Adequate symptom control is a problem for many people with asthma. We asked whether weekly email reports on monitored use of inhaled, short-acting bronchodilators might improve scores on composite asthma-control measures.TM (ACT), to assess asthma control at entry and monthly thereafter. After the first month, participants received weekly email reports for 3 months. The reports summarized inhaled bronchodilator use during the preceding week and provided suggestions derived from National Asthma Education and Prevention Program (NAEPP) guidelines. Paired t-tests and random-effects mixed models were implemented to assess changes in primary asthma endpoints.Through an investigational electronic medication sensor attached to each participant's inhaler, we monitored 4 months' use of inhaled, short-acting bronchodilators. Participants completed surveys, including the Asthma Control TestThirty individuals participated in the 4-month study; 29 provided complete asthma control information. Mean age was 36.8 years (range: 19–74 years); 52% of respondents were female. Mean ACT scores were 17.6 (Standard Deviation [SD]  = 3.35) at entry and 18.4 (SD = 3.60) at completion of the first month. No significant difference appeared between ACT values at entry and completion of the first month (p = 0.66); however, after participants began receiving email reports and online information about their inhaler use, mean ACT scores increased 1.40 points  for each subsequent study month. Significant decreases occurred in 2-week histories of daytime symptoms  and nighttime symptoms ; no significant change in activity limitation  was observed. Participants reported increased awareness and understanding of asthma patterns, level of control, bronchodilator use  and triggers, and improved preventive practices.Weekly email reports and access to online charts summarizing remote monitoring of inhaled bronchodilator frequency and location were associated with improved asthma control and a decline in day-to-day asthma symptoms. Despite improved understanding and the development of new medications, asthma remains a substantial and costly public health problem National Asthma Education and Prevention Program and Global Initiative for Asthma clinical practice guidelines recommend doctors monitor whether treatment is controlling symptoms and improving quality of life Questionnaires that capture composite asthma-control measures have become popular tools to help patients and their physicians determine level of control This pilot study's goals were 1) to investigate the use of a device to monitor objectively the time and location of inhaled bronchodilator use as a measure of asthma control, and 2) to determine whether information about inhaled bronchodilator use and feedback on asthma control via weekly email reports was associated with improved scores on composite measures of asthma control.The study was reviewed and approved by the University of Wisconsin Health Sciences institutional review board (protocol H-2007-0291) and all participants signed informed consent documentation.TM (ACT) We recruited adults (aged >18 years) from a variety of clinical and community settings on a rolling basis between February and April 2009. These adults had a diagnosis of asthma and used a prescribed inhaled short-acting beta-agonist (SABA). By the end of August 2009, all participants had participated for 4 months. At entry, participants completed questionnaires to collect data on their demographic and clinical characteristics, including asthma history, then-current status, and asthma management such as medical treatments, perceived triggers, healthcare utilization, and level of control using the Asthma Control TestWe collected data about the time and location of use of inhaled short-acting bronchodilators. This involved using a small and portable investigational electronic medication sensor that is attached to an existing inhaler housing by means of a hook-and-loop fastener strap . This alThe device also contained equipment to communicate via a wireless network and supporting electronics, including a simple user interface, a power switch, and a rechargeable battery. The device obtained and wirelessly communicated medication event information in real-time to a remote server. It did not require that the patient (or any other person) participate in obtaining or transmitting the information. Because the routine dose of some SABAs can vary from one to multiple inhalations, the device was not designed to record each inhalation of medication but rather to capture information about the occasion of medication use, following American Thoracic Society/European Respiratory Society guidance After completing the first month of the study, participants began to receive weekly e-mail reports displaying maps and charts of their usage. They also received access to an online interface with similar features. This online interface allowed participants to indicate when an inhaler was used prophylactically before exercise. The weekly e-mail reports included the time and location for each inhaled bronchodilator use and summarized use for the preceding week. Participants received a basic assessment of their asthma control based on the number of days of use in the preceding week, the timing of those events, and on thresholds from the National Asthma Education and Prevention Program (NAEPP) guidelines. Participants also received simple advice derived from the NAEPP guidelines about how to improve asthma self-management  [7].TM score and 2-week histories of activity limitation and days and nights with asthma symptoms. A score ≤19 (of 25) on the Asthma Control TestTM was considered to indicate uncontrolled asthma. We collected 2-week histories of asthma symptoms and activity limitation to capture continuous estimates of outcome measures in addition to the ordinal responses gathered by the Asthma Control TestTM. Exit questionnaires also assessed participant feedback that included the utility of the electronic communications, perceived changes in asthma awareness, and modifications in day-to-day asthma management.At the conclusion of each month, and on conclusion of the study in August 2009, participants completed questionnaires designed to detect any changes in the four primary study outcome measures: the Asthma Control TestTM score; two-week histories of activity limitation and days and nights with asthma symptoms) while participants were blinded to data collected by the medication sensor in the first month of participation. Linear mixed effects models with a random effect for subject assessed changes in primary asthma endpoints across the three subsequent months of enrollment  when participants were able to access their usage data online. Participants with one or more observations were included in the analysis.We used pairwise t-tests to evaluate the null hypothesis that there was no change in the four primary study endpoints . Regression diagnostics were performed using the MIXED_DX macro A total of 33 individuals were initially recruited for the study and 29 provided complete information on asthma control at entry. On average, participants had longstanding asthma, with a mean time since diagnosis of 25 years (standard deviation [SD]  = 16). Approximately 62% (18/29) of study participants entered with uncontrolled asthma; mean ACT score at study entry for uncontrolled participants was 15.4 (SD = 2.11). Mean ACT score overall was 17.6 (SD = 3.35).At study entry, participants reported 4.84 days (SD = 4.13) with asthma symptoms, 2.03 (SD = 3.35) nights with asthma symptoms, and 0.86 (SD = 1.38) days with activity limitations in the 2 weeks before entry . More thParticipants were enrolled for a total of 3,887 patient-days of observation between March and August 2009. Of these days, devices were turned on and available on 1,916 days –49% of patient-days. On the other days, devices were powered off or out of range of a wireless network.TM (score >19) used bronchodilators on 25% of the days their devices were active, compared with 37% among those with uncontrolled asthma.On average, participants used the devices on 27%  of patient-days in which the devices were active, and 13%  of patient-days overall. During the study period, the devices reported a total of 958 medication-use events, occurring on 520 patient-days. The median number of events per day was 1.35, with a mode of 1. The percentage of days on which individual participants used bronchodilators ranged from 1% to 83%. Overall, participants who entered the study with controlled asthma (11/29) as defined by the Asthma Control TestA total of 30 individuals provided information for at least one of the periodic (monthly) surveys. Results from the pairwise t-test  indicateAfter the first month that participants received weekly email reports, mean ACT scores increased 1.5 points to 20.1 (SD = 3.66). After a second month of reports (at exit), mean ACT scores increased an additional 1.1 point to 21.2 (SD = 3.36). At exitTM score. Receipt of weekly e-mail reports and online access to asthma event history was also associated with a significant decrease in number of days with symptoms  and number of nights with symptoms . We did not observe a significant change in the frequency of activity limitation reported by participants. Regression diagnostics indicated that linearity assumptions were adequately met for the primary analyses.On exit surveys, participants reported that the weekly email reports increased their awareness of the frequency and patterns of asthma symptoms. Participants suggested that the feedback helped improve perceptions of their asthma control and their ability to communicate to their physicians their level of control. As one participant described, “I learned that I used my inhaler more than I remember. I was able to see and relate to my doctor that my asthma is not under control.” Participants also reported that the receipt of information about the time and location where they used their inhaler helped to highlight locations and exposures to triggers that led to symptoms. “I've been more keen to note surroundings when I feel shortness of breath,” one participant said. “It opened my eyes to triggers I wasn't aware of in the past.” Some described subsequent attempts to minimize or avoid exposure. Finally, participants reported that weekly feedback helped to reinforce their use of controller medications. “I'm now using my controller medicine more regularly and on time,” one participant told us. “I noticed that my rescue inhaler use went down significantly when using a daily inhaler as well.”Participants reported that remote monitoring and feedback proved a practical aid. All but one of the participants indicated that the weekly e-mail reports were either very or somewhat useful in improving their asthma management; overall, 80% expressed an interest in continuing to track their inhaler use.The findings from this pilot study suggest that capturing data on inhaled bronchodilator use, incorporating that information with some basic asthma education, and putting it all into a simple, weekly e-mail report may be associated with improved asthma control. The majority of participants in this study experienced small, but statistically significant and steady increases in composite measures of asthma control and significant declines in the day-to-day burden of asthma symptoms. We observed a mean per month increase of 1.4 points in ACT score, or 2.8 points overall, slightly less than the Minimal Important Difference of 3 points This study provided patients with novel feedback about the ongoing management of their asthma. Today, many patients have inappropriately low expectations for their own disease control or are unaware that more can be done to prevent attacks and day-to-day symptoms. In one recent study, more than 4 in 5 individuals with poorly controlled asthma regarded their disease as well controlled or very well controlled A number of recent studies have demonstrated that internet- and mobile phone-based self-management interventions can improve asthma control and asthma-related quality of life The results of this pilot study are subject to numerous limitations. In particular, this study purposefully enrolled a small convenience sample of adult volunteers with relatively uncontrolled asthma that does not represent that general population with asthma or specific asthma phenotypes. Participants in the study did not undergo reversibility studies to confirm their reported diagnosis of asthma, nor were pulmonary function studies performed to quantify the severity of airflow limitation. Given the age range of participants, it remains possible that some persons may have also had undiagnosed chronic obstructive pulmonary disease. In addition, not all participants in this study provided complete information on all questionnaire outcomes at all time points. While attrition may have introduced some bias, persons who did not provide complete information or who withdrew did not differ significantly from those who completed the study. That many individuals continued to contribute information from the electronic medication sensor even when they failed to respond to asthma control questionnaires underscores the unique potential role and value of passively collected data in the remote monitoring of asthma control.This study was also limited by its relatively short duration and the lack of a control group to isolate the effect of time-varying covariates  known to influence asthma. As a result, it is not possible to know if participants would have improved regardless of the intervention . Subsequent research with a randomly selected sample and a randomized and controlled protocol of greater duration is underway to isolate and assess the effect and persistence of remote monitoring and feedback on management. Stratification by level of control at entry should also strengthen the analysis and interpretation This pilot study has identified a number of areas where additional research could improve the value of remote monitoring and patient feedback, and underscores the need for evaluation of its potential effects in a properly designed and conducted randomized-controlled trial. For example, more information is required to understand how participants interpret weekly feedback and how often they share reports with friends, family, and health care providers. Further research is also needed to determine whether providing patients with information about their patterns of inhaler use motivates them to adhere better to preventive therapy, or increases the probability that patients undertake environmental interventions or behavior changes to reduce exposures that cause attacks. Finally, we recommend additional research to identify which composite measure of asthma control, or combination of questions from available instruments, best captures current impairment and future risk when used in conjunction with information about rescue and controller medication use captured by remote monitoring devices.Remote monitoring of inhaled bronchodilator use may also be valuable for epidemiological research and public health surveillance. By aggregating individual patterns of inhaler use, epidemiologists and public health groups can use the accumulated data to strengthen their ongoing asthma surveillance activities, improve scientific understanding of disease triggers and progression in the community, and provide a novel source of data about the local burden of disease through which to examine the response to treatment and interventions. Data can also target and evaluate interventions designed to reduce asthma-related morbidity in the community. Monitoring the timing and location of use of inhaled bronchodilators provides instant, objective data on the burden and frequency of asthma symptoms in populations. With widespread use it may also reveal clusters of asthma exacerbations and help identify new epidemiological patterns and risk factors, particularly when used as part of focused investigations.Receipt of weekly email reports with asthma management guidance and access to online materials summarizing use of inhaled bronchodilators was associated with improved measures of asthma control. Such a system has the potential to improve the recognition and treatment of poorly controlled asthma. It could also represent an important improvement in asthma management, epidemiological research, and public health surveillance.Appendix S1Sample weekly email report.(TIFF)Click here for additional data file.


Dilated cardiomyopathy (DCM), characterized by left ventricular dilatation and systolic dysfunction, constitutes a significant cause for heart failure, sudden cardiac death or need for heart transplantation. Lamin A/C gene (LMNA) on chromosome 1p12 is the most significant disease gene causing DCM and has been reported to cause 7–9% of DCM leading to cardiac transplantation. We have previously performed cardiac magnetic resonance imaging (MRI) to LMNA carriers to describe the early phenotype. Clinically, early recognition of subjects at risk of developing DCM would be important but is often difficult. Thus we have earlier used the MRI findings of these LMNA carriers for creating a model by which LMNA carriers could be identified from the controls at an asymptomatic stage. Some LMNA mutations may cause lipodystrophy. To characterize possible effects of LMNA mutations on lipid profile, we set out to apply global serum lipidomics using Ultra Performance Liquid Chromatography coupled to mass spectrometry in the same LMNA carriers, DCM patients without LMNA mutation and controls. All DCM patients, with or without LMNA mutation, differed from controls in regard to distinct serum lipidomic profile dominated by diminished odd-chain triglycerides and lipid ratios related to desaturation. Furthermore, we introduce a novel approach to identify associations between the molecular lipids from serum and the MR images from the LMNA carriers. The association analysis using dependency network and regression approaches also helped us to obtain novel insights into how the affected lipids might relate to cardiac shape and volume changes. Our study provides a framework for linking serum derived molecular markers not only with clinical endpoints, but also with the more subtle intermediate phenotypes, as derived from medical imaging, of potential pathophysiological relevance. Dilated cardiomyopathy (DCM) is characterized by left ventricular dilatation and decreased contractility. DCM constitutes a significant cause for heart failure, sudden cardiac death or need for heart transplantation. At least 25% of DCM has been estimated to be familial Clinically, identifying subjects in an early phase of developing DCM would be important but it is often difficult and for example subtle abnormalities in echocardiography may easily be overseen. We have previously shown that specific parameters derived from the heart cine magnetic resonance (MR) images help identify LMNA carriers from the controls at an early stage We first investigated the differences in serum lipidomes and cine MR image data between the LMNA carriers not diagnosed with DCM, and matched healthy controls . The “LaWe applied Ultra Performance Liquid Chromatography coupled to mass spectrometry (UPLC/MS™) to study molecular lipids as previously described The cine MR images were analyzed from 11 LMNA carriers and their 11 matched controls (Groups 1 and 2). Surface models of the epicardium (1516 points) and the endocardium of the left and right ventricles (906 points each) containing all together 3328 points were mapped on the heart images. The heart surface models were also used to derive 86 variables that describe anatomical and physiological features of the heart, as described earlier i.e., increase of double bonds). Since LMNA mutations are known to be associated with lipid metabolism A total of 57 and 56 lipid variables were found significantly different when comparing Groups 1&2 and Groups 3&4, respectively . CompariP = 0.044 by Chi-square test; In order to further investigate the disease specificity of lipidomic changes detected in LMNA mutation carriers, we used the lipids from P<0.05 by Wilcoxon test). Linear correlation analysis between these image variables and the 57 significantly changing lipids  according to the change observed in Group 1, and the resulting heart model was generated. The change in the heart model was visually evaluated by computing the wall thickening and/or wall motion from end-diastolic shape to end-systolic shape and visualizing the results using color maps overlaid on the mean heart model.i.e. a lipid found diminished in LMNA mutation carriers as well as in DCM  who did not have any cardiac disease, recruited during year 2007. Clinical examination, ECG and echocardiography  were performed to each of the participants to exclude the possibility of cardiomyopathy or valvular disease. Venous blood samples were drawn for lipid analyses. DNA analyses were not performed.2 and EF<45% at time of diagnosis. All patients who had secondary causes of DCM were excluded. The subjects were asked to participate in the molecular genetics study of DCM during the years 2000–2008. In some patients the left ventricular function improved remarkably during medication. In the gene analyses carried out either by SSCP (single strand conformation polymorhism) -method or sequencing of the LMNA gene at the University of Kuopio, no mutations in the lamin A/C gene could be detected in these patients. Venous blood samples were drawn for lipid and metabolomic analyses and cardiac MRI studies were performed in year 2008.Group three comprises eight unrelated DCM patients with no LMNA mutation (three men and five women), who had been diagnosed with DCM and followed up at the Department of Cardiology of the Helsinki University Hospital. The diagnosis was based on clinical examination, ECG and echocardiography. Clinical data was obtained from the hospital records. Inclusion criteria were LVEDD>27mm/mGroup four consists of healthy controls, matched for age and sex with group three: eight previously healthy volunteers (three men and five women), recruited in 2008. The possibility of cardiomyopathy or valvular disease was excluded by clinical examination, ECG and echocardiography . Venous blood samples were drawn for lipid analyses. DNA analyses were not performed.Informed consent was obtained from the study participants, and the Ethics review Committee of the Department of Medicine, University of Helsinki, approved this study.The imaging was done as described previously a priori heart model, consisting of triangulated surfaces of the ventricles and epicardium, was deformed to fit both short- and long-axis MRI data. Because the same a priori model was used for each subject, the number of the surface points was identical in each case , and the point-correspondence existed between all the subjects and time frames. The surfaces were rigidly aligned in the same coordinate system to remove the position and orientation variations from the data. This enabled point-wise comparison of the cardiac motion in the study population. The heart surface models were also used to derive 86 MRI parameters that describe anatomical and physiological features of the heart, as described earlier The left and right ventricles and epicardium were semi-automatically segmented by a technician together with a radiologist from each time frame of the cine MRI series with a software tool developed for this purpose 4Ac, 0.1% HCOOH). The flow rate was 0.2 ml/min and the total run time including column re-equilibration was 18 min. Data were processed using MZmine software version 0.6 The lipidome was analyzed as described previously Calibration was performed as follows: all monoacyl lipids except cholesterol esters, such as monoacylglycerols and monoacyl-glycerophospholipids, were calibrated with lysophosphatidylcholine PC(17∶0/0∶0)  as an internal standard. All diacyl lipids except phosphatidylethanolamines were calibrated with phosphatidylcholine PC(17∶0/17∶0) (Avanti Polar Lipids), the phosphatidylethanolamines with PE(17∶0/17∶0) (Avanti Polar Lipids) and the triglycerides and cholesterol esters with triglyceride TG(17∶0/17∶0/17∶0) .q-order, partial correlations in which a set of q variables are controlled qpgraph and networks were visualized using Cytoscape (www.cytoscape.org) and yEd (www.yworks.com) graphical editors.We calculated the marginal correlations the Pearson correlations, which indicate the degree of linear dependency between a pair of variables. The partial correlations were calculated as the Pearson correlations, with the effect of a set of control variables removed. In case the set of control variables includes all other variables the partial correlation is “full” and it depicts the linear dependency between the pair of variables that cannot be explained by any linear combination of the other variables. In “omics” applications where the number of variables is usually much larger than the number of samples, like in our case here, it is not feasible to calculate the full partial correlations, but efficient algorithms have been developed to estimate them with restricted, elasticnet package of R. Elasticnet is convenient for our current application because the L1 and L2 penalties on the regression coefficients can be flexibly tuned by changing 1u and 2u respectively in Equation (1), which allows exploration of solutions ranging from simple models with no lipids at all to more complex ones that are either sparse, containing non-correlated lipids, or non-sparse with several correlated lipids included. Elasticnet solution is obtained by minimizingu1 and u2 are tuning parameters, y is the response variable and X a matrix containing the explanatory variables. In this study, each of the image parameters were separately explained by the whole lipid profile data and the tuning parameters were selected by minimizing the mean cross-validation error:p = (p(1), …., p(n)) is partition of the samples into n blocks and −p(i) refers to all samples but those that belong to the i-th block (which belong to p(i)). Here we randomly assigned the samples into n = 5 blocks for 100 times and calculated the average errors.In addition to correlation based analysis, regression methods were applied to elucidate dependencies between the MR image parameters and the lipid profiles in more detail. Due to the large number of variables compared to sample size the regression must be regularized and we chose to apply the elasticnet regression method The cross-validation errors were also used to assess whether the lipid profiles in general explained an image parameter. The criterion was defined as follows:→ The image parameter in question can be explained with the lipid profiles.emin is the minimum average cross-validation error, ei,min is the standard deviation of the cross-validation errors over the n blocks for the model that gives the minimum average error and econs is the average cross-validation error of the constant model. In other words, if the minimum mean cross-validation error plus one standard deviation of the error at minimum was lower than the mean cross-validation error of the constant model, i.e., a model that contained no lipids, then the image parameter was interpreted to be dependent on the lipid profiles. The final model for each image parameter was estimated using all samples and the pair of tuning parameters that corresponded to the minimum average cross validation error.Above i of the mean heart model.A mean heart surface model, i-th surface points of sample subject j and For each sample subject in Groups 1&2, the heart surface model's deviation from the mean heart model along the surface normal was computed:A linear regression model was computed for the values of a lipid and the shape parameters From the surface models observed, wall thickness along the surface normal was determined for each surface point of left ventricle for end-diastole and end-systole, and their difference was used as the wall thickening measure. Amount of wall motion along surface normal was computed from the location of a surface point in end-diastole and end-systole.Table S1vs. Group 2, and Group 3 vs. Group 4, respectively (two-sided t-test). Reported fold change is the median of Group 1 or Group 3 divided by the median of Group 2 or Group 4, respectively.Comparisons of mean values of lipid variables in Group 1 (PDF)Click here for additional data file.


During leisure activities young people are often exposed to excessive noise levels resulting in an increase of noise-induced symptoms such as hearing loss, tinnitus and hyperacusis. Noise-induced tinnitus is often perceived after loud music exposure and provides an important marker for overexposure as a temporary threshold shift that is often not experienced by the individual itself. As oxidative stress plays an important role in the pathogenesis of noise-induced hearing loss, the use of antioxidants to prevent hearing damage has recently become the subject of research.N-acetylcysteine (600 mg) and magnesium (200 mg) prior to leisure noise exposure in young adults. The primary outcome measure is the tinnitus loudness scored by a visual analogue scale (VAS). Secondary outcome measures are the differences in audiological measurements for the antioxidant treatments compared to placebo intake. Audiological testing comprising of pure tone audiometry including frequencies up to 16 kHz, distortion product otoacoustic emissions, transient-evoked otoacoustic emissions and speech-in-noise testing will be performed prior to and within 7 hours after noise exposure. By use of a mixed effects statistical model, the effects of antioxidants compared to placebo intake will be assessed.This study proposes a randomized, double-blind, placebo-controlled crossover trial to assess the effects of a prophylactic combination of As adolescents and young adults often do not use hearing protection while being exposed to loud music, the use of preventive antioxidant intake may provide a useful and harmless way to prevent noise-induced hearing damage in this population. Furthermore, when exposed to hazardous noise levels the protection provided by hearing protectors might not be sufficient to prevent hearing damage and antioxidants may provide additive otoprotective effects. Previous research mainly focused on occupational noise exposure. The present study provides a protocol to assess the usefulness of antioxidants during leisure noise activities.NCT01727492.The present protocol is registered at ClinicalTrials.gov:  Adolescents and young adults frequently expose themselves to hazardous noise levels during social events or through personal listening devices. This has led to an increase of noise-induced symptoms such as noise-induced hearing loss (NIHL) and noise-induced tinnitus (NIT) over the last few years ,2. AlthoOxidative stress plays a crucial role in the pathogenesis of NIHL and NIT. During excessive noise exposure, the outer hair cells endure metabolic depletion leading to accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which may ultimately lead to necrosis and apoptosis . While nUnder normal circumstances, the human cochlea contains molecules including vitamins, glutathione, enzymes and reactive transcription, which work together to form a complex and sophisticated defense mechanism against oxidative molecules . In caseN-acetylcysteine (NAC) [2+) [While antioxidant treatment for noise-induced hearing damage has often been the subject of animal research -17, wellne (NAC) -21 and mAC) [2+) ,23. As oAC) [2+) . Most huAC) [2+) . Hence, 2+ as prophylactic antioxidants in adolescents during leisure noise exposure. The present report will follow the guidelines expressed by Consolidated Standards of Reporting Trials (CONSORT).The present article proposes a protocol for a clinical study consisting of a double-blind, randomized, placebo-controlled trial with a combination of NAC and Mg2+) or a placebo, 1 hour prior to noise exposure. Each participant needs to repeat this procedure four times  and each participant will receive twice the antioxidants as well as the placebo. This study aims to investigate the combined effects of NAC and Mg2+ by use of four repeated treatments on the hearing of noise-exposed young adults.This study proposes a randomized, double-blind, placebo-controlled crossover trial. Participants need to spend at least 3 consecutive hours in an environment where loud music is played (>95 dB(A)) after taking either a combination of antioxidants . The aim is to always investigate groups  of peers going out together and therefore attending the clinic within the time range of 7 hours after noise exposure. It is assumed that the pressure of peers also participating in the study will reduce the risk of drop-out. In addition, at the stage of enrolment it will be determined which occasions the subjects will attend and test moments will be scheduled. The aim is to plan all treatments within the range of 3 months in order to reduce the risk of drop-out.2+, or a placebo drug (sugar pill). The antioxidant packets contain two pills of each 300 mg NAC and 100 mg Mg2+. Both pills, providing a total dose of 600 mg NAC and 200 mg Mg2+, should be taken orally 1 hour before noise exposure with a large glass of water. In 50%, the packets contain a placebo with an identical appearance and weight as the antioxidants. All participants (as well as the researchers) will be blinded to the order of antioxidants and placebo. The effects of NAC and Mg2+ at the respective doses of 600 mg and 200 mg will be analyzed as well as the effects of the combined product, for which no significant adverse effects have been reported in the past. Adverse events are defined as symptoms occurring after the administration of antioxidants or placebo that were not necessarily related to the intervention. Participants will receive a telephone number to report adverse events during the study and will be urged to come to the clinic in case of serious adverse events.Participants will receive an antioxidant combination of NAC and MgThe use of alcohol and tobacco is limited to a maximum of two alcohol consumptions and no to two cigarettes. Other psychoactive drugs are strictly prohibited.The inclusion criteria are as follows:• Age between 18 and 28 years old .• Experience of temporary tinnitus after leisure noise exposure in the past scoring at least 5 or more on a visual analogue scale (VAS) for loudness.• Willing to attend an indoor/outdoor music event with mean noise levels ≥95 dB(A) LAeq, 60 min for at least 3 consecutive hours.The exclusion criteria are as follows:• Middle ear pathologies such as otitis media and the perforation of the tympanic membrane and history of such pathologies.2+.• Known allergies for NAC or Mg• Adolescents using hearing protection.• Recreational noise exposure within 4 days prior to a study trial.The production of the antioxidant and placebo packets as well as the randomization of the ABAB protocol will be performed by an independent pharmacist. The packets of antioxidants and placebos will be labeled with a number corresponding with the numbers on the forms. The randomization file will be put into a sealed envelope and retained in a safe at the pharmacy. The randomization table will not be available for assessment by anyone else involved in the study. At the end of the study the envelope will be requested.t-test for mean differences was performed in which an α-level of 0.05 and a nominal power of 80% were used. Assuming a minimal difference of clinical effect of 0.5 between the pre- and post-noise measurements on the VAS, a non-parametric calculation showed a requirement of 21 participants.A power calculation was performed in order to make an estimation of the sample size needed to detect significant differences in the VAS between the experimental and control group by use of the software G-Power. A two-sided paired All data in this trial will be assessed with SPSS Statistics version 20.0  and SAS version 9.1.3 . Because multiple correlated measurements will be performed in the same participants, a mixed effects model will be applied. This analysis is preferable over more traditional approaches such as repeated measures analysis of variance (ANOVA) because of the advantages to deal with missing values. The main analysis will be fully specified before unmasking.The primary outcome measure will be the score on the VAS for tinnitus loudness, which will be measured pre-noise exposure (at the time of antioxidant intake) and 1 hour after noise exposure. The assumed expectation is a drop of 50% of the tinnitus loudness in the antioxidant group compared to the placebo group.The secondary outcome measure is the evaluation of a significant difference in test results of the audiological testing  when comparing the placebo results to the antioxidant trials.A series of audiological tests (described in the following paragraphs) will be performed prior to and within 7 hours after each noise exposure.Tinnitus loudness will be scored on a VAS from 0 to 10 , once at the time of antioxidant (or placebo) intake (1 hour prior to exposure) and once 1 hour after noise exposure. At both occasions the VAS will be undertaken in a quiet room.Pure tone liminar audiometry will be performed according to the current clinical standards  8253-1:2010) using a two-channel AC40 audiometer  in a silent room. Air conduction thresholds will be measured under headphones at 125 Hz, 250 Hz, 500 Hz, 1 kHz, 2 kHz, 3 kHz, 4 kHz, 6 kHz and 8 kHz. In addition, high frequency air conduction thresholds will be performed including 9 kHz, 10 kHz, 11,200 Hz, 12,500 Hz, 14 kHz and 16 kHz. When air conduction thresholds between 250 Hz and 4 kHz exceed normality levels of 20 dB HL, the bone conduction threshold will be measured on 250 Hz, 500 Hz, 1 kHz, 2 kHz, 3 kHz and 4 kHz in order to make a distinction between conductive and sensorineural hearing loss.Transient-evoked otoacoustic emissions (TEOAEs) as well as distortion-product otoacoustic emissions (DPOAEs) will be performed. TEOAEs are elicited with click sounds presented at an intensity level of 80 dB SPL and recorded over a frequency range of 500 to 4,000 Hz. DPOAEs are elicited by use of a set of two pure tone frequencies (f1 and f2) closely spaced and presented simultaneously at a level of 55 dB SPL for f1 and 65 dB SPL for f2. The largest and most robust distortion product is 2f1-f2 and can be detected in almost all normal ears. The amplitude of 2f1-f2 is optimized by use of the frequency ratio f2/f1 = 1.22. DPOAEs will be recorded in the frequency region from 1 kHz to 6 kHz.Speech-in-noise testing will be performed using the Leuven intelligibility sentence test (LIST)  in an adIn order to be able to respect the inclusion criterion of the minimal noise levels of 95 dB(A) LAeq, 60 min for 3 consecutive hours, all participants will be equipped with portable decibel meters . The decibel meter can be fixed onto a shirt or trousers but participants must be aware that the microphone entrance should remain free from overhanging clothing.Written consent will be obtained from each participant. This study protocol was approved by the ethical committee at Antwerp University Hospital in June 2012 with protocol number 12/18/172.According to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines, the authors declare that data that break the blind will not be presented prior to the release of mainline results. The breaking of the blind will occur at the end of the study. A clinical article will be written on the primary  outcomes of the study and results will be disseminated regardless of the magnitude or direction of effect. The present trial is not industry-initiated. As such, there are no publication restrictions imposed by sponsors.In addition, a full study report, anonymized participant-level dataset and statistical code for generating the results will be made publicly available no later than 3 years after the termination of the study for sharing purposes.As adolescents and young adults often do not use hearing protection while being exposed to loud music, the use of preventive antioxidant intake may provide a useful and harmless way to prevent noise-induced hearing damage in this population. Furthermore, when exposed to hazardous noise levels the protection provided by hearing protectors might not be sufficient to prevent hearing damage and antioxidants may provide additive otoprotective effects. Previous research mainly focused on occupational noise exposure. The present study will assess the usefulness of antioxidants during leisure noise activities.2+ levels in the individual. However, the investigators considered that it was very likely that the drop-out rate would be very high when participants needed to give a blood sample at every test moment, which would be eight in total. It is also assumed that it is the acute increase of NAC and Mg2+ prior to noise exposure that provides the extra otoprotective effects and not so much the naturally high or low levels.The authors acknowledge the limitation of not including blood samples in the present protocol in order to control for naturally varying Mg2+ as prophylactic antioxidants for noise-induced tinnitus and noise-induced hearing damage in young people performed in a randomized, placebo-controlled trial. Furthermore, both participants and investigators will be blinded for the sequence of antioxidants and placebo. Thus, this would not influence on one hand the participants in rating the tinnitus loudness and on the other hand the investigators in performing the audiological testing, limiting investigator bias as far as possible.To our knowledge, this study is the first to combine NAC and MgTo date, half of the needed participants have been included in the study. The procedure started in September 2012. The study is anticipated to be completed in April 2014.N-acetylcysteine; NIHL: Noise-induced hearing loss; NIT: Noise-induced tinnitus; RNS: Reactive nitrogen species; ROS: Reactive oxygen species; SNR: Signal-to-noise ratio; SPIRIT: Standard Protocol Items: Recommendations for Interventional Trials; SRT: Speech reception threshold; TEOAE: Transient-evoked otoacoustic emission; VAS: Visual analogue scale.ANOVA: Analysis of variance; CONSORT: Consolidated Standards of Reporting Trials; DPOAE: Distortion-product otoacoustic emission; LIST: Leuven intelligibility sentence test; Mg2+: Magnesium; NAC: The authors declare that they have no competing interests in the publication of the present protocol.AG conceived and designed the study, and undertook data collection and analysis, manuscript writing and final approval of the manuscript. BI conceived and designed the study, and undertook data collection and analysis, manuscript writing and final approval of the manuscript. KW undertook data collection and analysis, critical revision and final approval of the manuscript. PvdH conceived and designed the study, and undertook manuscript writing and final approval of the manuscript.AG is a third year PhD student at the University of Antwerp. BI is a seventh year medical student at the University of Antwerp. KW is a PhD statistician at the University Hospital Antwerp. PvdH is Head of the Department of Otorhinolaryngology and Head and Neck Surgery at the University Hospital Antwerp and Professor at the University of Antwerp.


Objectives: The purposes of this study were: (1) to show the impact of chronic low back pain (CLBP) on physical performance, fear avoidance behavior and depressive symptoms in older adults; (2) to describe the relationships between outcome measurements obtained in this study.Methodology: Ninety-one participants with or without chronic low back pain were included in this study. Only four tests in the Back Performance Scale were used to assess the physical performance of the participants. A Fear Avoidance Beliefs Questionnaire related to physical activity and the Geriatric Depression Scale were also used to examine each subject.Results: The level of performance shown by elderly adults with low back pain was worse than elders without low back pain in the sock test and the pick-up test (p < 0.05). Little correlation between the finger-to-floor test and fear avoidance behaviour related to physical activity was found (p < 0.05). There was little/poor correlation between all performance tests and depressive symptoms (p < 0.05).Conclusions: The findings indicate that CLBP decreases physical performance, but increases fear avoidance behavior and depressive symptoms in elderly adults. At the same time, it was determined that performance level of elderly adults with back pain was related especially with depressive symptoms. The prevalence of LBP in the elderly is not accurately known but is reported to range between 13% and 49%.3 Self-report measures for related physical functions assess the perceived physical status in subjects with low back pain.4 Some studies show a mismatch between self-reported measurements and observed disability.5,6 It was stated that the tests such as muscle strength and range of motion, which are used to measure the functional level in patients with low back pain, remain incapable of determining disability in daily life as they measure determining physical impairment.7,8 The majority of the tests used to assess functional level have aimed to measure trunk mobility and the coordination of speed and movement, in the last few decades in particular.8 Some tests such as progressive isoinertial lifting test, which evaluate the functional level for patients with low back pain require suitable space, time and equipment.9,10 Therefore, it is important to choose the most suitable tests among various functional tests in order to determine performance level of people with CLBP.Mobility is a vital factor in order to be able to perform the activities of daily living. One’s state of mobility affects his/her medical condition and state of well-being.4,9 The Back Performance Scale (BPS) is a useful instrument for determining the performance of the individuals with CLBP.9The assessment of trunk mobility in different activities better reveals the difference between subjects with and without CLBP.11The relationship between the psychological status and spinal pain has not been fully explained. One study found the incidence of depressive symptoms to be higher in community-dwelling elderly people with low back and neck pain compared to elderly people not experiencing without pain.Our main goal in this study, which was planned as a home-based study, was to determine the degree of mobility difficulty faced by older adults with CLBP during activities of daily living and to describe the impact of CLBP. We preferred to use performance-based measurements imitating the activities of daily living for this purpose.We investigated how LBP affects the performance-based functional level, fear avoidance behavior and depressive symptoms in older adults aged 65 and over living in Denizli, Turkey. At the same time, we attempted to detect the relationship between performance-based functional level and the other outcome measurements in the participants with CLBP.This is a home-based personal interview study. Participants aged 65 years or older living in their own residences were included in this study. All subjects were retired or had never worked. Inclusion criteria for the group with LBP were long-term pain (>3 months) and, for the controls (pain-free group), was having experienced no musculoskeletal pain in the last six months. All the subjects were independent with ambulation and activities of daily living. Exclusion criteria for both groups were orthopedic or neurological problems such as inflammatory rheumatic diseases, serious cardiovascular problems, visual impairment, communication problems and regular medication usage.One hundred and twenty two patients who met the inclusion criteria were evaluated out of a total of 167 subjects aged 65 years or older. Thirty-one subjects were excluded from the study because of missing data. Finally, a total of 91 subjects (56 subjects with and 35 without CLBP) composed the population of this study.This was a single-blind study. An examiner noted the demographics and completed the questionnaires. Demographic data were recorded with a prepared questionnaire (Table-I). All performance tests were supervised by another examiner. The second examiner did not know to which group the subject’s belonged. Ethics committee approval for the study was obtained from Ethics Committee of Clinical Researches, Pamukkale University (B.30.2.PAU.0.20.05.09/84).4,9 We used only the following four tests  which could be easily performed. The lifting test entails lifting a box , in which a sandbag with a weight of 5 kg was placed, from floor to the table (at a height of 76 cm.) repetitively for one minute.9 This test was not included due to the difficulty of carrying materials required for the test.The five BPS physical performance tests which involve trunk mobility and combined movement are useful to assess activity limitations in patients with back pain. These tests, which entail harmonious movement of body and lower limbs, begin from different starting positions such as standing and require different angular values of knees.9 Pain intensity was evaluated with the 10 cm Visual Analog Scale (VAS) . Subjects were asked to indicate their general pain intensity on the scale.Each BPS test was scored from 0 to 3 over the four-point ordinal scales according to the observed physical performance level.12,13 We used only the physical activity section from the FABQ since none of the subjects in our study were employed. The physical activities score was classified as low fear (0-14 points) or high fear (15 points or more) according to the median split value defined by Burton et al.14The fear avoidance beliefs were evaluated with The Turkish version The Fear Avoidance Beliefs Questionnaire (FABQ) was developed by Waddel et al.15,16 When the cut-off point was accepted as 14  for this scale, sensitivity and specificity were found as 0.90, 0.94, respectively.17 Therefore, the cut-off point was also accepted as 14 for this study.The Turkish version of the Geriatric Depression Scale (GDS) was used to determine depression in older adults.Statistical Analysis: Descriptive data were presented as mean ± standard deviation (SD) or as a percentage (%). The Kolmogorov-Smirnov test was used to examine the normality of the data. Student’s t test was used to compare demographic variables and FABQ-PA and GDS scores between older adults with/without low back pain. The chi-square test was used to compare 4 tests of the BPS between the two groups. The correlations between the scores of performance tests, self-report questionnaires, pain intensity and pain duration were determined using by Spearman correlation test. The correlations were interpreted as follows: 0.25 or less little if any relationship, 0.26–0.49 poor relationship, 0.50–0.69 moderate relationship, 0.70–0.89 strong relationship, 0.90–1.00 very strong relationship.18 P values less than 0.05 were considered statistically significant.There were 37 females (66.1%) and 19 males (33.9%) with CLBP. There were 14 females (40%) and 21 males (60%) in the controls without LBP. There were no differences between the groups in terms of age, weight, height, BMI and years in education (Table-I) (p>0.05). The mean pain intensity was 5.40 ± 1.93 cm  for older adults with CLBP. The other pain-related information for the CLBP group can be seen in Table-I.The level of performance shown by older adults with lower back pain was worse than elders without lower back pain in the sock test and the pick-up test (p<0.05). There were no significant differences in terms of finger-to- floor test and the roll- up test between two groups (p> 0.05). The scores of fear avoidance beliefs related to physical activity and depression were also found to be higher in older adults with CLBP (p<0.05) compared free-pain older adults (Table-II).No correlation was found between the individual test scores and pain severity, and pain duration in the group with CLBP. Little correlation was found between the finger- to- floor test and fear avoidance behavior associated with physical activity (p<0.05). Little/ poor correlation was found between the individual test scores and depressive symptoms (p<0.05) (Table-III).Our results point out that back pain among older adults influence specific functional activities about activities of daily living. Moreover it was determined that performance level among older adults with back pain is not related with pain and fear avoidance behavior (except to finger-to-floor) but related with depressive symptoms at low level. 19,20 On the other hand, we concluded that there is no significant difference between older adults with and without pain in the finger-to-floor and the roll-up tests. The finger-to-floor test is a test that entails shoulder and hip movement in addition to body mobility.21 At the same time, the roll-up test, which requires adopting a sitting position, is related to abdominal muscle strength.22 Flexibility and strength evaluation was not carried out as part of our study. There is a possibility that similar muscle strength and flexibility values may have affected outcomes in both groups. Our results obtained from the sock test and the pick- up performance tests are in conformity with literature.6,23,24 Reneman et al. found that the relationship between pain, pain-related fear and functional performance level is generally poor or not significant. Investigators reported that patients performed the functional tests despite their pain. 6 Geisser et al. determined that there is a meaningful relation between floor to waist lifting section and fear of Progressive Isoinertial Lifting Evaluation which is used in order to evaluate performance but there is not a meaningful relation between waist to shoulder section and fear.10 This result demonstrates that the applied test battery affects pain-related fear behavior. Magnussen et al, determined that there is no correlation between fear in low back pain and the total score of BPS. Authors explained this situation in the way that fear associated with low back pain is associated with the future rather than the current situation but BPS is not predictive.4 Our results show that despite low correlation, fear behaviour may occur due to physical activity and mental state influences performance level. Previous literature and our study show that many factors such as low educational level of the study population, brevity of the implemented tests, high motivation and chosen tests influence results.2,6,11,25There are a few studies which analyse the relation between performance level, fear avoidance behaviour and depressive symptoms in patients with low back pain.As far as we know, this is the first home-based study which was conducted in Turkey in this field which is a strength of our study. Moreover in this study we have analyzed the relation between performance based functional tests, fear avoidance behaviour and depressive symptoms in older adults with low back pain. This makes our study quite important.Limitation of the study: There are some limitations of this study. The most important limitation is excluding lifting test which is the most challenging item of The Back Performance Scale. On the other hand, that we obtained meaningful results in only two tests among four tests we have used which made it difficult for us to make comment about the effectiveness of BPS in determining activities of daily living and relative performance level. The fact that the test battery which we used only evaluates movements in the sagittal plane is regarded as the limitation of our study. On the other hand, Magnussen et al. found a high correlation between BPS and the Der Funktionsfragenbogen Hannover Questionnaire, which contains twisting and side bending works in addition to items similar to four items of BPS. Therefore, they surmmarized that evaluating movements in different planes in addition to BPS is not necessary in low back pain.4It is quite important to determine the level of inability in performing functional activities of older adults with back pain in their own home environment where they spend most of the time. For this aim, it would bring more accurate results to evaluate people in their own living environment based on observation. However, since only two out of four tests we have used in measurement have significant results show that more comprehensive studies which are suitable for the daily activities of older adults with back pain in home environment should be carried out. This helps us to understand how the CLBP interfers with daily living activities. Further studies are needed to confirm our finding.


ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.Menkes disease (MD) is caused by mutations in the  ATP7A gene. The severe classical form of MD is characterised by progressive neurodegeneration, connective tissue abnormalities, distinctive “kinky” hair and ultimately death in early childhood. Though most patients (90–95%) exhibit the severe clinical course, there are various milder atypical forms with longer survival of the affected patients . The Occipital Horn Syndrome (OHS), mainly characterized by connective tissue manifestations is the mildest form Menkes disease (MD) is an X-linked, multisystemic lethal disorder of copper metabolism caused by mutations in the ATP7A mutations (from single amino acid substitutions to large deletions and chromosome aberrations) has been identified in patients with classical or one of the milder MD forms ATP7A mutations are splice site mutations A broad spectrum of Splice site mutations are DNA sequence changes that alter or abolish correct mRNA splicing during the process of precursor mRNA maturation. Splice site mutations can result in the complete skipping of one or more exons, retention of introns, creation of a pseudo-exon, or activation of a cryptic splice site within an exon or an intron ATP7A gene including 33 novel mutations, identified in patients with classical MD, atypical MD or OHS. Studies of splice site mutations are highly relevant in order to identify a correlation between the effects of splice site mutations and patient phenotypes, to enable a prediction of the severity of the disease, and to design therapies. It has been proposed that MD patients might especially benefit from early copper therapy when some functional ATP7A protein is present in vivo, and 61 mutations with the bioinformatics tool, Human Splicing Finder (HSF). We compared the in silico predictions with in vivo results and with the observed phenotypes of the patients.This study presents a comprehensive overview of 61 splice site mutations in the The 33 novel splice site mutations were found in a cohort of MD patients who were referred to the Kennedy Center for molecular and/or biochemical diagnosis. Mutations were identified by PCR amplification and direct sequencing of the 23 exons using an ABI3130XL sequencer .ATP7A and designed to amplify those exon(s) that were expected to be affected by the mutation, and the flanking exons. The cDNA fragment spanning from exon 6 to exon 9 was PCR amplified from 8 patients . The cDNA fragment spanning from exon 12 to exon 16 was PCR amplified from patient 32. The cDNA fragment spanning from exon 18 to exon 23 was amplified from 3 patients (cases: 58–60). Primer sequences can be obtained upon request. The PCR products were separated on a 1% agarose gel. The relevant bands were purified, and sequenced using an ABI3130XL sequencer .Fibroblast cell cultures obtained from patient-skin biopsies were grown in a 1∶1 mixture of RPMI 1640 with 20 mM HEPES and a nutrient mixture of F-10 Hams medium, supplemented with 7.5% Amnio Max (Life Technologies), C100 supplement, 4% fetal calf serum, penicillin, and streptomycin. RNA was isolated with RNeasy , and single-stranded cDNA was synthesized with the High-Capacity cDNA Archive Kit in accordance with the manufactureŕs instructions . To investigate the effect of selected splice site mutations, RT-PCR was carried out with primers specific for ATP7A splice site mutations. All were analysed with HSF version 2.4 http://www.umd.be/HSF/). Only those mutations that are located in the defined splice site consensus sequences are included here. The consensus sequences are (C/A)AG|gt(a/g)agt and cag|G for the donor splice site and the acceptor splice site, respectively ATP7A transcript ENST00000341514 from Ensembl. A in the ATG start codon is defined as c.1. The relative strength of the splice sites obtained from the bioinformatics tool is given as the consensus value (CV), which ranges from 0 to 100. Splice sites with CVs higher than 80 are strong splice sites; less strong sites have CVs that range from 70 to 80. Splice sites with CVs of 65 to 70 are weak, as only a few of these sites are active We have compiled 61 ATP7A gene in patients with Menkes disease or Occipital Horn Syndrome, we identified a total of 33 novel splice site mutations. They are listed in When screening the coding region of the ATP7A transcript processing in 12 patients, from whom fibroblast cultures were available  . Other mutations lead to the use of a cryptic splice site  or to several different transcripts with a combination of exon-skipping and the use of a cryptic splice site . The majority of the reported cases lead to the production of out-of-frame transcripts and hence, probably to non-functional protein. However, in some cases , a limited amount of wild-type transcript is produced alongside the mutant transcript. Only in two cases, leading to atypical MD and OHS respectively , wild-type transcript could not be detected. No wild-type transcript was identified in any of the classical MD cases analysed .We determined the effect of the mutations on , 58–60) . In totaTo explore whether a bioinformatics tool can be used to predict the effect of the genomic mutations on mRNA processing, and possibly the severity of the disease, we compared the experimental results and the observed phenotypes with the predictions obtained with the Human Splicing Finder.The Human Splicing Finder results are listed in In contrast, among the mutations identified in the 6 atypical MD patients and the 6 OHS patients, only four mutations (33%) lead to a ΔCV reduction over 10% (or 7% for +4 mutations) . One of these mutations (16) also leads to a splice site CV below 70 and, as expected, no detectable wild-type transcript in the fibroblast material. In 67% of the mutations that lead to the mild phenotypes, the splice site CVs were above 70 in combination with ΔCV reductions below 10%, indicating that these sites are active . In agreIn 15 cases  the CV- and ΔCV-parameters contradicted in the prediction of activity of the splice site. Mutations at the +5 position in particular, are responsible for a large fraction of these cases, as they constitute half of the total inconsistent incidences. In fact, the predictions for 8 out of 9 +5 point mutations are inconsistent. This indicates that the determination of both the CV and the ΔCV is essential.As splice site mutations may lead to the use of cryptic splice sites, potential splice sites in the vicinity of wild-type splice sites were identified with HSF. Most cryptic splice sites are likely to be located +/− 100 bp on each side of the exon-intron boundary ATP7A in relation to their effect on transcript processing and patient phenotype. Of the 57 mutations identified in patients with known clinical phenotypes, 21% of the mutations lead to the milder phenotypes, atypical MD or OHS  allow the production of some amount of wild-type transcript, whereas a CV below 70 combined with a ΔCV reduction over 10% completely block the production of wild type transcript, 96% of the mutations identified in classical MD patients are predicted to have a significant effect on splicing with no production of wild type transcript. However, this is only the case for 33% of the mutations identified in patients with the milder phenotype. Overall, when using the CV parameter in combination with the ΔCV parameter, 51 of 57 mutations (89%) fit the hypothesis that mild phenotypes develop when wild-type transcript is produced, whereas classical MD is the phenotypic outcome when no wild-type transcript is produced . Only, iIn approximately one third of the represented exon-intron boundaries (8 of 26) the CV for one or more of the potential splice sites identified with HSF is higher than the CV for the un-mutated wild-type splice site. A similar observation has previously been reported in e.g. the β-globin intron 1 In case 2, HSF found several possible alternative donor splice sites , in the vicinity of the wild-type splice site. We have previously reported that a cryptic splice site at position +50 is used in the mutated sequence in this patient in silico predictions and in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript correlates with a milder phenotype. Notably, disease-causing mutations identified in patients with mild phenotypes might be difficult to distinguish from benign mutations, which do not cause disease. However, if the family history confirms beyond any doubt, that a splice site mutation is the cause of disease, HSF is very successful in predicting the phenotype.In conclusion, both the


Attention deficit hyperactivity disorder (ADHD) symptoms can be difficult to treat. We previously reported that a 20-session brain-computer interface (BCI) attention training programme improved ADHD symptoms. Here, we investigated a new more intensive BCI-based attention training game system on 20 unmedicated ADHD children  with significant inattentive symptoms (combined and inattentive ADHD subtypes). This new system monitored attention through a head band with dry EEG sensors, which was used to drive a feed forward game. The system was calibrated for each user by measuring the EEG parameters during a Stroop task. Treatment consisted of an 8-week training comprising 24 sessions followed by 3 once-monthly booster training sessions. Following intervention, both parent-rated inattentive and hyperactive-impulsive symptoms on the ADHD Rating Scale showed significant improvement. At week 8, the mean improvement was −4.6 (5.9) and −4.7 (5.6) respectively for inattentive symptoms and hyperactive-impulsive symptoms (both p<0.01). Cohen’s d effect size for inattentive symptoms was large at 0.78 at week 8 and 0.84 at week 24 (post-boosters). Further analysis showed that the change in the EEG based BCI ADHD severity measure correlated with the change ADHD Rating Scale scores. The BCI-based attention training game system is a potential new treatment for ADHD.NCT01344044ClinicalTrials.gov  Attention deficit hyperactivity disorder (ADHD), a childhood onset developmental disorder, is a chronic condition that can extend into adulthood We previously reported the results of this approach in a small controlled trial In our first study we found that intervention with a training program involving the BCI-based attention training game system improved parent-reported inattentive symptoms. We also noted that behavioural improvement was sustained 3 months after the intensive 20-session intervention. The initial study was 10 weeks in duration with 2 sessions per week. It also involved the use of a very simple game and a tethered connection to a computer. Since then, we have developed a new version of the device that is simple, uses dry EEG electrodes and is connected by Bluetooth to the computer. A new game that could be calibrated based on the performance of the child was also developed. In addition it was important to understand who would be the right candidates to benefit from this approach.Therefore, the purpose of this study was to investigate ifThe new device with a new game and a more intensive new training schedule 3 sessions per week over 8 weeks was acceptable to patients.There is preliminary evidence of efficacy in improving ADHD symptoms.There is any clinical predictor of response.There is any EEG change as a function of response.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see This study was approved by the ethics review boards of the Institute of Mental Health  and National Healthcare Group, Singapore . Written informed consent from parents and assent from children were obtained prior to study entry .This was a one-arm prospective study and we aimed to enroll 20 children, who would receive treatment with the BCI-based attention training program. The Consort Flow Diagram is shown in We invited the clinic’s psychiatrists to refer patients clinically diagnosed to have the inattentive or combined subtypes of ADHD based on the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-IV) for the study. During the screening phase, parents completed the Diagnostic Interview Schedule for Children Version IV (DISC-IV) A subject was eligible for inclusion in the study only if all the following criteria applied at pre-study screening:Subject’s age was within the age range of 6–12 years old;Subject had never received treatment with stimulant medication or Atomoxetine;The subject should satisfy the following criteria for the diagnosis of ADHD:DSM-IV-TR criteria for ADHD, either the combined or inattentive subtype, based on clinical assessmentDiagnostic Interview Schedule for Children (DISC), as completed by the parents;Written Informed Consent from parent and Assent Form from child were both obtained;Subject and the parent/guardian were willing to comply with study procedures and were able to return to the clinic for scheduled visits.A subject was not eligible for inclusion in the study if any of the following criteria applied at pre-study screening:Present or history of medical treatment with stimulant medication and Atomoxetine;Co-morbid severe psychiatric condition or known sensori-neural deficit e.g. complete blindness or deafness (such that they could not play computer games);History of epileptic seizures;Known mental retardation (i.e. IQ 70 and below);Predominantly Hyperactive/impulsive subtype of ADHD (i.e. no predominant inattentive symptoms).We recruited 20 participants for the study, including 16 males and 4 females. The mean age was 7.80 . There were 17 Chinese, 2 Eurasians and one Malay. Fourteen children were diagnosed to have the combined subtype of ADHD based on C-DISC, and the other 6 had the inattentive subtype of ADHD.The BCI system consisted of a headband with mounted dry EEG sensors  that transmitted EEG readings to the computer through Bluetooth-enabled protocol. The headband was worn around the forehead, with a grounding reference electrode clipped to the earlobe see . Two dryThe possible effects of noise or artifacts such as extraocular activity on the EEG were considered and reduced in the BCI system. Since the noise and artifacts were generally uncorrelated with the attentiveness and in-attentiveness conditions, they were filtered out in our machine learning algorithm that extracted only discriminative features from EEG between the two conditions. To further reduce the electrooculography artifacts, we added a virtual EEG channel, which was the differential potential between Fp1 and Fp2. As a result, the system was not affected by normal eye movements.Prior to playing the video game (CogoLand), which was the main training activity, each participant underwent individual calibration using a colour Stroop task on the BCI-based attention training game system. During calibration, the participant performed the colour Stroop task to develop an individualized EEG profile of the optimal attentive state. The colour Stroop task required one to use the mouse to click on the name of the colour in which a word was spelt, and not the colour that the word spelt. The BCI-based attention training game system analyzed the critical EEG parameters during the correct attempts, compared to that when the participant was relaxing, to derive an individualized EEG pattern representing the participant’s most attentive state A computerized 3D graphic game, CogoLand was developed specifically as the training game. In CogoLand, the participant controlled an avatar via the signals detected by the EEG electrodes. This was computed into a BCI ADHD Severity Measure, or BASM (see next section for details of its computation). The BASM was then transformed to a score ranging from 0 (minimum attention) to 100 (maximum attention), which was reflected on the computer screen. The participant would hence need to ‘concentrate’ in order to move the avatar, which would move at a speed proportional to the participant’s attention level as measured by the BCI-based attention training game. The ‘higher’ the concentration level of the participant was, the higher would be the speed of the avatar’s movement. There were three difficulty levels in CogoLand. The main goal of the first level was to make the avatar run around an island in the shortest time possible. The next two levels had an additional component where the child needed to collect a series of fruits floating in the air as the avatar navigated through a pre-determined route in a colorful town. The child would use a specific key on the keyboard to make the avatar jump to collect the fruits which would appear along the journey. The child was asked to collect as many fruits as possible within a given timeframe, after which the number of fruits collected was entered into a personal logbook. At the third level the child had to collect the fruits in an order presented on the screen. A short break was allowed between attempts. For each training session, the individual would complete 30 minutes of training, including the breaks.All raw EEG data obtained during calibration with the Stroop task was analyzed by the BCI system. It was screened to detect any abnormality in the EEG recordings, such as disconnected electrodes and saturated digital samples. Any abnormal EEG readings, including the readings within two seconds from the occurrence of the abnormality were excluded from analysis. The system then extracted discriminative rhythmic power features from the screened EEG using spatial-spectral filtering.The system examined an array of 8 frequency bands, continuously covering from 4 Hz to 30 Hz. This arrangement not only covered traditional EEG bands from theta to beta, but also had a finer grid of frequency bands. Band powers were computed using the following procedure. First, the EEG data was segmented into a continuous sequence of 2-second long time blocks; in each block, power spectrum was computed using a 256-point Fast-Fourier-Transform technique; a specific band power was calculated as the sum of the spectrum powers at all the discrete frequencies in the band; and the specific band power was the average value of the band power over the time blocks. The band power was calculated for each EEG channel separately, in addition to the differential potential between the two channels. The BCI system then selected the band power features for maximizing the separation between attentive and inattentive states according to the information theory. A regression function would be applied by the BCI system to transform the selected features into a BASM score, which represented the severity of the inattentive symptoms of ADHD at the time of EEG recording. The BASM score was inversely proportional to the severity of the inattentive symptoms and the lower the BASM score, the more inattentive the individual was.We used this BCI-based attention training game system for our intervention program (the BCI-based attention training program), which comprised of an intensive phase with 3 training sessions weekly for 8 weeks, followed by a maintenance phase with once-monthly booster training sessions for 3 consecutive months. At the end of every alternate training session starting from the second session, each participant would complete 2 short English and Mathematic worksheets consisting of multiple choice type questions on the computer. These worksheets were appropriate to their educational level and took approximately 10 minutes to complete. Each participant was instructed to concentrate like when they were playing the games, and their EEG was monitored during this period. Treatment was administered by 3 therapists trained to fit the headband and administer the BCI-based training program. All the therapists had obtained at least a graduate degree in psychology, and they administered treatment according to a standardized treatment protocol. Calibration was done at weeks 0, 4 and 8.th edition (ADHD-RS) At baseline, parents completed the 18-question ADHD Rating Scale, 4There were 17 (85%) subjects who completed the entire study. One boy dropped out before 4 weeks, as the parent felt there was no improvement in the child’s behaviour. Two other boys dropped out between 4 and 8 weeks due to difficulty adhering to the treatment schedule.We conducted Intention-To-Treat (ITT) analyses on the results and excluded the subject who dropped out before week 4 as there was no follow up data at all. We carried forward the last available observation  where appropriate. Multiple imputations using Markov chain Monte Carlo and a per protocol analysis including only subjects who completed the study were also conducted and the results did not show much difference from that based on the last observation carried forward method.There was no statistically significant change in parental observation of inattentive and hyperactive-impulsive symptoms on the ADHD-RS at 20 weeks compared to 8 weeks, or at 24 weeks compared to 20 weeks. When examining the ratings at 24 weeks, compared to the baseline score, there was significant improvement in parent-rated inattentive and hyperactive-impulsive symptoms (mean changes = −5.0 (5.8) and −5.7 (5.1) respectively and p≤0.01 for both IA and HI). These results appear to suggest that monthly booster training for 3 consecutive months after an intensive 8-week training did not significantly improve inattentive or hyperactive-impulsive symptoms further. The behavioural benefits from the intensive training at 8 weeks were sustained at 24 weeks. The child’s age and gender did not have any statistically significant effect on ADHD-RS scores in this study.We used EEG data from the calibration/re-calibration sessions (at week 0 and 20) and examined the BASM scores. We had to exclude the 3 participants who dropped out before week 8 and 3 other patients who had missing EEG data at Week 20. Thus, we analyzed a total of 14 participants from the original 20 recruited. When comparing the BASM scores at Week 0 and at Week 20, there was an increase in the mean score (standard deviation) from 60.9 (81.0) to 96.9 (64.7), although paired t-test showed that the change was not statistically significant (mean change = 32.5 (60.8), p = 0.067).Linear regressions showed that baseline IA, HI and COM scores statistically significantly predicted their respective changes of ADHD rating scale scores from week 0 to 8 (β (SE) = −0.7 (0.2), −0.9 (0.3) and −0.9 (0.3) respectively and p = 0.013, 0.008 and 0.007 respectively). Thus, a higher score on the ADHD-RS at baseline predicts greater improvement at week 8. We investigated the possible correlations between the change in BASM scores and the changes in scores on ADHD rating scale from Week 0 to Week 20. Correlation analyses performed in The intervention was also well tolerated. In this trial, the main side effect reported was headache, which affected two of the participants. The severity of the headache was reported to be mild and did not stop them from continuing with treatment.The present study evaluated the new version of the BCI-based attention training program, which included dry sensors and blue tooth technology in place of EEG leads with a game CogoLand, in the treatment of combined and inattentive subtypes of ADHD. Our results show that an 8-week intervention significantly improved inattentive symptoms of ADHD, based on a behavioural rating scale by parents. Among children with the combined subtype of ADHD, parents also reported a significant improvement in their hyperactive-impulsive symptoms on the ADHD Rating Scale. When these children received monthly training sessions subsequently, the behavioural improvements were sustained but did not further improve. Those with more severe symptoms were also the ones who showed greater improvement.This built upon our initial report on the training program’s positive effects on treating ADHD symptoms. Although shown to improve inattentive symptoms more than hyperactive-impulsive symptoms then, we now found significant treatment effects for both inattentive and hyperactive-impulsive symptoms of ADHD. It is possible that non-specific factors like behavioural contingencies, self efficacy and a structured training environment could have contributed to improvement in the hyperactive-impulsive symptoms. Children with both inattentive and combined subtypes, which are far commoner than the hyperactive-impulsive subtype, appear to improve with treatment.Additionally we also found that the change in BASM score correlated with the change in behavioural rating score by parents. This provided some evidence that the improvement with training, as reflected by the BASM score, might explain the improved ADHD symptoms reported by parents. The BASM score also appeared to be a good surrogate marker for observed inattentive behaviour. In many ways this is a refinement from previous work that looked at specific EEG bands. Neurophysiological studies have previously shown that children with ADHD exhibit specific patterns on the electroencephalogram (EEG) and this has been utilized clinically to diagnose, treat and even predict response to treatment with medication BCI-based attention training game system can offer several advantages over current evidence-based treatment options offered in most clinical practices. It has less adverse events compared to medication. Unlike behavioural management or parent training, there is no need for regular clinic visits which can be inconvenient. The system potential utility adds to the growing research in an area that was a precursor to this approach namely neurofeedback. Computer-based neurofeedback attention training programmes have been shown to improve inattentive symptoms There are some important limitations to our present study. This is an uncontrolled open-label trial and thus the parents who completed the behavioural rating scale were not blinded. This could have resulted in an exaggerated treatment effect. Unfortunately the non-response rate from the children’s schoolteachers was too high for the results to be interpreted meaningfully. A well-designed randomized controlled trial is needed to evaluate the efficacy of BCI-based attention training program in treating ADHD.Brain computer interface based attention training game can be a potential new treatment for ADHD. A randomized controlled trial to study the efficacy of this intervention and the neural mechanisms underlying the behavioral improvements is currently underway. It represents a novel treatment modality for ADHD, which not only has the potential for being used in combination with present evidence-based treatment, but also uniquely in a non-clinical setting.Protocol S1Trial protocol.(PDF)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.