies, we used Ianalysis . The resgeneity) . If heteA total of 288 potentially relevant citations, including 256 reports in English and 32 in Chinese, were retrieved after the initial database search using the search strategies described previously. The titles and abstracts of relevant articles were read by two authors independently. A total of 185 citations were excluded from analysis after the first screening based on abstracts or titles , leaving 103 citations for further full text evaluation. Upon further review, 73 articles were eliminated . Then after sensitive analysis as follows, three publications were removed. As a result, 27 eligible studies , 30–55 iThe basic characteristic descriptions of the 36 eligible studies are summarised in Table 2 values of 76.0%, and Figure REMARK was used a guideline rather than a scoring scale, so the assessment was a qualitative process rather than a quantitative one , signifying that the variation was due to heterogeneity rather than chance. To make a conservative estimate, a random-effect model rather than a fixed-effect model was used to account for the highly significant inter-study heterogeneity to summarise the prognostic value of miR-21 across studies. When all study populations were combined, dismal survival outcomes were observed with the overexpression of miR-21 .Highly significant heterogeneity was detected when all studies were pooled I = 76.0%,2 = 10.63%). The results showed that combined HR of the Asian population was 2.27 with I2 = 76.0%. We also tried to use other grouping terms to explore the prognostic role of miR-21, such as TNM stage, publication year, CEA  et al. However, no clinical significance could be found.Considering the substantial heterogeneity exhibited in the trials aggregated with respect to the overall survival, meta-regression and subgroup analyses were conducted to explore the heterogeneity of the covariates including the publication year, study location, number of patients, mean age, follow-up, cut-off value of miR-21, cancer category and stage (Table p =0.174) . In addition, the lack of detailed information about baseline characteristics as a result of non-standardised reporting likely contributed to diversity across the studies as well. Moreover, studies that do not extend their analysis beyond univariate survival analysis are therefore less valuable because these confounders between miR-21 and OS did not distribute equally in each group for the variations in author's clinical experience, adjustment method and the innate characteristics of different cancers. These issues contribute to inaccuracies associated with HR estimation, and therefore, the pooling of results may produce bias and heterogeneity. As a result, the diversification of adjusted factors across studies along with a statistical adjustment for the different clinicopathologic factors included in multivariate analysis might have contributed to the significant heterogeneity.In addition, traditional survival analysis techniques  rely on variable dichotomisation into high or low values or splitting variables into multiple bins. In addition, cut-off point selection itself could potentially influence the prognostic value of the proposed association . Due to p value of 0.174, the funnel plot provides some slight evidence of asymmetry between the included studies, which indicates that some epidemiological research bias exists. We attempted to minimise publication bias by performing the literature search as completely as possible using PubMed and Chinese Biomedicine databases, without language or date of publication restrictions. However, limitations still existed as the total number of included studies and the total sample size were relatively small. In addition, we did not extend the search to unpublished data that would likely include increased proportions of null results. Positive results tend to be more acceptable by journals, whereas negative results are often rejected or are not even submitted for review. As already highlighted, the negative studies reported less detailed results, making them unlikely to be evaluated. What's more, the language of publication also introduced bias because positive results tend to be published in English-language journals. Although our search was conducted without language restriction, all of the studies included in the meta-analysis were published in English. As is known to all, the line across the top of a funnel should be representative of the pooled effect.Although the Begg’s test suggested a Our study identified several limitations that must be addressed. First, inadequate sample size was a frequent problem in the studies included in our analyses, with only 15 of the 31 studies reporting outcomes from over 100 patients. While pooling data may in part address deficiencies in individual study sample size, smaller studies are more likely to generate heterogeneity, as we observed. Second, our study used data from published studies rather than individual patient data (IPD), which limited our ability to explore the potential for confounding by various demographic and clinical factors . By contrast, IPD based meta-analysis can be used to analyse all of the data in a consistent manner and includes data from unpublished studies. A careful collaborative reanalysis of the raw data from several good studies may be more valuable than a more superficial review that mixes good and poor studies . AdditioMiR-21 overexpression was found to be associated with decreased overall survival in patients with a wide variety of tumour categories in the present systematic review with meta-analysis. As this research is limited to patients who received surgical treatment without any adjuvant therapy, the miR-21 expression status is a direct outcome of cancer itself and reflected the impact of miR-21 on cancer progression. Therefore, MiR-21 expression is a potentially useful biomarker for predicting prognosis and is a promising prognostic tool to help clinicians make difficult therapeutic decisions. In addition, although we excluded studies that did not include sufficient survival data, we noted that some of them contain negative conclusions about the prognostic role of miR-21. Therefore, large adequately designed prospective studies, both observational cohorts and clinical trials that employ standard methodology, are now urgently needed to substantiate our conclusions. The exact role of miR-21 expression needs to be determined by an appropriate multivariate analysis taking into account the classical well-defined prognostic factors for each type of cancer. Additionally, international consensus is urgently required to mandate a homogeneous miR-21 assessment methodology, to enable comparisons and the combination of large, prospectively planned individual patient data meta-analyses. These types of studies may help determine if miR-21 expression might be more appropriate and better used in clinical decision-making for tumour patients.Additional file 1: Table S1: Data extraction from the eligible studies. (DOCX 41 KB)Additional file 2: Table S2: The PRISMA Guideline for reporting this systematic review and meta-analysis. (DOCX 26 KB)Additional file 3: Figure S1: Sensitivity analysis of all of the studies. (TIFF 5 MB)


Synthetic meshes are frequently used to reinforce soft tissues. The aim of this translational study is to evaluate tolerance and long-term MRI visibility of two recently developed Gadolinium-modified meshes in a rat animal model.Gadolinium-poly-ε-caprolactone (Gd-PCL) and Gadolinium-polymethylacrylate (Gd-PMA) modified meshes were implanted in Wistar rats and their tolerance was assessed daily. Inflammation and biocompatibility of the implants were assessed by histology and immunohistochemistry after 30 days post implantation. Implants were visualised by 7T and 3T MRI at day 30 and at day 90. Diffusion of Gadolinium in the tissues of the implanted animals was assessed by Inductively Coupled Plasma Mass Spectrometry.in vivo with experimental 7T as well as 3T clinical MRI. Mass spectrometry analyses showed that levels of Gadolinium in animals implanted with coated mesh were similar to those of the control group.Overall Gd-PMA coated implants were better tolerated as compared to those coated with Gd-PCL. In fact, Gd-PMA implants were characterised by a high ratio collagen I/III and good vascularisation of the integration tissues. High resolution images of the coated mesh were obtained Meshes coated with Gd-PMA are better tolerated as compared to those coated with Gd-PCL as no signs of erosion or significant inflammation were detected at 30 days post implantation. Also, Gd-PMA coated meshes were clearly visualised with both 7T and 3T MRI devices. This new technique of mesh optimisation may represent a valuable tool in soft tissue repair and management. Synthetic meshes have been routinely used for soft tissue reinforcement since their introduction in late 1950s ,3.Meshes can be classified according to their properties including pore size and fiber type , weight Those commonly used in clinical practice are made of: (i) macroporous polypropylene (PP), which is a non-absorbable material; (ii) polyester (PET); polytetrafluoroethylene (PTFE) and expanded PTFE, etc. , 7. RandIndeed, for POP repair a prevalence of 15 to 20% of short-medium-term mesh complications (more than 6 months) is reported. These include pain, discomfort, vaginal bleeding, abnormal discharge, and recurrence of the prolapse that may eventually necessitate implant removal ,11. For in vitro) and clinical (in vivo) MRI [Therefore, it is essential to optimise the mesh in order to improve tolerance and biocompatibility to the implant. Also, to improve management of complications and reduce recurrence of surgery, monitoring the position or a possible shrinkage of the implants would be a considerable advantage. Also, the possibility to visualise the mesh may be helpful for its removal following a complication. Magnetic resonance imaging (MRI) seems to be the most powerful imaging technique for the pelvic exploration ,15. Unfoivo) MRI . With thTo this purpose we used a rat animal model, which is one of the preferred animal model for mesh fascia repair research –20 and tIn vivo experiments were performed at Nîmes University Hospital. The study was approved by the appropriate Animal Research Ethics Committee (CEEA-LR-1010) and conducted according to European Union animal care guidelines (Directive 2010/63/EU). All rats were female, 9–10 weeks old and weighed more than 300 g (mean 355 g ± 40g). The animals were housed on a 12:12-hour light–dark cycle and food and water were available ad libitum. All animals were anaesthetised with 2.5% halothane (0.5 L/min) and ketamine (50 mg/kg) and all efforts were made to minimise suffering.A total of 66 Wistar rats  were used during this study; all rats were randomised to one of the 6 groups of the study see . For theMRI analyses were performed at day 30 and day 90: before being placed in MRI devices, all animals were sedated. At day 90, after all imaging analyses were performed, animals were euthanised. The subcutaneous plane was initially exposed and the scar tissue over the mesh was examined. The inner surface of the mesh was then inspected via a U-shaped incision. None of the animals died during surgery. Lastly 12 rats, in which no mesh was implanted, were used as negative controls for gadolinium toxicity measures . Rats were kept isolated for 3 days after operation then caged in pairs. Analgesic treatment was applied routinely once a day for 3 days after surgery by subcutaneous injection of Metacam 0.07 ml per 400 g bodyweight, as described before [Gd-PCL and Gd-PMA were coated onto PP mesh  as described before .Thirty days after surgery (D = 30) prostheses were explanted and the entire anterior abdominal wall (from the skin until the peritoneum) was harvested. For each test group  three biopsies were collected. Each specimen was divided into two parts along the longitudinal axis  MRI (Bruker Avance DRX system) was used to obtain high resolution 3D images of Gd-PCL and Gd-PMA coated meshes. Images were acquired with standard T1-weighted fast spoiled gradient recalled echo (FSPGR) .in vivo, coated implants were embedded in 1% w/w low gelling point agarose type VII  or inserted in muscle tissue within 20mL disposable scintillation glass vials  and then placed in 3T MRI  devices for analysis. Gradient echo sequence, routinely used in clinical practice for the exploration of the pelvis, was used. In order to assure reproducibility, the best sequences obtained for both 3T and 7T  were used to acquire all images. To verify the stability of the coated meshes, samples were stored up to one year at 37°C and tested for imaging at 4 months and one year, the size of the mesh was measured at every imaging assay. The reconstruction of images obtained was performed using the 3D reconstruction software OsiriX.To define and optimise sequences for visualisation of meshes The validation of the stability of the Gd-polymers was confirmed by assaying the diffusion of gadolinium by inductively coupled plasma mass spectrometry (ICP-MS) ,23. The The number of animals was based on published studies ,25. All The evaluation of the clinical tolerance on an animal model and the non-systemic diffusion of Gadolinium are essential features to meet the pre-clinical characteristics of the implant.At three months after implantation, the healing process was completely achieved for the animals implanted with Gd-PMA and PMA/PMMA meshes while rats implanted with Gd-PCL meshes displayed a noticeable erosion rate (100% (n = 10/10) Gd-PCL vs 0% (n = 0/10) Gd-PMA) as shown in Microscopic analysis of histology sections showed mild inflammation for the non-coated mesh and Gd-PMA coated mesh groups. For both these groups the tissue surrounding the implants was characterised by the presence of non-inflammatory collagen and important blood supply. For the Gd-PMA group in particular, substantial neoangiogenesis with capillaries and venules could be observed. For the non-coated mesh group, the tissue was also characterised by the presence of myofibroblasts with good mesenchymal organisation. On the other hand tissues surrounding Gd-PCL coated mesh were characterised by the presence of disordered collagen, persistent cellular inflammation and giant cell granulomas. Also, low levels of neo angiogenesis were visible .Immunohistochemistry showed similar patterns of smooth muscle α-actin, types I and III collagen between non-coated and Gd-PMA coated mesh groups. In particular, for the non-coated mesh group we could observe well organised blood vessels, which were no longer capillaries but mainly venules and arterioles. For the Gd-PMA group blood vessels were organised in an intermediary structure (few capillaries and mostly venules and arterioles). Tissues from Gd-PCL implanted rats show a high level of inflammation with few blood vessels mainly capillaries. Types I and III staining showed a good extracellular matrix organisation for the uncoated and Gd-PMA coated implants with a higher amount of collagen I compared to collagen III. For the Gd-PCL on the contrary, very low levels of collagen I were visible .For imaging of the coated meshes both experimental 7T and clinical 3T were used. Due to the very poor tolerance of Gd-PCL meshes, animals implanted with these implants were excluded from imaging assays.2. Coated meshes were visible with both 3T and 7T MRI techniques. All meshes were entirely a homogenously visible; indeed, the size of the mesh (image) corresponded to the size of the mesh in vivo.With both techniques we obtained a clear visualisation of a Gd-PMA mesh coated at a dose of 3.8 μg / cm3D reconstruction program allowed the optimisation of MR imaging by highlighting the contrast of the mesh and therefore allowing us to distinguish the implant within the different muscular layers Figs.  and 6.The ICP-MS evaluation after degradation of explanted meshes allowed to measure residual doses of gadolinium in surrounding tissues and in the organs metabolising Gd. The concentrations were measured in liver, kidney, tibia bone and skin of the animals implanted with coated meshes. No significant differences in mean levels of gadolinium were found in implanted group as compared to control group .With this study we sought to evaluate the tolerance and long-term MRI visibility of Gadolinium-modified polypropylene meshes for their use in soft tissue repair.in vitro visualisation of the mesh for at least one year [Appropriate modification of the meshes used for reinforcing the tissues in pelvic organ disorders enables their visualisation on magnetic resonance imaging . This teone year . Also, Gone year . With thone year . In thisGadolinium was not the only MRI contrast agent that may have been selected for this purpose. Fluorinated derivatives have been exploited before , but resThe MRI visualisation was obtained in vitro for both types of polymers (Gd-PCL and Gd-PMA), but the intensity of the signal observed was stronger with PMA than with PCL, probably because of its hydrophilic nature. As discussed above, the cutaneous and tissular integration was not clinically satisfactory in the case of the Gd-PCL implants confirming that Gd-PMA were more suitable for further investigation.The amount of gadolinium necessary for mesh imaging is correlated to the type of MRI, the tissues surrounding the area of the implants and their intrinsic contrasts. Results from previous studies helped to reinforce the idea that low doses of gadolinium are necessary (ranging from 1 to 20μg / cm2 according to the type of MRI used) .In vivo MR imaging raises the issue of the implant precise location and the risk of confusion with many artefacts due to the production of millimetre slices. The imaging artefacts, such as respiratory movements of animals, may hamper the image optimisation in abdominal position. On the contrary, the dorsal implantation of the mesh allowed us to precisely locate two very close (mm distance) implants. Nevertheless, these distances may entail more artefacts; discrimination of such artefact was however confirmed by accurate 3D images reconstruction  effects of the implants. This problem may be only partially overcome by testing the Gd-modified meshes on a bigger (ovine) model which allow a longer follow-up period.Nephrogenic systemic fibrosis (NSF) induced by Gadolinium-based contrast agent, has been described in rats, mainly in the dermic tissue . Hence, The last decades have seen a remarkable growth in the applications of MR imaging. The need for the visualisation of meshes used in pelvic floor surgery derives directly from the necessity to optimise the monitoring and care of patients. The stability of the Gd-complex coated meshes allows the visualisation over time of the implants. By linking the clinical symptoms to the observation of an anatomical region will result in the improved management of complications. Monitoring of the mesh would allow clinicians to assign responsibility for a post-operative symptom. In conclusion, the technique described above allows a non-invasive and long-term imaging of implants.


The frontal pole corresponds to Brodmann area (BA) 10, the largest single architectonic area in the human frontal lobe. Generally, BA10 is thought to contain two or three subregions that subserve broad functions such as multitasking, social cognition, attention, and episodic memory. However, there is a substantial debate about the functional and structural heterogeneity of this large frontal region. Previous connectivity-based parcellation studies have identified two or three subregions in the human frontal pole. Here, we used diffusion tensor imaging to assess structural connectivity of BA10 in 35 healthy subjects and delineated subregions based on this connectivity. This allowed us to determine the correspondence of structurally based subregions with the scheme previously defined functionally. Three subregions could be defined in each subject. However, these three subregions were not spatially consistent between subjects. Therefore, we accepted a solution with two subregions that encompassed the lateral and medial frontal pole. We then examined resting-state functional connectivity of the two subregions and found significant differences between their connectivities. The medial cluster was connected to nodes of the default-mode network, which is implicated in internally focused, self-related thought, and social cognition. The lateral cluster was connected to nodes of the executive control network, associated with directed attention and working memory. These findings support the concept that there are two major anatomical subregions of the frontal pole related to differences in functional connectivity.The online version of this article (doi:10.1007/s00429-014-0809-6) contains supplementary material, which is available to authorized users. The most anterior portion of the primate brain is often designated as a single brain region of granular cortex, broadly defined as Brodmann area (BA) 10 in humans , or theThe human BA10 likely comprises structurally and functionally heterogeneous subregions based on cytoarchitectonic studies in humans. Connectivity-based parcellations seem to correspond to subregions identified cytoarchitectonically  and orbital (10o)  and relative reward monitoring  characterization of the structural and functional connectivity of each of the subregions to the rest of the brain.We investigated the structural connectivity of the FPC to identify subregions within this area with an approach that has been used to investigate the cingulate, parietal, and temporoparietal junction cortices . Additionally, 10 non-diffusion-weighted scans  were acquired at the beginning of each run.All subjects provided informed consent to procedures approved by the UHN Research Ethics Board. Diffusion-weighted images were acquired for 35 healthy subjects  on a 3T GE MRI  system fitted with an eight-channel phased array head coil. Two sets of DWI data were acquired for each subject with 60 non-collinear, isotropic directions  with a factor of 2; 2) high-resolution  anatomical scan was also acquired for each subject using a 3D fast spoiled gradient-echo (FSPGR) sequence .Also, a whole brain . For the 5-min task-free scan, subjects were instructed to lie still, clear their thoughts and “not to think about anything in particular”, with their eyes closed.T2*-weighted functional MRI scans with an echo-planar pulse imaging (EPI) sequence were also acquired for every subject . We used the PALS th element within the matrix represents the correlation between the connectivity profile of voxel i and the connectivity profile voxel j. We then permuted the rows of the cross-correlation matrix using a K-means clustering segmentation algorithm, implemented in the ccops toolbox in FSL, for automated clustering to define K different clusters. This algorithm randomly selects a starting point in the matrix to cluster the voxels in the seed  that have similar connectivity profiles . It is possible that two separate regions cluster as a single cluster because of their interconnectivity (c.f. the similarity of their connectivity to the rest of the brain). Therefore, we included a scaled Euclidian distance matrix to the cross-correlation matrix as implemented in FSL  that are formed. Previous work has suggested that there are either two or three subregions in BA10 . The resulting tractograms were thresholded at 5,000 samples to demonstrate regions of differential connectivity. The tractograms were then binarised and summed to make a group probabilistic tractogram for each cluster  to spatially preprocess each subject’s functional data. These steps include realignment (motion correction), coregistration to a structural T1 image, normalization to the MNI standard brain, and spatial smoothing (6-mm FWHM Gaussian filter). Additionally, in the Conn toolbox, subjects’ anatomical T1-weighted images are segmented for grey matter, white matter and cerebrospinal fluid (CSF), and eroded  to later remove temporal confounds related to these tissue types (see below). The data were also temporally preprocessed to control for other potential confounds and to restrict the analysis to frequencies of interest (<0.1 Hz). These steps include using linear regression to remove potential sources of noise, including estimated subject motion parameters (3 translation components and 3 rotation components), BOLD signals in white matter and CSF areas. The additional white matter and CSF covariates are included using the anatomical component-based noise correction method  seeds within each hemisphere. Second-level group-level random-effects analysis was thresholded at p < 0.05 corrected for multiple comparisons with family-wise error correction with an extent threshold of 8 voxels. The final results were displayed on the FSL standard brain (MNI152_T1_2mm_brain.nii.gz).We performed seed-based resting-state functional connectivity  and determined the largest value for K where the clusters remained consistent amongst all 35 subjects , and anterior to the cingulate sulcus. Bilaterally, the lateral clusters spanned the middle frontal gyrus, between the superior frontal sulcus and the inferior frontal sulcus. All four of these clusters were consistent amongst all subjects. The three-cluster solution resulted in a medial, a rostral and a lateral cluster , the ipsilateral pallidum and putamen, and the pons. The medial cluster, but not the lateral cluster, was connected to the medial PFC, the cingulate cortex, the orbitofrontal cortex and the contralateral caudate nucleus.For the three-cluster solution, all three clusters had widespread connections. The parcels did have differential connectivity, albeit with some overlap. For example, tracts from the medial cluster uniquely projected to medial frontal brain regions, including the medial PFC (BA10), orbitofrontal (BA11) and anterior cingulate (BA 24/32) and subgenual cingulate (BA 25) cortices. The left rostral cluster uniquely projected to the anterior, mid and posterior regions of the cingulate cortex  the dorsomedial PFC . In the right hemisphere, the rostral cluster did not uniquely project to any brain regions. The lateral cluster projected to lateral frontal  and parietal cortical regions, as well as subcortical regions.In the three-cluster solution, the medial and rostral clusters are subdivisions of the medial cluster of the two-cluster solution. Given that the rostral cluster of the three-cluster solution did not have a unique pattern of structural connectivity distinct from the medial cluster, the three-cluster solution was not pursued further. Instead, the two-cluster solution was used for the purposes of resting-state functional connectivity analysis.It is critical to note that structural connectivity may or may not be reflected in functional connectivity. The individual nodes within networks of brain regions may lack direct structural connectivity identified with diffusion-weighted tractography , and yet the BOLD activity within these nodes may still be correlated, with the nodes co-activating in a reliable fashion to subserve a common function. Thus, the specific set of other brain regions with activity correlating with each of the FPC subregions could conceivably provide additional useful information regarding their respective functions. Therefore, we used resting-state fMRI to investigate the functional connectivity of the medial and lateral subregions identified in the two-cluster solution Fig. .Fig. 6DiWe found that the seeds in the two-cluster solution activated two distinct networks of regions in resting-state fMRI. Bilaterally, the medial clusters were significantly more functionally connected to nodes of the default-mode network see Fig. : namely,The aim of the current study was to investigate whether there are discernible structural subregions in the FPC based on white matter connectivity profiles. We used a data-driven approach based on probabilistic tractography to determine the connectivity of every voxel within BA10, and a clustering algorithm to parcellate the BA10 into subregions. Based on structural connectivity, we found two solutions that were reliably reproducible across 35 subjects: a two-cluster and a three-cluster solution. The two-cluster solution comprised a medial and a lateral cluster, whereas the three-cluster solution further divided the medial cluster into a more ventral and a more dorsal cluster . Structural connectivity of these clusters revealed that at the population level the two-cluster solution was more consistent than the three-cluster solution. Specifically, there were no unique tracts in half of the subjects for the rostral cluster in the right hemisphere in the three-cluster solution, whereas the two-cluster solution showed clear differences.The structural and functional heterogeneity of the FPC has been previously explored using a variety of methodologies . The oldest such treatment, electroconvulsive therapy (ECT), conventionally places electrodes over the lateral frontotemporal or parietal cortex. Although the effects of ECT on brain activity are widespread, the effectiveness of the treatment correlates best to the degree of reduction in frontopolar metabolism Below is the link to the electronic supplementary material.


Children’s after-school physical activity (PA) and sedentary behaviours (SB) are not well understood, despite the potential this period holds for intervention. This study aimed to describe children’s after-school physical activity and sedentary behaviours; establish the contribution this makes to daily participation and to achieving physical activity and sedentary behaviours guidelines; and to determine the association between after-school moderate- to vigorous-intensity physical activity (MVPA), screen-based sedentary behaviours and achieving the physical activity and sedentary behaviour guidelines.Children  wore an ActiGraph GT3X accelerometer. The percentage of time and minutes spent sedentary (SED), in light- physical activity (LPA) and MVPA between the end-of-school and 6pm (weekdays) was calculated. Parents  proxy-reported their child’s after-school participation in screen-based sedentary behaviours. The contribution that after-school SED, LPA, MVPA, and screen-based sedentary behaviours made to daily levels, and that after-school MVPA and screen-based sedentary behaviours made to achieving the physical activity/sedentary behaviour guidelines was calculated. Regression analysis determined the association between after-school MVPA and screen-based sedentary behaviours and achieving the physical activity/sedentary behaviours guidelines.Children spent 54% of the after-school period SED, and this accounted for 21% of children’s daily SED levels. Boys spent a greater percentage of time in MVPA than girls , but this made a smaller contribution to their daily levels . After school, boys and girls respectively performed 18.8 minutes and 16.7 minutes of MVPA, which is 31.4% and 27.8% of the MVPA (p<0.05) required to achieve the physical activity guidelines. Children spent 96 minutes in screen-based sedentary behaviours, contributing to 84% of their daily screen-based sedentary behaviours and 80% of the sedentary behaviour guidelines. After-school MVPA was positively associated with achieving the physical activity guidelines , and after-school screen-based sedentary behaviours were negatively associated with achieving the sedentary behaviours guidelines .The after-school period plays a critical role in the accumulation of children’s physical activity and sedentary behaviours. Small changes to after-school behaviours can have large impacts on children’s daily behaviours levels and likelihood of meeting the recommended levels of physical activity and sedentary behaviour. Therefore interventions should target reducing after-school sedentary behaviours and increasing physical activity. In spite of the evidence of detrimental health outcomes ,2, childThe after-school period, recently defined as the end-of-school to 6pm , has beeSubsequently, the aims of this study were to: 1) identify the percentage of the after-school period boys and girls spend in objectively measured sedentary behaviour (SED), light-intensity physical activity (LPA), MVPA and proxy-reported screen-based sedentary behaviours; 2) identify how the after-school period contributes to daily levels of SED, LPA MVPA and screen-based sedentary behaviour; 3) identify how the after-school period contributes to achieving the physical activity and sedentary behaviours guidelines among boys and girls (i.e. ≥60 minutes of MVPA/day and ≤120 minutes of screen-based sedentary behaviour/day); and 4) examine the association between after-school MVPA and sedentary behaviours and the odds of achieving the physical activity/sedentary behaviours guidelines during weekdays.This study used baseline data from the Transform-Us! intervention . Transform-Us! targeted children’s physical activity and sedentary behaviours and the methods for that intervention have been described in detail elsewhere . In brieEthical approval was obtained from the Deakin University Human Research Ethics Committee, the Victorian Department of Education and Early Childhood Development and the Catholic Education Office. Written parental consent and child assent to complete the assessments were obtained prior to participation.Children reported their age and sex and parent’s self-reported their highest level of education.Children were fitted with a GT3X Actigraph accelerometer  by trained researchers during class time and were informed that the monitors assess how active they are. Accelerometers have been shown to have acceptable validity among children . ChildreParents’ proxy reported their child’s behaviour as evidence suggests that children younger than 10 years cannot reliably report the duration of their activities . ParentsSpecifically developed excel macros and Stata code (State 12) were used to analyse the raw accelerometer data files. Data from the end-of-school  to 6pm were extracted for analysis. Non-wear time was considered ≥20 consecutive minutes of zero counts in line with the most commonly used definition as identified in a recent review of children’s accelerometer studies  and beca-1 which is the cut point that most closely represents sitting time among children [Previously established age-specific cut-points  were usechildren . This alchildren . The perMean daily time in after-school screen-based sedentary behaviours was calculated by summing the durations spent in each screen-based sedentary behaviours and dividing by five. The contribution of after-school screen-based sedentary behaviours to total daily screen-based sedentary behaviours was calculated as: (minutes in screen-based sedentary behaviours time during the after-school period/minutes in screen-based sedentary behaviours during the entire day)*100. The contribution that screen-based sedentary behaviours performed during the after-school period made to achieving the sedentary behaviours guidelines *100. T-tests were used to assess sex differences between the behaviour intensities. Logistic regression was performed to determine the likelihood of achieving the physical activity guidelines based on the percentage of the after-school period spent in MVPA. Logistic regression was also performed to determine the likelihood of achieving the sedentary behaviour guidelines based on the time spent in screen-based sedentary behaviours during the after-school period. Both models were adjusted for clustering by school and the covariates of age, sex and maternal education. There was no interaction between sex and achieving the physical activity guidelines, therefore all children were analysed together.The final sample consisted of 406 children who had valid survey and accelerometer data for the after-school period (68% of sample). Of these, 359 also had valid accelerometer data for the entire day . Three-hundred and eighteen parents  completed a survey. There were no differences in age or sex among the children with and without parent proxy-report data. During the entire day children spent 60.3% of their time SED, 29.2% in LPA and only 10.6% in MVPA. Girls spent a significantly greater percentage of time in LPA than boys, whereas boys spent a greater percentage of time in MVPA than girls during both the after-school period and the entire day. The after-school period accounted for more than one-fifth of children’s daily SED (20.8%) and 27.6% and 29.8% of daily MVPA for boys and girls respectively. The after-school period made a significantly larger contribution to boys’ daily SED than girls’ . Conversely, the period made a significantly larger contribution to girls’ LPA and MVPA than boys’ . After school, boys and girls performed 31% and 28% of the MVPA needed to reach the guidelines of ≥60 minutes of MVPA per day.Significantly more boys than girls achieved the physical activity guidelines (≥60 minutes of MVPA) on weekdays ; however, there were no differences in the percentage of boys and girls who achieved the sedentary behaviour guidelines (≤120 minutes screen-based sedentary behaviours) on weekdays (58.7% and 59% respectively). When comparing the percentage of children who achieved both the physical activity and sedentary behaviour guidelines (i.e. ≥60 minutes of MVPA and ≤120 minutes screen-based sedentary behaviours), more boys than girls achieved both guidelines on weekdays .Data from the parent proxy-report of after-school and daily screen-based sedentary behaviours is shown in Results of the logistic regression showed that the odds ratio of achieving the physical activity guidelines was 1.31 . Therefore, for each 1% increase in percentage of the after-school period spent in MVPA, children have a 31% increased likelihood of achieving the physical activity guidelines of ≥60 minutes of MVPA per day. Logistic regression also showed the odds ratio of achieving the sedentary behaviour guidelines was 0.97 . For each 1% increase in screen-based sedentary behaviours during the after-school period, children were 3% less likely to achieve the sedentary behaviour guidelines of ≤120 minutes in screen-based sedentary behaviours per day.This study highlights the importance of the after-school period for children’s physical activity and sedentary behaviours participation and achieving the associated guidelines. Results showed a 31% increase in likelihood of achieving the physical activity guidelines for every 1% increase in the after-school period spent in MVPA. If this finding was extrapolated for an average child in the current study, a 1% shift  would increase MVPA from 21 minutes to 22.5 minutes during an after-school period of 150 minutes (e.g. 3.30-6pm). We also conducted the analysis with minutes of after-school MVPA as the predictor. Results were very similar and therefore are not presented. After-school screen-based sedentary behaviours participation was associated with a reduced likelihood of achieving the sedentary behaviours guidelines. For every additional 10 minutes of screen-based sedentary behaviours, children are 30% less likely to meet the sedentary behaviours guidelines further highlighting the need to reduce after-school screen-based sedentary behaviours.Children perform the majority of their daily screen-based sedentary behaviours during the after-school period and almost reach the maximum recommended amount of daily screen-based sedentary behaviours during this period alone. Previous research has investigated if children meet or exceed the sedentary behaviour guidelines on a daily basis ,11. HoweAlthough less than 15% of children’s time after school is spent in MVPA, this contributed almost a third of children’s daily MVPA, highlighting the crucial role the after-school period plays in the accumulation of children’s daily physical activity. Although girls spent a lower percentage of the after-school period in MVPA, that percentage made a larger contribution to girls’ total daily physical activity (both LPA and MVPA) than boys’ after-school physical activity. This suggests that the after-school period is a key time of the day to target these behaviours among girls as they perform MVPA at lower levels than boys during other times of the day (e.g. recess and lunchtime ). In conPrevious studies have shown boys participate in more physical activity ,34 and sIn comparison to children in other studies and countries, the current sample spent a smaller percentage of the after-school period in SED, and a greater percentage of time in LPA and MVPA than Canadian 11 year olds . CompareThis is the first study to determine how important the after-school period is for children’s likelihood of achieving the physical activity and sedentary behaviour guidelines. In particular, findings highlight the large impact that very small changes in after-school physical activity can have on the probability of children meeting the physical activity guidelines (31% more likely to meet the guidelines for every 1% increase in time spent in MVPA during the after-school period). It is therefore important to develop interventions that target increases in children’s physical activity and reductions in sedentary behaviour and screen-based sedentary behaviours during the after-school period. For example, active homework strategies  may targThe response rate was 37% and as such it is not known if these children were representative of the broader sample. As parents are not with their child during some parts of the day (e.g. school hours), they may not be able to recall their screen-based sedentary behaviours during this time which may impact their reporting of daily screen-based sedentary behaviours duration. Further, the objective measure of sedentary time may include time when children are standing and not moving as it is not a direct measure of sitting. Also, the proxy-report measure did not ask specifically for the time engaged in specific sedentary behaviours while sitting. However, the collection of objective measures of physical activity and overall SED and proxy-report screen-based sedentary behaviours duration during the after-school period (when parents are often with their child) is a strength of this study as this provided the intensity and duration of after-school behaviours performed by young children. This information may be used to enhance the effectiveness of interventions which target after-school physical activity and sedentary behaviour. Future research should also examine other contextual factors that may be related to children’s after-school behaviours  and collect detailed information on the type of physical activity  and sedentary behaviours  they are performing to further tailor after-school interventions.After school, children perform 80% of the maximum daily recommendation for screen-based sedentary behaviour but less than 30% of the daily MVPA recommended for health. After-school MVPA is positively associated with achieving the physical activity guidelines and after-school screen-based sedentary behaviours are negatively associated with achieving the sedentary behaviour guidelines. However, more than half of the after-school period is spent in screen-based sedentary behaviours and little time is spent in MVPA. Interventions should target reductions in sedentary behaviour and increases in physical activity during the after-school period as the behaviours performed during this period make a large contribution to daily levels and to achieving the associated guidelines.