Motivation: Exome sequencing has proven to be an effective tool to discover the genetic basis of Mendelian disorders. It is well established that copy number variants (CNVs) contribute to the etiology of these disorders. However, calling CNVs from exome sequence data is challenging. A typical read depth strategy consists of using another sample (or a combination of samples) as a reference to control for the variability at the capture and sequencing steps. However, technical variability between samples complicates the analysis and can create spurious CNV calls.Results: Here, we introduce ExomeDepth, a new CNV calling algorithm designed to control for this technical variability. ExomeDepth uses a robust model for the read count data and uses this model to build an optimized reference set in order to maximize the power to detect CNVs. As a result, ExomeDepth is effective across a wider range of exome datasets than the previously existing tools, even for small (e.g. one to two exons) and heterozygous deletions. We used this new approach to analyse exome data from 24 patients with primary immunodeficiencies. Depending on data quality and the exact target region, we find between 170 and 250 exonic CNV calls per sample. Our analysis identified two novel causative deletions in the genes GATA2 and DOCK8.Availability: The code used in this analysis has been implemented into an R package called ExomeDepth and is available at the Comprehensive R Archive Network (CRAN).Contact: v.plagnol@ucl.ac.ukSupplementary Information:Supplementary data are available at Bioinformatics online. The improvement of DNA sequencing technologies in recent years has radically changed the identification of genetic variants associated with human diseases and in particular, rare disorders . Read deGATA2 gene and a single-exon homozygous deletion in the DOCK8 gene.Here, we evaluate two different exome sequence datasets and show that Gaussian assumptions generally do not hold. Technical variability at the library preparation, capture and sequencing creates noise that affects the numbers of reads matching to particular exons in a sample-specific manner. As a result, the observed variance exceeds what is predicted by a binomial model that affects the CNV calls. Motivated by this observation, we propose a modified and more robust statistical framework for CNV calling. We apply this model to provide guidelines for the construction of an optimized reference sequence dataset for CNV calling purposes, as well as realistic power estimates. We find that two main factors improve statistical power: increasing the read depth and controlling for any source of technical variability across samples at the capture and sequencing steps. We have developed and coded a new set of tools in an R package called ExomeDepth. We then illustrated its efficiency by discovering novel small causative CNVs in two patients with primary immunodeficiencies, a heterozygous deletion of two exons of the Supplementary Table S1 and Section 3). An overview of a normalized measure of read depth [matching fragments per million reads and per kilobase, FPKM, . This beta-binomial model significantly improved the fit  and increasing the bias (by adding exome samples to the reference in spite of being less correlated). In Dataset 1 A we founSupplementary Fig. S2), a difference also commonly observed for all array-based CNV assays. Both datasets lacked power to detect single-exon heterozygous duplications , but larger duplications could be identified. For example, in Dataset 1, and to some extent in Dataset 2, a three-exon heterozygous duplication typically can be detected . Homozygous deletions are naturally easier to detect than heterozygous deletions. Although quantifying the power is complicated by the arbitrary parameterization of the background level of mapping reads, we found that under most realistic assumptions, an expected number of reads greater than 30 mapping to an exon in the test sample was sufficient to identify a homozygous deletion in either of the two datasets.ad depth C and D. ad depth B–D compaad depth C and D. Supplementary Fig. S3). We found the total number of CNV calls to be relatively stable over the range of parameters considered , only increasing sharply for a prior expectation of 1000 CNV calls genome-wide. The ExomeDepth default parameter uses a relatively stringent prior expectation of 20 CNV calls per exome sequence. With this choice, ExomeDepth called a median number of 213 CNVs per sample in Dataset 1, including 62.3% deletions. Consistent with the more limited power in Dataset 2, ExomeDepth identified a lower median number of 177 CNV calls in this dataset (62.9% of them deletions), in spite of a 31.5% larger target region (50 Mb versus 38 Mb). CNVs called by ExomeDepth in Datasets 1 and 2 included a median number of five exons and a median length of 10.6 kb. About 10% of the CNV calls were longer than 100 kb and 0.1% were longer than 500 kb.The probability for the hidden Markov chain to enter a cn ≠ 2 state sets the sensitivity/specificity balance of ExomeDepth. We parameterized this value using the expected number of CNV calls for an exome sequence , .Table 1To estimate the false negative rate, we used an additional dataset of 12 high-depth exome samples (1000 Genomes Project) for which an independent experiment generated CNV calls using a high-density Nimblegen CGH array [. Second, ExomeDepth detected 75.2% of the known exonic CNVs in the 12 exome samples from the 1000 Genomes Project (CNVs identified by the independent aCGH experiment). This was markedly higher than the fraction of CNVs identified by exomeCopy (52.8%) and ExomeCNV  containing 26 probes in the GATA2 gene region. In this patient we validated a heterozygous deletion of 6 kb that included GATA2 exons 6 and 7 .We then investigated if any of the newly discovered rare CNVs in our data affects genes that previously have been involved in primary immunodeficiencies. In patient P1 ExomeDepth identified a heterozygous deletion of the consecutive exons 6 and 7 of the quantile A. Indepe 6 and 7 B. We theDOCK8 gene . This CNV call had a posterior probability >99.99%. Complete absence of reads mapping to exon 8 is indicative of a homozygous deletion. We sequenced the deletion breakpoints and identified the exact boundaries of a 3197-bp deletion . The clinical presentation is consistent with previous reports involving homozygous mutations in the DOCK8 .In another patient (P2), ExomeDepth identified a deletion of a single exon 8 of the We investigated 24 patients who suffered from severe and/or disseminated recurrent infections, and have been diagnosed with primary immunodeficiencies (PIDs). Of these patients, 13 were of European descent and 11 patients were of Asian descent, of which, 5 originated from consanguineous families. All material from patients was obtained with informed consent from adults and from the parents of children who participated in the study in accordance with the Declaration of Helsinki and with approval from the ethics committees .www.novocraft.com). Single sample summary statistics are provided in Supplementary Table S1.We isolated DNA samples from blood or peripheral blood mononuclear cells (PBMCs). Exome sequence data have been generated in two batches. Dataset 1 comprises exomes from 15 patients and Dataset 2 comprises exomes from 9 patients. For exome target enrichment Agilent SureSelect 38 Mb and 50 Mb kits have been used for samples in Datasets 1 and 2, respectively. Samples in both datasets have been sequenced using Illumina HiSeq with 94-bp paired-end reads. Reads were aligned to the hg19 reference sequence using the software novoalign (Exon locations were defined using the Ensemble database release version 57 (human genome build hg19). We only considered autosomal chromosomes (chr1–22) to avoid the additional complication of gender . Analysis of the GC content for each exon uses the same Ensembl release and the Perl Ensembl API tool. We used the R package Rsamtools to extract the read count information from the individual BAM files. All reads were paired-end. We only included consistent paired reads (i.e. both reads located <1000 bp away from each other and in the correct orientation) and with Phred scaled mapping quality ≥20. The location was defined by the middle location between the extreme ends of both paired reads. Exons closer than 50 bp were merged into a single location owing to the inability to properly separate reads mapping to either of them. After merging close exons, we considered a total number of 229 056 autosomal exons.i denotes the exon and the covariate i: the proportion of reads mapping to the test sample for deletions/duplications is computed based on the expected proportion for normal copy number and assuming a read ratio of 0.5 (for a deletion) or 1.5 (for a duplication). A motivation for our work is the fact that this binomial model does not fully capture sample specific biases. We propose instead the robust beta-binomial model (3)Supplementary Fig. S1). We therefore, modified the model to allow the parameter Supplementary Figure S1 describes this fitting process in more details.However, an analysis of the data showed that a single a and b [mean We fitted the binomial logistic model described in e models B and C, a-priori, i.e. the probability of transitioning from cn = 2 to cn = 1 or cn = 3 is n = 229 056 is the total number of exons. We set the default model such that, from the hidden state cn = 2, the probability to move into a deletion state is the same as the probability to move into a duplication state. In a deletion/duplication state, the underlying Markov chain has a default probability 0.5 to revert back to cn = 2 and a probability 0.5 to remain in the same deletion/duplication state. To provide a set of calls for each sample, we use the maximum likelihood Viterbi algorithm. Each version of our statistical models in For each exon, our beta-binomial model generates a likelihood value under three distinct scenarios . To combine the likelihood across multiple exons we used a hidden Markov model. Each step of the hidden Markov state corresponds to one exon in the human genome. This model serves the double purpose of merging CNV calls across exons, as well as specifying a prior probability of observing a CNV for each exon. This prior probability is coded into the transition probability of the hidden Markov chain between the normal copy number state (cn = 2) and either of the copy number variable states (cn = 1 or cn = 3). We parameterized this before using the expected number ne of CNVs Supplementary Figure S2. Following the parameter estimation step, we can generate for each CNV, and given the test and reference read count data X and Y, a Bayes factor BF = P = Owing to the common particular interest in discovering loss-of-function variants we considered a heterozygous deletion as a typical scenario for our power estimations . The hetWe downloaded from the 1000 Genomes a dataset of 12 high-depth exome samples generated using the Solid sequencing technology (single-end reads), for which an independent experiment generated CNV calls using a high-density Nimblegen CGH array , the aggregegate reference set  consisted of the remaining exomes from the same dataset. We then applied the classify.eCNV function with default parameters setting the admixture rate to 0 (because we are not concerned by a mixture with tumour DNA). Finally, multi.CNV.analyze was used to obtain the final list of merged calls from the list of exonic CNV calls. For exomeCopy the read count data was estimated using the R built-in functions provided by this package. For each sample, the background noise was estimated on the basis of GC content and the read count data from the other exomes in the same dataset. We fitted the negative-binomial model and the hidden Markov chain using the steps recommended in the package vignette and the default parameters. For the exomeCopy analysis, the recommendation to split long exons into smaller units  increased the number of CNV calls and the concordance with DGV dropped, strongly suggesting that this step did not improve the overall accuracy. We therefore used our set of exon delimited regions to compute the read count.We have developed a novel CNV calling methodology using read depth information from exome sequence data and implemented it within an R package called ExomeDepth. This allowed us to maximize the statistical power to detect even small CNVs in the presence of technical variability inherent to high-throughput DNA sequencing technologies. As a consequence, compared with other tools, we found the greatest improvement for datasets that show more technical variability across samples. Although ExomeDepth is designed to be used as a standalone software, the contruction of a reference set is a problem shared across all read depth CNV calling algorithms for exome data. Therefore, additional refinements proposed by other CNV calling algorithms to improve calling accuracy could potentially be applied after ExomeDepth has identified the optimum aggregate reference set.Because ExomeDepth assumes that the CNV call is not present in the reference sample, it is best suited to call rare CNVs. Nevertheless, our analysis of exome samples from the 1000 Genome Project indicates that it can call common CNVs as well, even though some power is lost when the allele frequency is high. Our computations indicate that the power to detect rare CNVs is maximized for a reference:test ratio of ∼10:1. Hence, while we find no obvious benefit in using a very large dataset (> 100 exomes), it is essential to generate exome data in batches of six or more samples.Our analysis shows that a Gaussian model for the read count data is not appropriate for exome sequence data, which is likely to explain the discrepancy with the observed larger number of CNV calls generated by ExomeCNV. The discrepancy with exomeCopy is more surprising, because exomeCopy fits a robust negative binomial model related to our model. Additionally, in the exomeCopy analysis, we used the optimized reference set identified by the ExomeDepth analysis. The default parameters of the exomeCopy hidden Markov chain were also similar. Comparing both methods, we find that the main difference is that exomeCopy essentially takes a profile likelihood approach: it uses the median normalized read depth at each exon to account for the variability in exon capture efficiency, which is a nuisance parameter. However, the median read depth can be unreliable, if the sample size is small and/or the data are noisier (e.g. in Dataset 2). Instead, in ExomeDepth we used a logistic model, which deals with that nuisance parameter by conditioning on the total read count for each exon. We hypothesize that exomeCopy would show results more similar to ours, if the exon-specific parameters were estimated within the negative binomial model rather than prior to model fitting. However, the number of exons, and therefore, the number of parameters to estimate, would be large which makes it difficult in practice.In our study the statistical power to detect CNVs varied extensively between two datasets and was largely determined by sample-to-sample variability within datasets that emerged either at the exome capture or at the sequencing step. This feature of the data cannot be detected by commonly used single-sample quality metrics: it is the correlations across samples rather than the single-sample summary statistic that are relevant. Owing to its essential role for CNV calling, we argue that a measure of sample-to-sample consistency  should be provided by sequencing facilities when exomes are analysed in sufficiently large batches.GATA2 and DOCK8 deletions illustrates the power of ExomeDepth to identify even heterozygous and small CNVs comprising just one to two exons. We conclude that reducing technical variability between the samples and using bioinformatics tools that maximize statistical power of CNV detection, such as ExomeDepth, will allow efficient CNV identification and will increase the value of the future exome sequencing experiments.Finding of the


In Denmark the placement of chest tubes is routinely performed by the surgical specialties, namely thoracic, orthopaedic and abdominal surgeons. At our emergency department (ED) we have trained emergency physicians in the placement of chest tubes in patients presenting with a pneumothorax. We postulate that patient discomfort associated with dyspnoea from a pneumothorax can be mitigated by shortening the time from diagnosis to definitive therapy. Our study is retrospective and observational in design, and evaluates the placement of chest tubes by an emergency physician in the ED when compared to a surgeon performing the same procedure in the operating room (OR).All adult patients admitted to the hospital from our ED between 1-JAN-2010 to 31-APR-2011 with the diagnosis of pneumothorax were retrieved.The time from pneumothorax confirmed by X-ray to placement of chest tube (ΔtDrain) was calculated. The two groups were compared using two tailed unpaired student’s t-test.Thirty patients were admitted to the hospital from our ED with the diagnosis of pneumothorax. Of these 30 patients, 13 were excluded: five were misdiagnosed, four were observed without treatment, two had delayed insertion of chest tubes following progression of the pneumothorax, and two had chest tubes inserted at the ED with the assistance of surgeons.Of the 17 patients included, 12 had chest tubes inserted by surgeons in the OR, and 5 by emergency physicians at the ED. No complications were described. The mean ΔtDrain was 4:01 hours (range 00:46-9:31) when chest tubes were inserted by surgeons in the OR, which was significantly longer than ΔtDrain of 1:54 hours (range 0:45-3:20) when inserted by emergency physicians at the ED (p=0.04).This study is limited by the few numbers of patients studied, its retrospective design, and is unable to detect possible differences in the frequency of complications between the two study groups. Nevertheless it clearly indicates that ΔtDrain might be diminished substantially when chest tubes are inserted by emergency physicians at the ED. We therefore suggest that insertion of chest tubes should be mastered by emergency physicians, and that it should be a standard procedure at the ED.


Fmr1 KO mice during the Erasmus Ladder task. Fmr1 KO mice showed deficits in associative motor learning as well as avoidance behavior, both of which were rescued by intraperitoneal administration of Fenobam. While the Fmr1 KO mice did benefit from the treatment, control littermates suffered from a significant negative side effect in that their motor learning skills, but not their avoidance behavior, were significantly affected. On the basis of these studies in the FXS animal model, it may be worthwhile to investigate the effects of mGluR inhibitors on both the cognitive functions and procedural skills in FXS patients. However, the use of mGluR inhibitors appears to be strongly contraindicated in healthy controls or non-FXS patients with intellectual disability.Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. Patients with FXS do not only suffer from cognitive problems, but also from abnormalities/deficits in procedural memory formation. It has been proposed that a lack of fragile X mental retardation protein (FMRP) leads to altered long-term plasticity by deregulation of various translational processes at the synapses, and that part of these impairments might be rescued by the inhibition of type I metabotropic glutamate receptors (mGluRs). We recently developed the Erasmus Ladder, which allows us to test, without any invasive approaches, simultaneously, both procedural memory formation and avoidance behavior during unperturbed and perturbed locomotion in mice. Here, we investigated the impact of a potent and selective mGluR5 inhibitor (Fenobam) on the behavior of  FMR1)  (Fragile X syndrome (FXS) is the most common genetic form of mental impairment , affectin (FMRP) . Besidesn (FMRP) , the symα-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors may facilitate long-term plasticity and is stimulated by the synthesis of novel proteins after the activation of mGluRs  using the multifunctional, motor-cognitive assay on the Erasmus Ladder.A ladder rung task provides comprehensive assessment for skilled limb movements in mice  to test hybrid mice with 50% FVB/Ant and 50% C57Bl/6 contribution. Because the FVB/Ant strain is pigmented and devoid of the genetic predisposition to retinal degeneration of the FVB/N strain, these mice show clear visual evoked potential in the presence of normal eye histology and improved performance in the Morris water maze test (Fmr1 KO(2) line, unlike the first generation of Fmr1 KO model, does not express any FMRP and lacks detectable Fmr1 transcripts (N-(3-chlorophenyl)-N’-urea], which is a clinically validated non-benzodiazepine anxiolytic drug, is a selective and potent mGluR5 receptor antagonist acting at an allosteric modulatory site shared with 2-methyl-6-(phenylethynyl)-pyridine (MPEP) (Fmr1 KO and wild-type (WT) mice were assigned to different groups: one treated with Fenobam in MC and another one treated with the vehicle (MC only). In addition, we also tested the performance of Fmr1 KO and WT littermates without any application as control.Fenobam [The Erasmus Ladder is a fully automated test for detecting motor performance, associative motor learning deficits and cognitive phenotypes in mouse models. The Erasmus Ladder consists of a horizontal ladder in between two shelter boxes, which are equipped with a bright white LED spotlight in the roof and two pressurized air outlets . Both, light and air stimuli are used as cues for departure. In addition, one of the air outlets is used to control the speed of the mice and to prevent them from leaving the shelter box at the opposite side and crossing the ladder at unwanted moments . The ladder has 2 × 37 rungs for the left and right side. All rungs are equipped with pressure sensors (produced at Erasmus MC), which are continuously monitored and which can be used to register and analyze the walking pattern of the mouse instantaneously. Moreover, based upon the prediction of the walking pattern, the rungs can be moved up or down by a high-speed pneumatic slide (Pneumax) with a maximum of 13 mm at any moment in time. The computer system , which runs the real-time system recording sensor data, adjusts air pressure, predicts future touches, calculates interventions, repositions slides and stores data, operates in a fixed cycle of 2 ms. Details of the device and its operations have been published (During the first 4 days (‘unperturbed sessions'), mice were trained with the even-numbered rungs on the left side and the odd-numbered rungs on the right side in a descended position so as to create an alternated stepping pattern with 30-mm gaps. Mice were trained to walk the ladder for 72 runs per day. We calculated the number of missteps that were sensed by the descended rungs, and steptime, which is defined as the time needed to place one of the front paws from one rung to the other (i.e. onset of touch until onset of following touch). Associative motor learning trials (‘perturbed sessions') started on day 5 using a 15 kHz tone as conditioning stimulus  and a rising rung as the US (ascending 12 mm). The interstimulus interval was fixed at 285 ms. To keep this time period constant, we observed in real-time the speed of the mouse and calculated which rung should rise. Mice typically learn that increasing walking speed avoids being hit by the rung, so mice will decrease their pre-steptime (nearest steptime before the onset of the US) and post-steptime  through the sessions.Apart from motor coordination deficits, the Erasmus Ladder is able to detect cognitive phenotypes in mice. Cognition is tested by determining the capability of mice to respond to the cues of departure (light or air) and to modify this response under certain circumstances. Thereby, unperturbed sessions that are neutral in the beginning of the experiment , turn inanova) or two-way repeated measures anova, as appropriate. If a significant difference was found, post hoc analysis was performed using Fisher's least significant difference (LSD) test, unless stated otherwise. In total, 39 Fmr1 KO and their 41 WT littermates were measured in the Erasmus Ladder. Four mice were excluded from the analysis of motor performance and motor learning because the computer failed capturing several post-steptime values that could have changed the averaged post-steptime values. Three mice were excluded from the avoidance behavior analysis because they were not measured properly.Off-line analyses of motor coordination and avoidance behavior were performed using custom written software in Labview  and the results were stored in a relational database (MySQL). Statistical tests were performed with SPSS Statistics . Data were compared using one-way analysis of variance (Fmr1 KO mice (n = 35) and their WT littermates (n = 41) did not show significant differences in steptimes   (data not shown).To rule out the potential caveat that possible differences in motor learning or avoidance discrimination are partly due to differences in motor performance, we started out by testing the overall motor performance level on the Erasmus Ladder. Unperturbed sessions (s1–s4), that were used to evaluate motor performance capabilities, showed that s anova) . In addin = 17), which learned to adjust their walking pattern to the CS over the sessions, Fmr1 KO (n = 16) mice did not learn to do so  . The Fmr1 KO mice did not show any motor learning during the perturbed sessions  (Fmr1 KO mice did not show a clear reduction in post-steptimes compared with the first perturbed session (t8post: 557.97 ± 72 ms vs. t5post: 663.84 ± 82 ms). The difference in post-steptimes between WT and Fmr1 KO mice did not depend on a difference in steptimes before the onset of the CS–US stimuli as shown by the pre-steptime values which were not statistically different  (In contrast to WT mice (s anova) . Alreadyy anova) . Even dus anova) .Fmr1 KO and WT mice. The interaction genotype × treatment was statistically different . Fmr1 KO mice treated with Fenobam (n = 10) showed faster post-steptime responses during the perturbed sessions than those that were injected with vehicle (n = 9)  (n = 11)  . The overall level of motor performance remained intact during the perturbed sessions in that there was no significant interaction genotype × treatment neither in the number of    Fenobam or MC as vehicle to both oc test) . Moreoveoc test) . This ims anova)  nor in ps anova) .n = 13) was severely impaired. In this group, post-steptimes increased significantly compared with animals that were not treated  or that received vehicle   (data not shown).In contrast, the motor learning in Fenobam-treated WT (oc test) . Again, n = 41) and Fmr1 KO (n = 32) mice used light and air as cues to leave the box and to start to walk on the ladder at a similar level  . These changes reflect avoidance behavior in order to delay the exposure to the perturbation. In Fmr1 KO mice this avoidance behavior did not occur; they did not show any change in their reactions, neither to the light nor to the air stimuli   . Fmr1 KO mice treated with Fenobam decreased the percentage of occasions that they reacted to the light stimulus with respect to vehicle-treated Fmr1 KO mice from 29.78% ± 8.72 to 4.46% ± 0.73  and significantly increased their percentage of responses to the air stimulus from 57.77% ± 10.11 to 82.35% ± 4.17  during perturbed sessions; moreover, they were not significantly different from vehicle-treated WT mice  .During unperturbed sessions WT (y anova) . Howevery anova) ; in liney anova) . Importaoc test) . This imFmr1 KO mice to treatment with Fenobam while performing locomotion conditioning tasks showed that the Erasmus Ladder can be used to test different types of learning simultaneously and to assess the impact of different drugs at a medium to high throughput level. Our main findings are that Fmr1 KO mice show deficits in both associative motor learning and avoidance behavior and that Fenobam, which is a selective mGluR5 inhibitor, can rescue both deficits. In addition, we show that Fenobam treatment of WT mice showed profound side effects in the motor coordination task. Together, these findings are in line with the mGluR hypothesis, and they offer, as will be discussed below, impetus to potential therapies of both motor and cognitive symptoms in FXS patients.Subjecting Fmr1 KO mice showed a marked deficit in procedural memory formation in that they did not reduce significantly their post-steptime response during the perturbation training sessions. Given that there were no differences in steptimes during the unperturbed sessions, we can conclude that the higher values of post-steptimes of Fmr1 KO mice were not due to motor performance deficits. The deficits in locomotion conditioning on the Erasmus Ladder agree with the findings by Fmr1 KO mice and FXS patients show deficits in classical delay eyeblink conditioning. The abnormalities of the Fmr1 KO mice in locomotion conditioning were rescued after administration of 30 mg/kg of Fenobam, 30 min before the learning task started. In fact, Fmr1 KO mice were able to reduce significantly their post-steptime response to an auditory CS, achieving values that were close to those of WT mice. As application of vehicle  did not significantly affect motor behavior in the Fmr1 KO mice, it is parsimonious to conclude that the therapeutic impact of Fenobam in Fmr1 KO mice on procedural memory formation was not due to the induction of stress or other sham effects that might have been induced by the injection itself.Fmr1 KO mice also showed a marked deficit in avoidance behavior in that they did not show the normal waiting reaction inside the box; they did not delay the exposure to the suddenly occurring aversive stimulus, i.e. the perturbation due to the rising rung. WT mice tend to remain inside the boxes as long as possible during the unpleasant perturbation sessions. They usually do not leave the box when the light is turned on and they need the air stimulus significantly more often to force them out of the box as compared to the unperturbed performance task. Fmr1 KO mice instead did not modify their responses to the light and air departure cues after they were transferred from the unperturbed to the perturbed sessions. It appeared as if they did not fully perceive and/or did not know how to react to a stressful situation. This type of apathetic reaction might point toward a decrease in anxiety and/or a decrease in fear memory. This possibility is in line with other FXS mouse model studies in which a general lower level of anxiety and/or general deficits in fear memory formation were observed (Fmr1 KO mice (observed . ImportaFmr1 KO mice improved both their procedural memory formation and avoidance discrimination after administration of Fenobam, WT mice showed a clear negative side effect in that their procedural memory formation was severely impaired. Similarly, Jacob et al. reported impairments in the passive avoidance test, the Morris water maze and contextual fear conditioning following administration of similar dosages of Fenobam (While Fmr1 KO mice show deficits in both procedural memory formation and avoidance discrimination and both deficits can be rescued with Fenobam. On the basis of this mouse model study, it can be inferred that the use of mGluR inhibitors may be beneficial for procedural memory formation and avoidance discrimination in FXS patients, but it appears contraindicated for healthy controls or non-FXS patients with intellectual disability.Testing locomotion conditioning and avoidance discrimination in mutant mice can be performed reliably and noninvasively with the use of the Erasmus Ladder, and the impact of drugs on these tests can be screened at a medium to high throughput level.


The presence of an extra whole or part of chromosome 21 in people with Down syndrome (DS) is associated with multiple neurological changes, including pathological aging that often meets the criteria for Alzheimer’s Disease (AD). In addition, trisomies have been shown to disrupt normal epigenetic marks across the genome, perhaps in response to changes in gene dosage. We hypothesized that trisomy 21 would result in global epigenetic changes across all participants, and that DS patients with cognitive impairment would show an additional epigenetic signature.APP gene itself, to see whether there were changes in DNA methylation in this population. Using the Illumina Infinium 450 K Human Methylation Array, we examined more than 485,000 CpG sites distributed across the genome in buccal epithelial cells.We therefore examined whole-genome DNA methylation in buccal epithelial cells of 10 adults with DS and 10 controls to determine whether patterns of DNA methylation were correlated with DS and/or cognitive impairment. In addition we examined DNA methylation at the TSC2 gene that has previously been associated with Alzheimer’s disease pathology. We found no enrichment on chromosome 21 in either case, and targeted analysis of the APP gene revealed weak evidence for epigenetic impacts related to the AD phenotype.We found 3300 CpGs to be differentially methylated between the groups, including 495 CpGs that overlap with clusters of differentially methylated probes. In addition, we found 5 probes that were correlated with cognitive function including two probes in the Overall, our results indicated that both Trisomy 21 and cognitive impairment were associated with distinct patterns of DNA methylation. Down syndrome (DS) occurs in approximately 1 out of every 600 live births in the US and is the most prevalent genetic cause of developmental disabilities -4.Postmortem studies show that from the age of 40 upward, individuals with DS are at much higher risk than the general population of having neuropathological changes that meet the clinical criteria for Alzheimer’s Disease (AD) -7. TheseAPP promoter is specifically hypomethylated in brain tissues from AD patients [One mechanism by which cells may respond to changes in gene dosage is altered DNA methylation. DNA methylation is one of a group of epigenetic modifications to the genome which affect the ability of specific genes to be expressed but do not modify the sequence of the genome itself. One of the best-characterized effects of DNA methylation is its contribution to the inactivation of one entire X chromosome in females, which restores dosage equality with XY males. Methyl groups are added to CpG dinucleotides, and these modifications in turn recruit chromatin remodeling complexes which alter the structure of the surrounding chromatin, either increasing or decreasing the availability for the gene to be expressed. Changes in DNA methylation are associated with both normal aging and with Alzheimer’s Disease -16. Addipatients . Previoupatients -20.We hypothesize that altered DNA methylation on chromosome 21 and across the genome may be associated with accelerated cognitive aging in DS. To that end, we examined a cohort of 10 adult participants with DS and 10 age- and sex-matched controls. We evaluated cognitive function and collected cheek swabs from each participant. Genome-wide DNA methylation patterns were analyzed using the Illumina 450K Human Methylation Array, which interrogates over 480,000 CpG dinucleotides in the genome, including over 4000 on Chromosome 21 itself, and were correlated with scores related to cognitive function.This study was approved by the University of British Columbia Clinical Research Ethics Board. Participants with DS were recruited from the Down Syndrome Research Foundation (DSRF), in Burnaby, B.C. Informed consent was provided by either a parent or guardian, and assent was obtained from the participant. Age and gender matched control participants were recruited from the staff and students at the Child and Family Research Institute (CFRI) in Vancouver, B.C. A total of 20 adults between the ages of 27–46 years of age, 10 with DS  and 10 controls  participated in this study. All participants were financially compensated for parking and travel costs.The Dalton Brief Praxis Test (BPT) is an abbreviated, 20-item version of the Dyspraxia Scale for Adults with Down’s Syndrome, a 62-item cognitive test of praxis. It scores the ability to perform simple, highly practiced, voluntary movements in response to a verbal command or imitation, and therefore measures verbal comprehension and motor co-ordination and is used to monitor changes in cognitive function. The BPT was administered by a trained research assistant.Buccal (cheek) swabs were collected from each participant using standard protocols, and DNA isolated using the Isohelix Buccal DNA isolation kit  as per manufacturer’s instructions. DNA was then purified and concentrated using the DNA Clean & Concentrator kit . Approximately 750 ng of DNA was used for bisulfite conversion using the Zymo Research EZ DNA Methylation Kit . Sample yield and purity was assessed after each step using a Nanodrop ND-1000 . After bisulfite conversion, 160 ng of DNA was applied to the Illumina 450 K methylation array, as per manufacturer’s protocols .in silico nonspecific binding to the X or Y chromosomes [Data was subjected to stringent quality control before being normalized in R . First, omosomes . Togetheomosomes . ComBat omosomes ,25. Corri across all samples, aij are the projection values of each probe i onto the eigenvector j. The top ranked PCs can often be correlated with known traits in the cohort such as tissue type, cellular composition, or disease state. Because PCs are linearly independent, a particular PC’s contribution to each probe’s methylation pattern can be subtracted out without altering the information contained in the pattern arising from all the others. For this dataset, PCA was performed on the normalized data set twice; first on the 10 DS and 10 control samples and a second time with ten additional blood samples added. These blood samples were from unrelated healthy individuals of the same approximate ages. As described in more detail in the result section, the initial PCA without the blood samples revealed an unusual clustering of samples in the first PC, possibly indicating blood contamination of the buccal swabs. The second PCA including blood samples showed that indeed, some of our buccal swabs had scores more similar to blood than other buccal swabs for PC1. Since the dependency of methylation on tissue represented by that PC1 for this study is a confounder, its contribution to each probe was subtracted out, yielding methylation data that no longer has variation due to tissue differences. A dataset k is subtracted out is constructed as Principal Component Analysis (PCA) decomposes the measured methylation patterns into a set of linearly independent principal component (PC) patterns that are ranked according to how much variance in the data they explain. The methylation pattern of each probe All statistical analysis on normalized and corrected data was performed using R statistical software (version 3.0). Probes with DNA methylation levels significantly different between DS and control participants were identified first using the R limma package’s moderated t-tests with empirical Bayesian variance method and Benjamini-Hochberg correction to control the false discovery rate at 0.01 . SignifiAPP probes specifically were performed in R using a 2-sided Spearman correlation, and p-values were corrected with Benjamini-Hochberg or Bonferroni correction, as noted. For whole-genome correlations, probes were considered significantly correlated with BPT if the Benjamini-Hochberg corrected p-values were below 0.01 and the range of highest to lowest beta values was greater than 10%. T-test were performed using the base t-test function in R [Correlations between BPT scores in DS participants and DNA methylation levels both for all probes and for the ion in R .All statistical analysis was performed on transformed M-values . All valSignificant probe accession names were input into the DAVID online GO clustering tool . Only tht-test p-value of 0.90 showed good matching of cohort ages. Praxis scores were not significantly correlated with age in the DS cohort (Spearman’s correlation p = 0.55).Scores on the BPT ranged from 35 to 80 for the 10 participants with DS, and all 10 control participants received a score of 100  Figure a. Age waPrincipal component analysis (PCA) was used to determine the sources of variance across all samples and probes. Initial PCA revealed that the 5 participants with low BPT scores had markedly different scores for the first PC, which accounted for 64.8% of the variation, when compared to controls or the remaining DS participants Figure b. This aIn order to analyze the true differences between the DS participants and controls, as well as the differences correlated with BPT, it was important to eliminate this possible cell type-related confounder. To control for this, we subtracted from our data set the effects of PC1 from the PCA that included the blood samples (see Methods). This eliminated the variance due to tissue contamination while leaving any real DNA methylation differences between DS and control and any correlations with cognitive impairment. After subtraction we repeated the hierarchical clustering and noted that the distance between samples was reduced, reflecting the reduced variation in the modified data set  . We founTFAP2B had 26 out of a total of 45 probes on the array significantly different between DS and control, 13 of which overlapped with Bump Hunter clusters, and all of which were more methylated in DS by a range of 10-35%. The significant probes are located between 1.5 kb upstream of the TSS to 8 kb downstream. This gene was found in a GWAS study to be associated with type 2 diabetes, and was further characterized to be involved in glucose uptake and insulin resistance in adipocytes [DLX5 and its near neighbor DLX6-AS had a total of 42 probes in the DMPs, with 28 of these in the BH-DMPs. DLX5 and DLX6 have been identified as being important in neural crest differentiation, including GABAergic neurons of the developing forebrain and craniofacial development, particularly jaw development [TNXB has 23 probes significantly different between DS and control, all of which are also found in the BH-DMPs. This gene is an extracellular matrix protein responsible for Ehlers-Danos syndrome, which has been hypothesized to have clinical overlap with DS [CPT1B with 12 CpGs in the DMP and BH-DMP lists, is a carnitine palmitoyltransferase specifically expressed in mitochondria of skeletal muscle and associated with metabolic syndrome and lipid deposition [Within the DMPs and BH-DMPs, we found a number of genes known to have functions related to the pathology of DS. ipocytes ,33. DLX5elopment ,35. TNXB with DS . Finallyposition ,38. Reflt-test for differences between DS and control at a p-value of 0.05, for a final total of 4 probes , one of which had less than 5% methylation in all samples, and the remaining three were all located in intron 1  that are differently methylated in fetal trisomy 21 skin and muscle, and five genes  that are differentially methylated in trisomy 18 skin [HOXA2, CPT1B, and GRM6 were found in all three studies  gene is present on our hit list, there were 106 total adhesion-related genes in our DAVID hit list. DSCAM has been proposed to contribute to the congenital heart disease feature of DS, and it is possible that mis-regulation of a large number of adhesion-related genes through epigenetic modification may explain the increased risk in the DS population [The fact that an epigenetic signature that includes genes with functions related to the biology of DS is strong support for validity of our approach. The top four CpG clusters as mentioned in the Results section were related to diabetes, lipid metabolism, neural crest and craniofacial development, and connective tissue, all of which are connected to clinical features of DS. Our DAVID analysis further supported this, with enrichment clusters overlapping these functions as well as regulation of apoptosis and skeletal development. The massive enrichment for genes involving adhesion in our hit list for differentially methylated probes is interesting as, while a single CpG site in the Down Syndrome Cell Adhesion Molecule (pulation . It is apulation ,19. It cRND1 is a Rho GTPas that functions to promote dendritic cell growth and axon guidance, and loss of RND1 expression suppresses axon formation in hippocampal neurons [RND1 was positively correlated with BPT, meaning that lower levels of DNA methylation were associated with more severe cognitive impairment. If DNA methylation suppresses RND1 expression, the opposite pattern might be expected in DS participants, but without knowing the mechanistic relationship between RND1 expression and DNA methylation, it is difficult to interpret. The other gene, TSC2, had two CpGs in an island that overlaps with exons 27, 28, and 29 that showed decreasing DNA methylation with cognitive impairment. TSC2 is involved in mTOR signaling, which regulates levels of tau in mouse neuronal models, resulting in neuropathological symptoms which are alleviated when mTOR signaling is reduced [TSC2 gene product, are decreased in brain samples of adult males with DS and AD-like symptoms, and a mouse knockdown of Tsc2 showed an increase in tau-positive axon formation in hippocampal neurons [TSC2 was shown to result in an increase in expression [TSC2 in DS participants is associated with the decreased protein expression, which results in an increased probability of accumulation of tau through mTOR regulation, which predisposes these patients to AD-like disease. Thus RND1 and especially TSC2 are interesting targets for future studies of epigenetic alterations in cognitive impairment.Given that only five probes in four genes were found to be both correlated with BPT scores in DS participants and different between DS and controls, it is impossible to assess functional enrichment. Two of these four genes, however, have known functions in neural development or degeneration.  neurons ,42. We f reduced ,40,46,47 neurons ,49. In cpression . On the pression . We can Cross-sectional studies such as these are important to discover associations between DNA methylation and cognitive impairment. Given our relatively small sample size, we were surprised to find such clear differences between our study groups, but in the future, it will be important to perform larger and more powerful longitudinal studies on patients with DS and AD to track the dynamics of DNA methylation changes during cognitive decline. Post-mortem brain tissue analysis would also shed further light on the relationship between epigenetics and cognitive decline in these patients. Together, these data will help illuminate whether DNA methylation is changed with cognition, whether altered DNA methylation is a predictive factor for cognitive decline, or whether the two processes occur simultaneously.DS: Down syndome; AD: Alzheimer’s disease; GO: Gene ontology; BPT: Brief praxis test; PCA: Principal component analysis; DMP: Differentially methylated probe; DMR: Differentially methylated region; BH-DMP: Bump-hunter differentially methylated probe.The authors declare that they have no competing interests.MJJ performed the data analysis and drafted the manuscript. PF performed the PCA and PC1 subtraction. KW collected the samples and performed the BPT assessment. SN, LMM, and JLH processed the samples, performed the arrays, and did preliminary data analysis. EE conceived the PCA analysis and assisted with statistical analysis. MC participated in the study design and edited the manuscript. NVB conceived the study and design, recruited the participants, and contributed to analysis and interpretation of the data. MSK conceived the study and design, and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1755-8794/6/58/prepubTable of correlation coefficients and p-values for correlations of PCs after subtraction of PC1 and BPT scores or presence of DS.Click here for fileTable of CpG probes that are significantly differently methylated between DS and control participants.Click here for fileTable of CpG probes that are significantly differently methylated between DS and control participants and overlap with clusters found to be significantly differently methylated by Bump Hunter.Click here for fileDAVID results for probes that are significantly differently methylated between DS and control participants.Click here for fileTable of probes that are significantly correlated with BPT.Click here for fileOverlap of our DMPs with DMPs from the Kerkel study .Click here for fileOverlap of our DMPs with DMPs from the Jin study .Click here for file


Preemptive therapy with ganciclovir (GCV) based on the results of a cytomegalovirus (CMV) antigenemia assay is a standard strategy for preventing CMV disease after allogeneic hematopoietic cell transplantation (HCT). However, the appropriate threshold of antigenemia-positive cells for deciding when to start GCV remains unclear.This retrospective study included 80 recipients who received HCT from an alternative donor between 2007 and 2011. In 2009, we switched the threshold from 3  to 20  antigenemia-positive cells per two slides for preemptive therapy after HCT from an alternative donor.Early CMV disease within 100 days after HCT was observed in one patient in the 20A group. Antiviral agents including GCV, val-GCV, and foscarnet were given in 17 (71%) and 36 (64%) patients in the 3A and 20A groups, respectively (p=0.23). In 13 (23%) patients in the 20A group, the initiation of preemptive therapy was avoided because of the change in the cutoff value for CMV antigenemia. However, the total dose of GCV was not different between the two groups. The use of steroid was significantly associated with CMV antigenemia of at least 20 positive cells among patients with low-level antigenemia at the first detection.The increased threshold up to 20 positive cells for starting preemptive therapy was not associated with a significant increase in CMV disease, but the total dose of GCV was not reduced and there was one early CMV disease in the 20A group. We should explore how to identify patients who are at high risk for increased antigenemia among patients with low-level antigenemia, but at least, preemptive therapy should not be withheld in patients who are already receiving systemic steroid. Cytomegalovirus (CMV) infection is one of the major complications after allogeneic hematopoietic cell transplantation (HCT). Monitoring of CMV reactivation with a CMV antigenemia –4 or a rTakenaka et al. monitored CMV infection and CMV-associated disease in patients who received related or unrelated donor bone marrow transplantation using CMV antigenemia assay without any prevention or preemptive therapy for CMV . They shWe previously performed a randomized controlled trial to compare plasma real-time PCR and the antigenemia assay for monitoring CMV reactivation in bone marrow transplantation from HLA-matched unrelated donors . In thatWe retrospectively collected data regarding adult patients who received allogeneic HCT from alternative donors at our center from June 2007 to December 2011. A CMV antigenemia-guided preemptive approach for CMV disease had been applied in all of them. Alternative donors included all donors other than HLA-matched related donors. Unrelated PBSCT was not available in Japan during the study period. Exclusion criteria were as follows: patients who received HCT from cord blood or HLA haploidentical donors, patients who received alemtuzumab in the conditioning regimen, patients who failed to achieve neutrophil engraftment, and donor and recipient pairs who were CMV antibody-negative. The mismatched related donors in this study included all the donors who were HLA 1 allele, 1 antigen, or 1 antigen and 1 allele mismatched. These patients were not included in the haploidentical transplantation protocol and treated with a method similar to that for HLA-matched transplantation .5 peripheral blood leukocytes were attached to a slide using a cytocentrifuge and fixed with formaldehyde. The cells were sequentially immunostained with monoclonal antibody C10/11, which raised against CMV pp65 antigen, and reacted with goat alkaline phosphatase-labeled anti-mouse immunoglobulin . These cells were analyzed under a light microscope and the results are presented as the sum of the number of positive cells per two slides. An antigenemia assay was performed at least once a week after engraftment. The criterion for engraftment was the achievement of peripheral blood neutrophil count of 0.5 x 109/L or more for 3 consecutive days. Preemptive therapy with GCV or valganciclovir (VGCV) was started when more than the threshold level of CMV-positive cells were detected. In April 2009, we changed the threshold number of antigenemia-positive cells from 3 to 20 per two slides in HCT from alternative donors, whereas it was consistently 20 in HCT from HLA-matched related donors.A CMV antigenemia assay using MoAb C10/C11 was performed as described previously . BrieflyAs preemptive therapy, the induction dose of GCV or VGCV was 5 mg/kg/day or 900 mg/day, respectively. Preemptive therapy with a low initial dose of GCV has been shown to be effective even in high-risk patients in a previous study . In caseWe divided alternative donor recipients into 2 groups according to the two different cut-off values of CMV antigenemia-positive cells for preemptive therapy: the 3A-group and the 20A-group. “A” stands for alternative and indicates the donor type. We also used this higher threshold in patients who underwent HCT from HLA-matched related donors (20R-group).We retrospectively analyzed the incidence of early CMV diseases, defined as CMV diseases occurring before day 100 of HCT. The diagnosis of CMV diseases was made by histopathological examinations. However, CMV retinitis could be diagnosed when characteristic retinal changes were found by ophthalmoscopy. The diagnostic criteria for specific CMV infections have been explained elsewhere .U-test and Kruskal-Wallis test for continuous variables. The effect of steroid on the development of CMV antigenemia of at least 20 positive cells per two slide was evaluated with Cox proportional hazards modeling while treating the use of systemic steroid as a time-dependent covariate. Statistical significance was defined as a 2-sided P-value of <0.05. All statistical analyses were performed with EZR , which is a graphical user interface for R . More precisely, it is a modified version of R commander (version 1.6-3) that was designed to add statistical functions that are frequently used in biostatistics [The numbers of days to events were calculated from the date of HCT. Comparisons between groups were performed with Fisher’s exact test for categorical variables and the Mann–Whitney atistics .This study was approved by the institutional review board of Jichi Medical University. Written informed consent was obtained from each patient to be stored in the database and used for research.There were 24 and 56 patients in the 3A and 20A groups, respectively. Overall, the median age was 45.5 years. The 20A group included significantly more patients with HLA-mismatched unrelated donors and tended to be older  and to receive more bone marrow graft  and 36 (64%) patients in the 3A, and 20A groups, respectively p=0.62, . The medThree patients received GCV or foscarnet for possible virus infection, regardless of the CMV antigenemia level. In one case, the therapeutic dose of GCV was started for respiratory failure of unknown etiology because viral pneumonia could not be denied. Eventually, the cause of lung infiltration was considered to be body fluid retention associated with tacrolimus. The number of CMV antigenemia-positive cells was one at that time. In the other two cases, therapeutic doses of GCV or FCV were started for the disturbance of consciousness of unknown etiology because viral encephalopathy could not be denied. In the first case, CMV antigenemia-positive cells were not detected at that time. The etiology of altered mental status could be tacrolimus-induced encephalopathy, but it was not clear even later. In the second case, CMV antigenemia positive cells could not be checked at the onset because the neutrophil count was too low to perform the assay. The cause of disturbance of consciousness was considered to be bacterial meningitis and posterior reversible encephalopathy syndrome. If we excluded these 3 patients, GCV or VGCV was given in 17 (71%) and 34 (61%) patients in the 3A and 20A groups, respectively (p=0.45). The cumulative dose of GCV was also not significantly different between these groups (data not shown).Preemptive therapy was withheld in 35 patients in the 20A group at the first detection of CMV antigenemia of between 3 and 19 positive cells per two slides. In 13 of these 35 patients, preemptive therapy was avoided because the number of positive cells did not reach 20 (AVOID group). The other 22 patients received preemptive therapy a median of 7 days later, since the number of antigenemia-positive cells eventually reached at least 20 (DELAY group). The cumulative dose of GCV in the DELAY group was 135 mg/kg/day , which was higher than that in patients in the 3A group who received GCV at the first detection of CMV antigenemia of between 3 and 19 positive cells per two slides , although this difference was not statistically significant.There were no significant differences in the patient characteristics between the AVOID group and the DELAY group, including sex, background disease, CMV status, conditioning regimen, use of TBI or ATG, or the presence of grade 2 to 4 acute GVHD, except that the DELAY group included significantly more patients who used systemic steroids within 100 days after HCT  and the number increased to 96 on day 35. Preemptive therapy was started immediately, but the number of CMV-positive cells jumped to 456 on day 42 and the dose of GCV was increased to 10 mg/kg. The patient became febrile from day 42 and the oxygenation level became worse. The patient died on day 45. The autopsy showed inclusion bodies of CMV in the lungs and the intestine.With regard to late CMV disease, one case of CMV enteritis (day 164) and one case of retinitis (day182) were observed in the 20A group. Both were treated with GCV and improved.We also collected data of 39 patients who received HCT from HLA-matched related donors (20R-group) for comparison with the 20A-group. The incidence of CMV reactivation (46%), GCV/VGCV/foscarnet use (36%), and the median cumulative dose of GCV  in the 20R group were significantly lower than those in the 3A and 20A groups (data not shown). Early CMV disease was not found in the 20R group.We retrospectively analyzed the safety of increasing the cutoff value of CMV antigenemia-positive cells from 3 to 20 in preemptive therapy for CMV disease. As expected, 23% of the patients with a high cutoff value could avoid GCV administration without a significant increase in early CMV disease. However, the cumulative dose of GCV in the 20A group was not significantly reduced compared to that in the 3A group. This could be explained by the relatively high cumulative dose of GCV used in the DELAY group.It is difficult to decide upon an appropriate cutoff value for preemptive therapy. The current study showed that the unnecessary administration of preemptive therapy could be avoided by increasing the cutoff value in a subset of patients with positive antigenemia, but other patients eventually required preemptive therapy with at least a one-week delay. Although there was no statistically significant evidence that delaying in starting preemptive therapy induced an increase in incidence of CMV disease, delaying treatment provided no benefit to patients, since the cumulative dose of GCV was not decreased. Therefore, it is important to be able to identify patients who are at high risk for a subsequent increase in antigenemia-positive cells. There was no difference in the background characteristics between the AVOID group and the DELAY group. However, the use of systemic steroid was associated with a significantly higher incidence of CMV antigenemia of at least 20 positive cells per two slides, with a hazard ratio of 4.63. Therefore, preemptive therapy should be started as soon as systemic steroid is started, whereas patients who do not require systemic steroids could be good candidates for withholding preemptive therapy in the presence of low-level CMV antigenemia. In fact, more than half (56%) of the patients who did not receive steroid could avoid preemptive therapy with an increase in the threshold of antigenemia-positive cells.Although there was no significant difference in the incidence of CMV disease, one patient in the 20A group developed early CMV disease. He developed interstitial pneumonia before the first detection of positive antigenemia, and CMV disease was diagnosed by autopsy. Therefore, we could not determine the onset of CMV pneumonia. This patient developed severe acute GVHD and was heavily treated with high-dose corticosteroids, and therefore, the development of CMV pneumonia might be a later event. However, in such patients, antiviral agents should be started earlier.In conclusion, the increased threshold up to 20 positive cells for starting preemptive therapy was not associated with a significant increase in CMV disease. However, there was one case of early fatal CMV disease that was possibly related to the increase in the cut-off value. In addition, delaying the start of preemptive therapy did not result in the decreased use of GCV. On the other hand, subsets of patients could avoid the unnecessary use of GCV. We should explore how to identify patients who are at high risk for increased antigenemia at the first detection of low levels of positive cells. At least, preemptive therapy should not be withheld in patients who are already receiving systemic steroid.


Although it is well known that any material degrades faster when exposed to an aggressive environment as well as that "aggressive" cannot be univocally defined as depending also on the chemical-physical characteristics of material, few researches on the identification of the most significant parameters influencing the corrosion of metallic object are available.A series of ancient coins, coming from the archaeological excavation of Palazzo Valentini (Rome) were collected together with soils, both near and far from them, and then analysed using different analytical techniques looking for a correlation between the corrosion products covering the coins and the chemical-physical soil characteristics. The content of soluble salts in the water-bearing stratum and surfacing in the archaeological site, was also measured.The obtained results stress the influence of alkaline soils on formation of patina. Cerussite, probably due to the circulation of water in layers rich in marble and plaster fragments, was the main corrosion product identified by X-ray Diffraction (XRD). Copper, lead and vanadium were found in soil surrounding coins. By measuring conductivity, pH and soluble salts content of the washing solutions from both coins and soils, we could easily separate coins coming from different stratigraphic units of the site.Data were treated by cluster and multivariate analysis, revealing a correlation between part of the coins and the nearby soil samples. Coins are important evidence in terms of history, art and economy. Besides the composition of the alloy, what is usually revealed by written documents appears in form of effigies, short inscriptions and symbols useful. Taking into account what above said, it can be surely stated that coins are particular and important findings during archaeological investigations as source of documentation, understanding and knowledge of mankind evolution. However, coins are also test samples of metal very sensitive to the effect of deterioration processes, even more if intensified by polluted environments. Their state of conservation, obviously enough, depends also on normal wearing processes before burial as well as on all chemical-physical changes of the burial environment ,2. ObvioPreservation of buried coins is mainly affected by a limited set of variables, that are those favouring corrosion . A moderAcidic and salt-rich sediments would also favour the corrosion of metals. While low pH values normally cause thermodynamic instability of the outer corrosion layers (oxides and hydroxides), high salt contents would provide highly conductive electrolytes.In order to obtain information on the corrosion process on buried coins, we used non invasive and mini-invasive analytical techniques on a series of Roman bronze coins  and associated soil samples, taken both near and far from the find spots of the coins, but anyhow in the same archaeological area .In a preliminary research, 23 coins had been studied with scarce results due to a defective sampling procedure and to a very bad state of conservation and consequent impossibility of numismatic identification.As a second step, we studies 12 coins and associated sediments coming from the same site but excavated in March 2009. The innovative part of the present research lies in the sum of complementary analytical techniques applied, in the particular pre-treatment of soil samples and in the statistical data treatment.Analyses of coins and soil samples by Scanning Electron Microscopy coupled to Energy Dispersive Spectroscopy (SEM/EDS) detected both typical soil elements  and alloy constituents . In partEnergy Dispersive X-ray Fluorescence analysis (ED-XRF) confirmed the relatively high lead content of the coin's alloy. Comparing the results with those of SEM/EDS analysis, arsenic was not detected, but small percentages of silver are on record. Both techniques found tin in low percentage values, except for sample Pt8-US1012, which showed ca.10% of this metal.Fig. C in Additional file 3) and litharge (PbO) [2O) and tenorite (CuO) [XRD tests frequently identified cerussite (PbCOge (PbO) . Copper te (CuO)  , can alter data structure and thus seriously biasing interpretation . After cApplying the same procedure on compositional data we looked for a set of expected correlation. For example, in Figure 4-2 content found in the solutions coming from the washing of coins and near-to-coin soil samples; the same plot is shown in Figure As an example of the treatment of chromatographic data, Figure The scatter plot of matrix A, after column centring is shown in Figure - outlier is due to a high content of this element in the patina of coin 14-us10b.Figure In order to obtain full information from all the measured variables, we decided to implement a multivariate statistic analysis.-, Cl-, NO3-, SO4-2, PO4-3) measured in the washing solutions of coins (W) and soils samples near to (N) and far (S) from coins. In five cases we obtained grouping clusters evidenced by colours.The complete five layer matrix is available in .XLS format (version 97) in the ADF. Figure Furthermore, the dendrogram enhances strong similarities between burial soil samples and coins (i.e. W-a14us10b/N-a14us10b) thus fully confirming the effect of the soil chemistry on the composition of patinas.An analogous dendrogram appears in Figure Among the measured variables iron was excluded, due to its normally high concentrations in soils and concentration levels around the LOD in the bulk of coins. Unfortunately, the coins' bulk composition analysis is affected by the patinas, because, even for analytical purposes, these latter could not be removed as due to obvious ethical problems, the artefacts could not be scraped and radically cleaned. Furthermore, the use of the rodent 4 mm HSS iron wheel may have changed the iron content of the cleaned surface patches of the coins.In Figure When archaeologists find in a dig a coin or another interesting findings, their first interest is to use them to date the associated layers or the context.This immediate interest sometimes results in poor sampling strategies affecting the successive analytical procedure.This is why in the present study we could use only 12 coins; really, for the first series of coins the corresponding near and far soil samples lack so being no useful for a meaningful statistical treatment of archaeological  and analA complete analytical procedure is reported in Figs. A and B of Additional file Chemometry allows to individuate similarity among similar or different objects. Aim of our data treatment was to search for similarity among coins, their patina and the surrounding soil, while search of similarity among coins goes beyond the aim.The Hierarchical Cluster Analysis (HCA), after a suitable pre-treatment of the data matrix, was used as Exploratory Data Analysis.Variables in the matrix (see ADF) are put in columns; each one refers to a chemical element identified by compositional analyses of coins or to a measured parameter such as pH, conductivity, etc.Objects, i.e. coins, are put in the matrix rows , plus thBetter results were obtained with a 3D matrix containing 5 layers: the first one contains data from analyses of washing solutions of soils sampled far from coins; the second one reports analogous data for soils surrounding coins  plus elemental composition data; data of patina analyses filled the third and fourth layer and refer to the washing solutions and the elemental composition respectively; the last layer lists the chemical elements constituting the coin bulk.Obviously, only the second layer is complete, as it was possible to analyse both the solid phase  and the washing solutions (data on the soluble matter).The first column of each layer contains the alphanumerical label defined by the archaeologists.The data matrix could contain even more data, but we choose to construct a completely filled matrix avoiding statistic methods like Least Square Estimation  or otherThe less invasive pre-treatment method useful to enhance the difference among objects is probably the column centring that can be obtained by subtracting the arithmetic average of the column from each value in the same column. A discussion about the use of other "central values" like median, geometric mean, harmonic mean, mode, and so on goes beyond the aim of this paper.By autoscaling or column centring we removed the numerical difference among variables  allowing a self organization of the data in clusters using a simple method for distance and agglomeration.Generally pre-treatment like autoscaling or centring is done after the matrix unfolding but we preferred to do scaling on the original matrix (5 layers) and then divide it in two new matrices. The first one includes the unfolded layers 1,2 and 3, where variables are data coming from the analyses of the washing solutions; the column number remained 7 (plus the label one) while rows increased to 3 × 12 (plus the label one). In the same way we obtained the second matrix joining layers 2, 4 and 5 . Obviously, in the new matrices, data from the original layer 2, exceeding the variables present in the other two, were eliminated.The HCA was used to find similarities among objects; it works as a bottom-up procedure based on the distances among objects: the first cluster is assigned to the two nearest objects, the second one is assigned to other two of the remaining objects showing the least distance and so on.As our hypothesis bases on the effect of the soil characteristics on the corrosion process of the coins, we expected to obtain "a series" of clusters, equal to the number of coins, each one connected to bigger clusters as a function of the different soil characteristics.All data were stored using the software Lotus 123 ver. 9.8, the same software was used to obtain all the average values calculated from measurements on different points of each coin, to draw graphs of Figure Datalab software was used for all the statistical calculations on the matrix; data scaling, HCA by different agglomerative methods and distances as well as to draw the dendrograms; it is an evolution of Inspect  that, inThe distance values, in HCA, can be strongly affected by differences in scale among the dimensions from which the distances are calculated. We chose a simple classic method for scaling and we did the unfolding process after the studentisation on layers, i.e. on the columns of the original matrix.You may want to square the standard Euclidean distance in order to place progressively greater weight on objects that are further apart, by R.L. Carter [The only method we used to calculate distances for the HCA is the squared Euclidean distance; it doesn't use the typical Euclidean root square, which in such cases could alter values, in particular it can increase them with the strongest effect on the greatest distance.  [In complete linkage, the distance between two clusters is the maximum distance between an observation in one cluster and an observation in the other cluster, by SAS online documentation [For the Cluster Analysis, among all possible grouping methods, we used Average Linkage (ntation)  for the entation  for the Coins were found during the excavation carried out under Palazzo Valentini, which is located in the centre of Rome, very close to the Forum Traianum. Both coins and soils were sampled in nine different stratigraphic units (SU) located in three different rooms  at an height ranging from 0 to -1.7 m from the building cellar, corresponding to a mean deep of about 7 m from the street level.Soils far from the coins but coming from the same SU were sampled in order to obtain data not influenced by the contact coin-soil.In order to improve the analytical knowledge of the site, the water-bearing stratum surfacing in the archaeological site was sampled in two different points inside the same rooms.®E . Removed corrosion layers were then prepared for XRD analysis i.e. ground in a agate mortar and layered on a suitable emery glass holders.Coins were washed by ultrapure water (20 mL) in a Elmasonic S30H ultrasonic bath for 10 min and then pH and conductivity were measured. The procedure was repeated until a constant conductivity value was measured for three successive washings; this criterion was adopted to establish a complete cleaning and desalting of the coins. The total content of salts was successively determined by IC. Taking into account the ethic rules of restoration, the corrosion layers were removed from coins by means of a dental piezo-electric scaler Before EDXRF and SEM/EDS analysis three small areas of the coin was more deeply cleaned by rotary rodents tools in steel irons.Soil samples were dried in oven at 105 °C for half an hour and then sieved at 2 mm; 0.6 g were pressed at 10 atm for 15 min, using a Perkin Elmer 12T hydraulic laboratory press (0-12 ton), to obtain pastilles for SEM-EDS and XRF analysis. Other sieved portions were ground in an agate mortar; aliquots of 250 mg were put in 25 mL deionised water and treated in a Elmasonic S30H ultrasonic bath to extract soluble salts for the IC analysis.Electronic microscopy coupled to X-ray microanalysis (SEM-EDS) was carried out by means of a Zeiss Evo 60.Instrument setting was optimised for each single analysis  looking For a preliminary view of sample's morphology and distribution of the corrosion patina a low magnitude image (70x) by secondary electrons (SE) was used; basing on this, the most interesting and representative zones were then explored under higher zoom conditions .Distribution maps of the average atomic density were obtained as image by the backscattered electrons (BSE), enhancing the heavier elemental composition of the patina compared to that of the alloy.On interest points of both zones, with and without patina, EDS microanalysis was performed on single grains and crystals or on small areas .The same procedure was carried out for soil samples in order to identify common elements in soil and coins so obtaining data useful to search for any correlation by the successive multivariate analysis.In total 78 measurements were carried out: 29 on cleaned coin areas, 28 on coin patina, 18 on soil samples.A portable EDXRF was used for X-ray fluorescence measurements .The X-ray tube was set at tension and current equal to 36 KV and 0.12 mA respectively; 300 s was adopted as exposure time.The obtained spectra were processed by McUiICR software and for quantitative analysis the function single peak area - 2 Gauss was used. Further 7 different reference standards were used.For X-ray diffraction analysis (XRD) a Seifert MZ IV with copper anticathode (λCuKα = 1.542 Å) was used, applying a tension of 40 KV and a current of 30 mA. Scan angle 2θ was set between 3° and 70° with a 0.02θ sampling step and 2 s exposure time.The salt content of coin washing solutions, soil extracts and groundwater samples was determined by using a Metrohm 761 Compact IC equipped with a Metrosep A Supp4 column (61006430 - Size: 5.0 × 250 mm Particle size: 9.0 μm) and a 20.0 μL loop.3) by Sigma (purity certified by producer: 99.7-100.3%) and sodium carbonate (Na2CO3) by Merck, (purity certified by producer: 99.88% +/- 0.05%); the final concentration was 1.7 mmol/L and 1.8 mmol/L respectively. Deionised water (0.8 μS conductivity) was obtained by means of a Millipore Direct-Q 3 UV deionizer and used for sample and reagent preparation. First of the analysis, samples were treated in a Universal 32R centrifuge (by Hettich Zentrifugen) at 4000 rpm, for half an hour at 4°C; successively they were filtered through disposable membranes Minisart, 0.2 μm in PTFE .Eluent was prepared starting from sodium hydrogencarbonate  reports some other values measured but not used in this work (pH of the bearing stratum water and so on). Figures A and B describe, as flow charts, the analytical procedures adopted to obtain data relative to both coins and soils. Figure C shows, as an example, the SEM image and the EDS spectra of one of the coin. Tables 1-3 list some information on coins such as the finding location in the archeological site, image, dimensions and so on. Tables 4-5 list the main physical characteristics of soils while table 6 summarises the corrosion products found by XRD.Click here for file


However, the scores in the domain of treatment burden and emotional functioning increased significantly. There was no significant difference in QoL and lung function between patients participating in the exercise programme (n = 24) and non-participants (n = 14). A 12-week individually tailored unsupervised aerobic exercise programme where HR monitors were used significantly affected VO2max. Improvement in QoL could not be demonstrated in this study.It was hypothesised that increased exercise capacity is related to improved quality of life (QoL) in patients with cystic fibrosis (CF). A 12-week individually tailored unsupervised aerobic exercise programme was offered to 42 patients with CF. At the start and at the end of the exercise programme, data on QoL, current exercise habits and preferences, anthropometric data, exercise test, and lung function test were collected. Adherence was observed by a heart rate (HR) monitor. A total of 24 patients accepted to be enrolled in the exercise programme and 14 completed the programme. Another 14 patients declined to be enrolled in the exercise programme but completed the Cystic Fibrosis Questionnaire for Adolescents and Adults (CFQ-R 14+). Four patients did not want to participate at all. The 14 patients completing the exercise programme had a significantly increased VO Cystic fibrosis (CF) is the most common hereditary disease in Caucasians, and no curative treatment has yet been found. In Denmark, life expectancy is now a median of 42 years .Approximately 1 in 4,700  live birDuring the last decade, focus has been on exercise as an important element in the treatment of CF. Exercise has thus been incorporated in many standard treatment regimes, and exercise capacity is more often used as an outcome variable in intervention studies. Exercise capacity is currently used as an important parameter of disease status and progression , and it Therefore, we designed a 12-week intervention study consisting of an individually tailored and unsupervised aerobic exercise programme to investigate the association between increased exercise capacity and changes in QoL in patients with CF. The exercise programme comprised everyday life activities adapted to the preferences of each individual patient. The patient was personally responsible for activating an HR monitor for every exercise session, indicating intensity, frequency, and duration and thus adherence to the programme.It is our clinical experience that it can be difficult to motivate patients with CF to incorporate regular exercise into their everyday life. QoL is potentially negatively affected by the large burden of treatment and the complexity of the treatment evolving over years as CF progresses . It was It was hypothesised that a 12-week individually tailored and unsupervised aerobic exercise programme in patients with CF could increase exercise capacity and thus QoL.Patients from the CF Centre at Aarhus University Hospital, Skejby, between 14 and 50 years of age, were invited. In our CF registry we found 42 eligible patients, and 24 accepted to participate in the study. A total of 14 of the 18 patients who declined participation filled in the Cystic Fibrosis Questionnaire for Adolescents and Adults (CFQ-R 14+).1% <60, lung transplantation, debut of asthma or diabetes in the intervention period, and/or physical or psychological disability preventing participation.Exclusion criteria were FEVAll patients attended the CF Centre regularly, and during the study period the patients continued their regular therapy, including airway clearance therapy using a positive expiratory pressure mask, enzymes, vitamins, and other medical treatment.Written informed consent was obtained from patients or parents. The study was approved by the Central Denmark Region Committees on Biomedical and Research Ethics, file number 20060052.The study period was 12 weeks. Measurements were performed at the start and at the end of the study. The pretests and posttests were identical.Measurements were performed by the same assessors involved in the delivery of the intervention, and the patients were initially acquainted with the test procedures.QoL was measured by using the Danish validated version  of the dThe CFQ-R 14+ consists of 49 self-reported items within 12 domains: 1) physical functioning (8 items); 2) vitality (4 items); 3) emotional functioning (5 items); 4) eating disturbances (3 items); 5) treatment burden (3 items); 6) general health perception (3 items); 7) social functioning (6 items); 8) body image (3 items); 9) role limitations (4 items); 10) weight problems (1 item); 11) respiratory symptoms (6 items); and 12) digestive symptoms (3 items). Answers were reported on a four-point scale rating frequency, difficulty, or truth, and selecting one of four statements that described the patient's situation. The scores ranged from 0 to 100 and higher scores equalled higher QoL.To assess the patients' level of current physical activity, we asked them to fill in a short questionnaire . Furtherz score was calculated on the basis of Danish data on children and adults  and standing height using a stadiometer. The measurements were used to calculate the body mass index (BMI). For comparison between the different age groups, a BMI d adults .2max was determined as the peak oxygen uptake every 10 seconds throughout the test. VO2max was normalised to body mass (VO2max/kg)- The HR was monitored  continuously every 5 seconds during the test, and the highest value was recorded. After the test, efforts were considered to be at a maximum level when three of the following four criteria were met: 1) total exhaustion; 2) HR above (220 - age) -10% 3) respiratory exchange ratio (the ratio between the measurements of VCO2 and VO2) above 1.11; and 4) the VO2 curve levelled off with a surge of less than 0.15 L/min from the last increment until the patient stopped the test.Exercise capacity was measured as peak oxygen consumption rate (L/min). Before starting the incremental maximum exercise test, the patients warmed up on a voluntary level for 5 minutes on the cycle ergometer . To reach the patients' maximum in 5-10 minutes, step-wise increments of 0.4 kilopond (kp) every 1.5 minutes were made, starting on individual levels from 0.8 to 1.8 kp, depending on the physical fitness of the patient. The patients were asked to choose a cadence between 60 and 85 revolutions per minute and to keep it during the test. The maximum level was reached when the patient could no longer maintain the cadence or to volitional fatigue. Patients were verbally encouraged throughout the test. The patients wore a close-fitting mask connected to an online respiratory gas exchange analyser  and computer system. Expired air was sampled continuously, and oxygen consumption and carbon dioxide production were measured every 10 seconds. VO1% was performed by using a Pneumotach spirometer . The pretest on lung function was performed on the day the patient entered the study, and the posttest on lung function was performed the day the patient ended the study.FEVTo optimise adherence, each patient had the opportunity to select one or more physical activities for their individually tailored exercise programme. The patients chose different activities such as bicycling (including exercise bike), running, gymnastics, swimming, and dancing. Some patients performed more than one activity.2max was obtained  a warm-up phase of 5 minutes and 2) a training phase of at least 25 minutes. If the patients could not endure intensity above 70% of their HR maximum, they were allowed to stop the HR monitor and have a short break. HR was continuously monitored every 5 seconds by an HR monitor  to control training intensity. Exercise data were stored in the HR monitor as an exercise dairy and an evidence of adherence.The patients were offered supervised training at the CF Centre once every second week, but no patients accepted this offer. We made motivational phone calls to all patients every week in the beginning of the exercise programme, and subsequent follow-up was planned individually. The motivational phone calls followed a nonstandardised semistructured interview guide. After the posttest exercise, data were transferred to a computer for analysis.p-value of ≤ 0.05 was considered statistically significant. A Wilcoxin signed-ranks test was conducted to evaluate the impact of the exercise programme on CFQ-R14+ scores. By a Kruskal-Wallis test, CFQ-R 14+ scores of group 1 were compared with the CFQ-R 14+ scores of group 0 before starting the exercise programme and with group 2 (see classification of groups in Results).Pretest values for different groups were analysed by a Mann-Whitney test. Dichotomous variables were tested by a chi-square test. Pretest and posttest values were compared by using a paired t test. A n = 10); group 1 = patients completing the exercise programme (n = 14); and group 2 = patients only filling in the CFQ-R 14+, non-participants (n = 14). Clinical characteristics of the 24 patients in the exercise programme (groups 0 + 1) are shown in The patients were classified as follows: group 0 = dropouts from the exercise programme (Ten (group 0) of the 24 patients did not complete the exercise programme mainly due to practical reasons . These 10 patients were not posttested. Fourteen (group 1) of the 24 patients were pretested, completed the exercise programme, and were posttested.Engagement in vigorous activities in patients completing the exercise programme (group 1) did on average not meet the intended frequency and duration. Frequency was on average 2.31 times a week instead of three times a week, and duration was on average 79 minutes a week instead of 90 minutes a week. The patient exercising with the lowest intensity exercised with a 75% of maximum HR on average, and the patient exercising with the highest intensity exercised with an 86% of maximum HR on average  exercising more than the recommended 70% of maximum HR. As shown in p = 0.74) was found in FEV1% between participants in the exercise programme (groups 0 + 1) and non-participants (group 2). Similarly, there was no significant difference (p = 0.55) between participants completing the exercise programme (group 1) and non-participants (group 2).Fourteen (group 2) of 18 patients filled in the CFQ-R14+ but did not participate in the exercise programme. As shown in 2max, and their VO2max/kg improved, though not significantly.As seen in p = 0.013; and in the domain of emotional functioning from the scores before the intervention  to the postintervention scores  p = 0.021  to the post-intervention scores   = 0.021 . The etaThe CFQ-R 14+ scores of group 1 before the exercise programme were compared with the CFQ-R 14+ scores of group 0 (drop-outs) before the exercise programme and with the CFQ-R 14+ scores of group 2. No differences were found in any of the comparisons.The internal consistency of each scale showed Cronbach's α coefficients ranging from 0.42 to 0.92 with coefficients <0.70 in the domains of role limitations (0.67); body image (0.62); social functioning (0.43); and digestive symptoms (0.42).z scores .To our knowledge, this is the first study in patients with CF investigating the response to an individually tailored and unsupervised aerobic exercise programme involving use of HR monitors. As far as we know, this is the first time when VO1% between participants in the exercise programme (group 0 + 1) and non-participants (group 2). This may indicate that the patient's lung function did not seem to play any role in the willingness to participate in this study. Our individually tailored and unsupervised exercise programme had no positive or negative effect on FEV1%. The expected decline in lung function in patients with CF is likely to be between 2% and 3% annually. This suggests that studies shorter than 12 months may be unable to detect any difference in lung function (We did not find a significant difference in FEVfunction . Consequ1% above 60%) can increase their VO2max significantly by participating in a 12-week individually tailored and unsupervised aerobic exercise programme. Our results are consistent with the results of other studies in both children and adults. 2max but not in VO2max/kg following a 12-week anaerobic exercise programme in children with CF.We showed that patients with CF with a relatively good lung function  did, on average, not meet the intended frequency and duration. However, the intensity of the lowest and the highest exercising patient was between 75% of maximum HR on average and 86% of maximum HR on average, both patients exercising with an intensity considerably higher than the recommended 70% of maximum HR. This suggested that the recommended intensity on 70% of maximum HR was not the reason for the high dropout level in our study. It can be debated whether the intensity was individualised. The intensity in relative terms was the same for all patients, whereas the absolute intensity was based on maximum HR of each patient. It may be that motivation, adherence, and QoL could increase as intensity increased and the duration of the exercise decreased.2max would increase QoL and thereby the inner motivation to do exercise. A study by Arias Llorente, Bousono Garcia, and Dìaz Martin (2008) concluded that adherence has shown to improve with treatments that are perceived by patients as more important or influencing their QoL to a greater extent. Approximately 50% of people with chronic conditions do not adhere to treatment, and adherence reduces with the complexity of treatment. Previous studies in CF have found that adherence to chest physiotherapy and exercise is lower than other treatments  and participants were informed about this. At our CF Centre we encourage patients to gradually take responsibility of their disease when they become adolescents. We try to engage patients through continuous  dialogue regarding treatment to enable the patient to take ownership of their treatment. Our belief is that we can achieve more by voluntariness of our patients and by their inner motivation. We had hoped that an increase in VOeatments .Despite the proven benefits of exercise, the degree of physical activity generally undertaken in the CF population remains an issue of concern. The development of positive attitudes toward and a regular schedule for activity early in life may be important in determining an active style into adulthood . EarlierOur patients were offered supervised training at the CF Centre once every second week but no patients accepted this offer. We made motivational phone calls to all patients every week in the beginning of the exercise programme and subsequent follow-up was planned individually. Nearly all the patients had one supervised exercise session during the intervention. Our strategy was to improve adherence by regular contact between us and the patient, not to supervise the programme as such but simply to show interest, enquire about the patient's well-being, and allow any queries to be answered. This method has been used previously as a part of an overall strategy to improve compliance in an exercise study by We assumed that the short intervention period and individually tailored programme based on willingness to participate could lead to a relatively high adherence. However, several factors may influence adherence to any treatment regime, including a patient's willingness to participate in regular exercise over a period of time. These can be factors related to the disease process itself, gender, age, and external factors such as social support, perception of competency, self-esteem, enjoyment of activity, and factors associated with the patient's behaviour such as motivation and choice . The rea2max could improve QoL. We found a statistically significant increase in scores of the domain of treatment burden and emotional functioning. The magnitude of the differences in the means were very large . The increase in the scores of the domain of treatment burden was surprising. The explanation might be that the patients did not see the exercise program as a treatment burden, and the exercise program might have made them feel more energetic though this was not reflected in other domains such as vitality. Change of behaviour starts as a mental process, and this study indicated that changes did occur in patients who completed the exercise programme (group 1) by an improved emotional function score.Our study failed to show that increased exercise capacity expressed as VOIn this study the internal consistency of the CFQ-R 14+ scales measured by Chronbach's α failed to reach the recommended 0.7 in four of the scales. Three of these scales also showed a weak internal consistency in the validation of the CFQ-R +14; the fourth scale (role limitation) only showed a slightly lower Chronbach's α (0.67) than recommended.The fact that the questionnaire failed to show an overall improvement in QoL might be explained by the complexity of the CFQ-R 14+. Retrospectively, we could not expect improvements in QoL after a 12-week intervention within the physical domains in CFQ-R 14+ such as eating disturbances and weight problems. Furthermore, group 1 had a high score in several of the physical domains in the pretest, leaving little room for improvement. The CFQ-R 14+ consists of 49 self-reported items within 12 domains, and the tool might have been too complex to show whether increased exercise capacity could result in increased QoL for patients with CF.Two previous studies  on exercOther intervention studies on exercise have measured QoL as a secondary outcome but with different measurements. In terms of degree of limitation in activities of daily living (ADL), 2max or VO2max/kg in 12 or less than 12 weeks of exercise .Third, in our exercise test we applied stepwise increments with fixed increment of 0.4 kp in determining the peak oxygen uptake. We are aware that some laboratories use continuous load increase to avoid one heavy load at one occasion. Stepwise increments could possibly cause some patients to finish the exercise test too early. However, in the present study all peak oxygen uptake measurements fulfilled three of four criteria  - 10%, respiratory exchanges ratio above 1.11, and/or the VOFourth, the high dropout rate and thereby small sample size have weakened the conclusions. However, this has shown that the degree of encouragement and supervision in this programme may not have been sufficient and has to be individualised to achieve adherence to exercise as part of CF treatment.2max and QoL, and our study was not designed as a follow-up study due to resources.Finally, due to lack of follow-up, we do not know if the patients in group 1 obtained a lifestyle change at the end of the study period. 2max can be achieved with an individually tailored and unsupervised aerobic exercise programme using HR monitors. A frequently postulated benefit of any physical exercise is an enhanced feeling of well-being. Thus, it was hypothesised that exercise capacity would be related to QoL in patients with CF. Improvement in QoL could not be demonstrated in this study.In conclusion, this study has shown that an effect on VORegular exercise is a very important factor in maintaining good health. This is even more important in patients with chronic diseases such as CF. Therefore, we still recommend that patients with CF increase or maintain as good a physical fitness as possible.