Streptococcus anginosus) seem to characterize general dysbiotic states of the microbiome rather than connections with a specific disease. Our results in modelling features of the “healthy” microbiome can be considered a first step toward defining general microbial dysbiosis. The software framework, microbiome profiles, and metadata for thousands of samples are publicly available at http://segatalab.cibio.unitn.it/tools/metaml.Shotgun metagenomic analysis of the human associated microbiome provides a rich set of microbial features for prediction and biomarker discovery in the context of human diseases and health conditions. However, the use of such high-resolution microbial features presents new challenges, and validated computational tools for learning tasks are lacking. Moreover, classification rules have scarcely been validated in independent studies, posing questions about the generality and generalization of disease-predictive models across cohorts. In this paper, we comprehensively assess approaches to metagenomics-based prediction tasks and for quantitative assessment of the strength of potential microbiome-phenotype associations. We develop a computational framework for prediction tasks using quantitative microbiome profiles, including species-level relative abundances and presence of strain-specific markers. A comprehensive meta-analysis, with particular emphasis on generalization across cohorts, was performed in a collection of 2424 publicly available metagenomic samples from eight large-scale studies. Cross-validation revealed good disease-prediction capabilities, which were in general improved by feature selection and use of strain-specific markers instead of species-level taxonomic abundance. In cross-study analysis, models transferred between studies were in some cases less accurate than models tested by within-study cross-validation. Interestingly, the addition of healthy (control) samples from other studies to training sets improved disease prediction capabilities. Some microbial species . In particular, shotgun metagenomics provide data at unprecedented species- and strain- levels of resolution. Several large-scale metagenomic disease-associated datasets are also becoming available, and disease-predictive models built on metagenomic signatures have been proposed. However, the generalization of resulting prediction models on different cohorts and diseases has not been validated. In this paper, we comprehensively assess approaches to metagenomics-based prediction tasks and for quantitative assessment of microbiome-phenotype associations. We consider 2424 samples from eight studies and six different diseases to assess the independent prediction accuracy of models built on shotgun metagenomic data and to compare strategies for practical use of the microbiome as a prediction tool. The human microbiome constitutes the whole set of microbial organisms associated with the human host. It has been shown to be crucial for human health and for the development and maintenance of the immune system and for several metabolic activities –3. SigniNext-generation DNA sequencing technologies permit comprehensive profiling of the microbial communities from human-associated samples, and have now been sufficiently widely employed to enable meta-analysis for discovering patterns common to independent studies. Meta-analysis has been broadly adopted in other genomics applications, such as for analysis of microarray or RNA-seq data, where multiple studies have been performed for a similar purpose including identifying gene expression signatures of specific human cancers. The general objectives of meta-analysis include proposing new classifiers , compariThe most common and cost-effective approach for microbiome characterization to date targets the 16S rRNA gene as taxonomic marker . Meta-anImproved resolution and lower variability of shotgun metagenomics hold the promise to provide improved generalization of microbial signatures over 16S rRNA sequencing . Meta-anIn this study we uniformly process 2424 shotgun metagenomic samples from eight studies to assess the independent prediction accuracy of models built on metagenomic data and to compare strategies for practical use of the microbiome as a prediction tool. The software framework and the microbiome profiles for thousands of samples are made publicly available.Methods). Our multi-level validation strategy includes the assessment of microbiome models on single cohorts, across stages of the same study, across different studies, and across target outcomes and conditions . The software and validation framework is publicly available at http://segatalab.cibio.unitn.it/tools/metaml and was applied on a total of 2424 publicly available metagenomic samples from eight large-scale studies (see Table 1 and Methods). All samples were processed with MetaPhlAn2 .Lasso  and ENetTwo main kinds of analysis were performed in this work, i.e., cross-validation and cross-stages/studies. For cross-validation studies, prediction accuracies were assessed by 10-fold cross validation, repeated and averaged on 20 independent runs. We underline that model selection and feature selection are done using only the training set thus avoiding overfitting problems. In the cross-stages/studies case, all the samples of the first stage/study are considered for training and thus used to generate the classification model including the model selection and feature selection steps. The generalization of the model is evaluated by applying it on the samples of the independent stage/study. In all the cases, the results obtained on the original classification problem were compared with those obtained by a random classifier (denoted in the paper as SVM-Shuffled and RF-Shuffled). For such purpose, we applied the same setting after shuffling randomly the labels of all the samples.Several different metrics were taken into account to evaluate classification performances. First, we considered the OA, which is the percentage of correctly predicted samples. From the confusion matrix three main metrics were computed: i) the precision ; ii) the recall ; iii) the F1 score, which is the harmonic mean of precision and recall, i.e., F1 = 2*/. These three metrics  can be computed for each class separately. For brevity, we report in the paper only the average values: after calculating the metrics for each class, their average values, weighted by the number of samples per class, are computed. For binary classification problems, class posterior probabilities were used to plot the ROC curve, which represents the true positive rate  against the false positive rate . From the ROC curve, we computed the widely-used AUC statistic, which can be interpreted as the probability that the classifier ranks a randomly chosen positive sample higher than a randomly chosen negative one, assuming that positive ranks higher than negative. The AUC ranges in , where 0.5 corresponds to random change.1ij and AUC2ij are the AUC of two classifiers in fold i of repetition j, and σj is the standard deviation of the AUC1ij−AUC2ij across i = 1…10 folds in repetition j. Similarly, p-values were obtained from the t-statistic obtained with mean difference and standard error smoothed over the 20 repetitions:In the comparison among classifiers, prediction accuracy was assessed by 10-fold cross-validation, repeated and averaged on 20 independent runs. The same folds were used for all classifiers, i.e. training and test sets were identical for each classifier. In this way, the difference in performance of two classifiers could be calculated directly as the difference in AUCs (or any other metric) within each test fold. Mean difference and standard error were calculated for each 10-fold CV, then averaged across the 20 repetitions for smoothing. 95% confidence intervals on the difference in AUC performance of two classifiers were calculated using the t-distribution with df = 9, i.e.:In terms of feature selection, we reported the list of the 25 most important features found by RFs. For each feature, we considered also the relative importance score, which is a real number in the range  with features that sum to 1. Feature selection is done for each run independently, and we report the average results.We initially considered a total of 2571 publicly available metagenomic samples (from eight main studies/datasets) that were reduced to 2424 after pre-processing and curation (see next sections). These are all the human-associated shotgun metagenomic studies with more than 70 samples and read length bigger than 70nt available as of January 2015. Six studies were devoted to the characterization of the human gut microbiome in presence of different diseases. Cirrhosis included 123 patients affected by liver cirrhosis and 114 healthy controls . ColorecFig 2). Before applying MetaPhlAn2 the samples were subject to standard pre-processing as described in the SOP of the Human Microbiome Project [The entire analysis was done by taking into account two types of features: species-level relative abundances and presence of strain-specific markers. These features were extracted from the metagenomic samples using MetaPhlAn2  with def Project  without The experimental evaluation can be summarized into five main steps: 1) cross-validation analysis was done on the six disease-association datasets for evaluating the capabilities of metagenomic data for disease classification; 2) cross-stage studies were performed on the cirrhosis and T2D datasets in order to test the generalization of the model on independent collection batches from the same study; 3) in terms of T2D, the analysis was extended by taking into account also samples from completely distinct cohorts; 4) cross-studies were also done to model the features of the “healthy” gut microbiome for use as a dysbiosis prediction model for syndromes where few or no training samples are available; 5) cross-validation and cross-study analysis were applied to deal with different classification problem such as gender and body site discrimination. We note that all the investigated classification problems, excluding the body site discrimination, represented binary classification problems. Moreover, most of the analysis was done in terms of disease classification, in which the objective was to discriminate between “healthy” and “diseased” subjects.-2) and “obese” (BMI ≥ 30 kg m -2) subjects for a total of 253 samples. Individuals having an intermediate BMI  were excluded. All the 344 samples of the T2D dataset were considered to discriminate between “healthy” individuals and “T2D” patients. The same classification problem was investigated in WT2D, in which 96 samples were taken into account after excluding impaired glucose tolerance women.Cross-validation analysis  in gut samples see “”. We conFinally, the developed tool was applied to non-disease-based classification problems  software is open-source, written in Python and available online at http://segatalab.cibio.unitn.it/tools/metaml together with all the data used and discussed in this work.The MetAML Click here for additional data file.S2 TableFig 5). The IntCorr is the integrative correlation [The AUC is computed by training the model on one stage of T2D and testing it on WT2D (same results in relation  between (PDF)Click here for additional data file.S1 Figp-values for the null hypothesis of no difference in AUC by 10-fold cross-validation, assessed by paired t-test, for disease discrimination on six different datasets using species abundance and marker presence as microbiome features. Comparisons that are statistically different  are highlighted in red. Average values with margins of error for AUC are reported in Fig 2.The table reports the (TIFF)Click here for additional data file.S2 FigFig 3 and S3 Fig.The corresponding prediction performances are reported in (TIF)Click here for additional data file.S3 FigSpecies abundance and marker presence were used as microbiome features. The best value for each dataset and feature type  are in bold, and the overall best values for each dataset are circled. Enet and Lasso denote their use as classifier applied on the entire set of features; RF(or SVM)-FS:Enet(or Lasso) denotes RF (or SVM) used as classifier on the reduced set of features given by Enet (or Lasso). The margins of error are reported in parenthesis.(TIF)Click here for additional data file.S4 FigMost important discriminating species (left) and markers (right) identified by RF for disease discrimination in (a) IBD, (b) obesity, (c) T2D and (d) WT2D cross-validation studies. In the left panels, for each species reported on the vertical axis, the top bar (in blue) corresponds to the feature relative importance (with standard deviation reported with error bars) and the two bottom bars refer to the average relative abundance for healthy (in green) and diseased (in red) samples. In the right panels, for each marker the top bar is coloured according to the corresponding species and the two bottom bars refer to the average marker presence.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigFeatures and data sets were clustered using correlation similarity.(TIF)Click here for additional data file.S7 Fig(a) Cross-validation and (b) cross-study analysis for gender discrimination in multiple datasets. In bold: the best value for each scenario and feature type ; Circled: the absolute best value for each scenario.(TIF)Click here for additional data file.S8 Figa) Cross-validation results by considering one sample per subject or more than one sample per subject but each from a different body site. (b) Normalized confusion matrix when using RF on species abundance.((TIF)Click here for additional data file.S9 Figa) obesity, (b) T2D and (c) WT2D datasets are overestimated if the most relevant features  are pre-selected. Cross-validation prevents this problem.Prediction capabilities for disease discrimination in the ((TIF)Click here for additional data file.S10 Figa) Most relevant species (left) and markers (right) identified by RF by cross-validating on the entire set of samples. In the left panel, for each species reported on the vertical axis, the top bar (in blue) corresponds to the feature relative importance (with standard deviation reported with error bars) and the two bottom bars refer to the average relative abundance for healthy (in green) and diseased (in red) samples. In the right panel, for each marker the top bar is coloured according to the corresponding species and the two bottom bars refer to the average marker presence. (b) Hierarchical clustering on the feature relative importance score determined by running RF on species abundances in a "leave-one-dataset-out" cross-study validation [(lidation . For eac(TIF)Click here for additional data file.


Kr) block in human ventricles with acute regional ischemia.Acute ischemia is a major cause of sudden arrhythmic death, further promoted by potassium current blockers. Macro-reentry around the ischemic region and early afterdepolarizations (EADs) caused by electrotonic current have been suggested as potential mechanisms in animal and isolated cell studies. However, ventricular and human-specific arrhythmia mechanisms and their modulation by repolarization reserve remain unclear. The goal of this paper is to unravel multiscale mechanisms underlying the modulation of arrhythmic risk by potassium current (IK(ATP)) activation. Prolongation of refractoriness by 4% caused by 30% IKr reduction counteracts the establishment of macro-reentry and reduces the VW for reentry (by 23.5%). However, a further decrease in repolarization reserve (50% IKr reduction) is less anti-arrhythmic despite further prolongation of refractoriness. This is due to the establishment of transmural reentry enabled by electrotonically-triggered EADs in the ischemic border zone. EADs are produced by L-type calcium current (ICaL) reactivation due to prolonged low amplitude electrotonic current injected during the repolarization phase.A human ventricular biophysically-detailed model, with acute regional ischemia is constructed by integrating experimental knowledge on the electrophysiological ionic alterations caused by coronary occlusion. Arrhythmic risk is evaluated by determining the vulnerable window (VW) for reentry following ectopy at the ischemic border zone. Macro-reentry around the ischemic region is the main reentrant mechanism in the ischemic human ventricle with increased repolarization reserve due to the ATP-sensitive potassium current (IElectrotonically-triggered EADs are identified as a potential mechanism facilitating intramural reentry in a regionally-ischemic human ventricles model with reduced repolarization reserve. Pro-arrhythmic mechanisms of acute ischemia have been extensively investigated, although often in animal models at the single cell or tissue level rather than in human ventricles. Seminal studies by Janse et al. in pig and dog hearts highlight the complexity of the pro-arrhythmic and spatio-temporally dynamic substrate in acute ischemia . HeterogElectrotonic current flowing due to gradients in membrane potential in heterogeneous tissue has been hypothesized as a potentially-important pro-arrhythmic factor in acute ischemia . One potPro-arrhythmic mechanisms including both reentry and EAD formation strongly depend on the balance of currents during repolarization, and the specific degree of redundancy in repolarizing currents, termed repolarization reserve, available. This is affected by a variety of causes including inter-subject differences in ion channel density, drug effects and mutations. Individuals with reduced repolarization reserve are known to be at higher arrhythmic risk, particularly in the presence of a pro-arrhythmic substrate such as ischemia due to coronary heart disease as shown in clinical studies . FurtherInvestigating the pro-arrhythmic substrate of acute ischemia and its alteration by potassium block experimentally is very challenging. Of particular difficulty is mapping the three-dimensional volume of the heart with enough spatio-temporal resolution to investigate complex and irregular intramural propagation patterns through the ventricular wall. Microelectrode recordings and optical mapping have both been used in animal studies and they both suffer from a different set of limitations. The former exhibit low resolution, whereas the latter provides recordings only of the tissue surface and with photon scattering averaging effects , p. 2007In this study, we investigate the mechanisms of increased arrhythmic risk in the heterogeneous substrate caused by acute myocardial ischemia for different degrees of reduced repolarization reserve using an anatomically-based biophysically-detailed human ventricles model. We hypothesize that in human ventricles with low repolarization reserve, gradients in membrane potential across the ischemic border zone promote the occurrence of electrotonically-triggered EADs that contribute to the establishment of transmural micro-reentry. By analyzing high spatio-resolution simulation data, we unravel the three-dimensional mechanisms of EAD formation and propagation dynamics in the acutely-ischemic human ventricles, their modulation by repolarization reserve and their potential contribution to increased arrhythmic risk.22.1K(ATP)) ) . The TP0K(ATP)) . An expa2.2Size and location of ischemic regions exhibit great variability. We represented the ischemic region in our model as affecting ∼40% of the left ventricle, which is within the range of 13–72% reported by in vivo human hearts  of 800 ms, by stimulating the endocardium (S1) to mimic the activation sequence in m) and short APD  conductance in the TP06. The simulations yield a 5% and 9% prolongation in QT in the pseudo-ECG, which is similar to the 7% and 12% increase found by Fossa et al. in healthy patients, 2–4 h after being given 160 mg and 320 mg of sotalol respectively  was evaluated after reaching steady state at each CL and calculated as the shortest CI to induce sustained propagation in the tissue. Conduction velocity (CV) was calculated at steady state from the time interval separating two activation times at two points in the center of the tissue separated by 2 mm. One-dimensional simulations were conducted to characterize the effect of reduced repolarization reserve in NZ and ICZ tissue for varying degrees of I33.1via the NZ and BZ around the ICZ . The main anti-arrhythmic mechanism of decreasing IKr is that prolonged ERP favors conduction block both in the NZ and ICZ at short CIs, therefore leading to either bidirectional block just after premature excitation or failure to sustain retrograde conduction in the ICZ. While the patterns of macro-reentry were similar when IKr reduction was increased to 50%, the anti-arrhythmic activity of IKr diminished, as shown by an increased width of the VW . The causes of this increase in the VW with a further reduction in repolarization reserve are investigated below.Decreased repolarization reserve by 30% I3.2Kr block was caused by transmural reentry supported by electrotonically-triggered EADs near the ischemic BZ, as illustrated in via the lateral BZ, from endocardium to epicardium  with fully depolarized epicardial cells (black traces) close to repolarizing endocardial tissue (green traces). The voltage gradients result in electrotonic current flowing towards the endocardial cells, and this facilitates the development of electrotonically-triggered EADs  and 30% and 50% reduction . In all three cases, propagation first proceeds around the ICZ, and then through the ICZ once its tissue has recovered. For 0% and 30% IKr reduction, propagation completely engulfs the ventricles and reentry fails to be established. However, in the case of 50% IKr reduction, prolonged refractoriness in the ICZ leads to conduction block close to the BZ, followed by endocardial propagation and EAD formation  reactivation and EADs if injected during the repolarization phase. Results presented in Kr block (CaL current and its activation gate are the main driving force (m ≤ −20 mV) (Kr block. Low amplitude current applied at early stages of repolarization favors the occurrence of EAD formation compared to late repolarization stages (K(ATP) activation and elevated extracellular potassium concentration ([K+]o). The latter decreases the excitability of the cell and therefore the likelihood of regenerative EAD-like behavior.In order to investigate the mechanisms underlying the formation of EADs in the acutely-ischemic human ventricles, we simulated the effect of a current injected during the repolarization phase on the human ventricular ionic dynamics and AP under normal and ischemic conditions with various degrees of repolarization reserve. Based on the conditions shown in Kr block , Fig. 6.ng force  is not in stages B. Fourthn stages B. Therefn stages . This is4Kr causes an anti-arrhythmic prolongation of refractoriness, which counteracts macro-reentry formation. However, it simultaneously causes a pro-arrhythmic reduction in repolarization reserve, which promotes EAD formation and transmural reentry. Arrhythmic risk in regional ischemia is therefore determined by a fine balance between macro-reentrant mechanisms driven by heterogeneity in refractoriness around the ischemic region, and micro-reentrant patterns, electrotonically-triggered EAD formation and transmural reentry. Reactivation of the L-type calcium current is the primary mechanism underlying EAD formation by prolonged low amplitude current injected during repolarization. Our investigations were primarily mechanistic, and thus conducted using a single human ventricles model given the computational demands. We identify key factors that will vary between individuals, and that may explain inter-subject differences in arrhythmic risk in acute ischemia modulated by repolarization reserve.High-resolution simulation data obtained with an anatomically-based model of the regionally-ischemic human ventricles reveal, for the first time, the electrotonic trigger of EADs and their contribution to transmural reentry and increased arrhythmic risk under conditions of reduced repolarization reserve. In our human ventricular model, EADs develop as a consequence of complex patterns of propagation, repolarization and current flow in the heterogeneous ischemic area, whereas they do not develop spontaneously in single cell or in homogeneous tissue with the same membrane kinetics. Simulations show that reduced I4.1in vivo and ex situ human experimental studies including: human ventricular activation time , while prolonging the ERP in both regions, therefore reducing the chances of retrograde propagation and reentry. This leads to a decrease in the VW for 30% IKr reduction but the trend becomes different with 50% IKr decrease. The main reason is the emergence of a newly identified pro-arrhythmic mechanism revealed by our simulations: transmural reentry sustained by electrotonically-triggered EAD development.Our simulation results show that IEven though Janse et al. also reported the occurrence of intramural reentry in some of their acute ischemia experiments in pig and dog, they were unable to resolve the underlying mechanisms due to the low resolution of their microelectrode recordings . Lukas a4.3CaL and 80% reduction of IKr, as in EADs have long been identified as a potentially important pro-arrhythmic player  and theyOur simulations identify a more likely pro-arrhythmic mechanism of EAD formation and reentry, sustained by the heterogeneous ventricular substrate , Fig. 6.4.4Kr reduction) indeed reduces the propensity for macro-reentry formation. However, the concomitant reduction of repolarization reserve is pro-arrhythmic through electrotonically-triggered EAD formation and establishment of transmural reentry. Our results therefore indicate that quantification of arrhythmic risk requires evaluation of both the heterogeneity of the tissue substrate and repolarization reserve, which are closely inter-related. Furthermore, evaluation of EAD mechanisms with pharmacological interventions requires consideration of the electrotonic trigger via injection of an external prolonged low amplitude current, as it would occur in tissue, rather than by only looking for spontaneous EAD formation.The mechanisms highlighted in our study reveal the complex balance of electrophysiological properties that determine pro-arrhythmic risk in acute regional ischemia. Macro-reentry is favored by both temporal and spatial aspects including ERP and CV, in both the NZ and ICZ, as well as the size of the ICZ and the occurrence of ectopic excitation. EAD formation leading to transmural reentry is determined by repolarization reserve and electrotonic current flowing from the heterogeneous substrate of the BZ. The anti-arrhythmic prolongation of ERP (by IK(ATP) block in human hearts with cardiomyopathy, which was shown to reduce heterogeneity and promote spontaneous termination of ventricular fibrillation , as theillation .Kr block. This still enabled us to unravel key mechanisms determining pro-arrhythmic risk in ischemia and to demonstrate how mechanisms suggested in single cell or thin tissue layers could occur in the three-dimensional volume of the human ventricles. Our findings suggest that inter-subject differences in arrhythmic risk may be explained by differences not only in the ischemic region, but also in repolarization reserve, and further studies may be designed to examine patient-specific scenarios.The agreement with extensive experimental data supports the credibility of our regionally-ischemic human ventricles model and simulations, which also provide the flexibility required to evaluate the electrophysiological action of a range of pharmacological compounds and electrical therapies. The mechanistic understanding arising from such simulations could help in accelerating improvements in safety and efficacy of new anti-arrhythmic treatments. Furthermore, given the computational expense of the simulations in this study, we considered only one size, location and severity of the ischemic region and the membrane kinetics of the TP06 with 3 degrees of I4.5CaL) and the Na–Ca exchange current (INCX) are the major currents potentially meeting this positive feedback criterion” . Further studies should investigate the potentially important implications of β-adrenergic stimulation in modulation of the pro-arrhythmic substrate characterized in our study. Additional sources of heterogeneity such as inter-ventricular differences in APD could also contribute to reentry in acute ischemia, especially if the ischemic region is located near the septum (Our study focuses on the quantification of the vulnerable window during which an ectopic beat can trigger a reentry, as the first condition for the establishment of ventricular tachycardia in regional ischemia. Our results however indicate that sustained ventricular tachycardia may require additional factors such as the occurrence of more than one ectopic beat and this could be the focus of further investigations as in e septum .Please see also related communications in this issue by None.Wellcome Trust Fellowship in Basic Biomedical Sciences [grant number 100246/Z/12/Z to B.R.], an Engineering Physical Sciences Research Council doctoral scholarship to S.D. [EPSRC ref: EP/G03706X/1], the Canadian Institutes of Health Research [MOP 142424 to T.A.Q.] and the Nova Scotia Health Research Foundation [MED-EST-2014-9582 to T.A.Q], and a Marie Skłodowska-Curie Individual Fellowship from the H2020 EU Framework Programme for Research and Innovation [Proposal No: 655020-DTI4micro-MSCA-IF-EF-ST to E.Z.].This work was supported by a


Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer’s disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. TGC) and codon158 (TGC), APOE3 comprises cysteine at codon 112 (TGC) and arginine at codon 158 (CGC) while APOE4 contains arginine at both codon 112 (CGC) and codon 158 (CGC). There are functional differences in their binding ability to lipids and receptors among different isoforms of apolipoprotein E [APOE gene encodes the apolipoprotein E, which is involved in the transportation of cholesterol and lipids in blood circulation and central nervous system. Apolipoprotein E also mediates lipoprotein internalization and degradation via receptor-mediated endocytosis pathway and plays a crucial role in lipid homeostasis. There are three major isoforms of APOE gene, E2, E3, and E4, which are determined by the genetic polymorphisms at codon 112 (rs429358) and codon 158 (rs7412). APOE2 has a cysteine at both codon 112  analysis, fluorescent resonance energy transfer (FRET)-based melting curve analysis, TagMan genotyping method, high-resolution melting (HRM) analysis, and PCR direct sequencing –29. ThesDNA samples used in this study were taken from our previous collection for series of the molecular genetic study of schizophrenia. All the subjects were Han Chinese from Taiwan. In brief, genomic DNA was prepared from peripheral blood cells using Gentra Puregene Blood kit according to the manufacturer’s instructions . This study comprised DNA samples of 711 patients with schizophrenia and 665 control subjects collected from for our serial genetic study of schizophrenia. The study was approved by the Medical Research Committee of National Health Research Institutes of Taiwan, and written informed consent was obtained from each participant and their guardians after the whole procedures were fully explained.We developed a melting curve-based allele-specific PCR method to genotype APOE polymorphisms. The method mainly followed the principle of PCR Tm-shift SNP genotyping method described by Wang and colleagues with modification . In brie® BigDye Terminator Cycle Sequencing Ready Reaction Kit Version 3.1 , according to the manufacturer's protocol. The sequences of the PCR primers and the size of PCR product for sequencing are listed in PCR-based Sanger sequencing was used as the reference method to validate the authenticity of the melting curve-based allele-specific PCR method of APOE genotyping. APOE-Seq-F and APOE-158-R primers were used to generate PCR amplicon covering the codon 112 (rs429358) and 158 (rs7412). A 20 microliter PCR mixture containing 100 ng genomic DNA, 0.5 micromolar of each of the APOE-Seq-F and APOE-158-R primers, 0.0125U of KOD FX DNA polymerase , 0.1 mM dNTP, and 1X KOD FX buffer was prepared. After initial denaturation at 95°C for 5 minutes, 30 cycles of PCR were performed at 95°C 30 seconds, 63°C 30 seconds, and 72°C 30 seconds. Aliquots of PCR product were processed using a PCR Pre-Sequencing Kit  to remove residual primers and dNTPs following the manufacturer's protocol. The purified PCR products were subjected to Sanger sequencing using the APOE-Seq-F as the sequencing primer and an ABI PRISMTo verify the accuracy of the melting curve-based allele-specific PCR for APOE genotyping, 300 DNA samples selected at random from 711 patients with schizophrenia and 665 control subjects were genotyped using the melting curve-based allele-specific PCR method and the Sanger sequencing method simultaneously. The genotyping results were compared between the two methods.After validation of the melting curve-based allele-specific PCR for APOE genotyping, we applied this method to conduct a genetic association study of APOE with schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects. Differences in the genotype and allele frequency between the two groups were assessed using a chi-squared test. A P value less than 0.05 was considered statistically significant.To test the usefulness of the melting curve-based allele-specific PCR for APOE genotyping with DNA from different preparations, we first tested on whole blood samples collected on Guthrie filter paper  for the screening of congenital errors of metabolism. A 3 mm diameter disc was punched from each Guthrie card and collected in the PCR tube. Each disc was immersed in a 50 microliter mixture of methanol and acetone (1:1 V/V) and dried at 60°C for 30 min. After drying, the disc was incubated in 50 microliter water at room temperature for at least 2 hours. Aliquots (4 microliter) of the solution were used for the melting curve-based allele-specific PCR of APOE genotyping method and the Sanger sequencing-based APOE genotyping method as described.We further tested whether the whole blood lysate without DNA purification can be directly used in the melting curve-based allele-specific PCR method. Venous blood samples were collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, and frozen at -20°C freezer until use. After thawing and vortexing, the mixtures were centrifuged briefly. Aliquots (2 microliter) of crude blood lysate were first used directly as DNA template for the melting curve-based allele-specific PCR of APOE genotyping analysis as described before. Then, aliquots (2 microliter) of crude blood cell lysate were subjected to PCR amplification to generate an amplicon of 307 bp using primers APOE-Seq-F and APOE-158-R as described before. The 307 bp amplicons were subsequently subjected to APOE genotyping using the melting curve-based allele-specific PCR method and Sanger sequencing method as described.The representative results of the melting curve-based allele-specific PCR genotyping of the codon 112 (rs429358) using purified genomic DNA are shown in After genotyping a total of 300 DNA samples using both the melting curve-based allele-specific PCR method and the Sanger sequencing genotyping method simultaneously. We obtained a 100% concordance rate between these two methods. Representative sequencing results of are shown in The melting curve-based allele-specific PCR method was applied to a genetic association study of APOE and schizophrenia in a sample of 711 patients with schizophrenia and 665 control subjects from Taiwan. The genotype and allele frequencies of these subjects are listed in A total of 10 Guthrie cards containing blood spots collected from 10 individuals were tested in this study. The storage duration of these cards ranged from 18–20 years. Aliquots of solution that contained the dissolved genomic DNA from Guthrie blood spot were directly subjected to APOE genotyping using the melting curve-based allele-specific PCR method. We were able to determine the APOE genotype of these ten individuals using this method (data not shown). The authenticity of the genotype of these ten individuals was verified using Sanger sequencing method that used aliquots of the same DNA solution collected from Guthrie card. In the experiment of using crude blood cell lysate without purification as DNA template for APOE genotyping. A total of 10 samples collected from 10 individuals were tested in this study. At first, we were not able to directly determine the APOE genotyping using the melting curve-based allele-specific PCR method. Then, we PCR amplified the crude blood cell lysate using the primer pairs APOE-Seq-F and APOE-158-R and PCR conditions described in method section. As shown in In this study, we developed an APOE genotyping method based on the principle of Tm-shift SNP genotyping method described by Wang and colleagues . In thisIn the validation study, a 100% concordance rate was obtained by the melting genotyping method and the Sanger sequencing method in a sample of 300 subjects, indicating that the melting curve-based allele-specific PCR assay is as accurate as Sanger sequencing method. But our method is faster and more cost savings than the Sanger sequencing method. Furthermore, when our method was applied to the genetic association study of APOE polymorphisms and schizophrenia in our population, the genotyping of 711 patients with schizophrenia and 665 healthy control subjects was completed in one month by one research assistant, suggesting this is an efficient APOE genotyping method.Nevertheless, no association of APOE genotypes with schizophrenia was observed in this study. Several case-control studies have been conducted on the genetic association of APOE and schizophrenia. The results have been inconsistent and equivocal in different studies , 31. In In this study, we also demonstrated that blood samples collected on Guthrie blood filter paper that were used in the screening program of congenital errors of metabolism can be used directly in the melting curve-based allele-specific PCR APOE genotyping assay. Based on this result, we suggest that APOE genotyping can be added to the items of newborn screening programs to detect infants with a high risk of Alzheimer’s disease, cardiovascular diseases, and type 2 diabetes. Thus, preventive procedures may be adopted to avoid or delay the onset of these diseases in their later life.In the attempt of using total cell lysate of peripheral blood cells without DNA purification for APOE genotyping, we failed to use the crude lysate as DNA template directly for the melting curve-based allele-specific PCR APOE genotyping. The causes might be due to some materials present in the crude blood cell lysate that may inhibit PCR and interfere with the fluorescence detection. Further troubleshooting is needed to overcome this issue. Despite this, we were able to obtain PCR amplicon from the crude blood cell lysate using the sequencing primers APOE-Seq-F and APOE-158-R. The amplicon was used successfully for downstream melting curve-based allele-specific PCR and Sanger sequencing-based APOE genotyping. Thus, although indirectly, the use of crude blood cell lysate in the melting curve-based allele-specific PCR APOE genotyping represents a fast and cost-saving method that does not need DNA purification from blood cells. Hence, this is a convenient method and cost saving method that can be used in a large-scale population study.In summary, we developed a melting curve-based allele-specific PCR method for APOE genotyping that is efficient, convenient, and accurate and has the flexibility to suit different sample size. The method is versatile and robust that different preparations of DNA can be used in this method. Thus, the method should potentially have a broad range of applications in clinical and research laboratories.


Process optimization deals with various parameters and statistical methods to guarantee consistent cell grow than product quality. Even if high throughput systems can handle these parameter combinations in parallel experiments, the heuristic restriction of boundaries can result in stepwise iterations with many experiments. This can make the way from process development to process establishment even more troublesome, since academia or start-up research facilities might not have the possibility to perform these experiments. The optimization of complex biotechnological production processes with approved Design of Experiment (DoE) methods is therefore time-consuming and cost-intensive. The use of DoE tools in combination with an appropriate growth model might be a valuable tool to develop and to test fed-batch strategies in silico before experiments are carried out in the laboratory. To approve this concept, a two-step growth process with media exchange followed by a fed-batch with an optimized feeding profile was designed using DoE tools in silico.The model cell line CHO-XM-111CCOS-837)was cultivated in two steps with different chemically defined growth media  according to the DECHEMA guideline for the evaluation of single use bag materials,3.Model 7was cultTo increase there solution of the response surface,the parameter space was reduced step wise starting with a wide distribution of data points.In this way,optimal parameter ranges can be identified by determining the boundaries of the next iteration from the contour plot.From the third iteration a numerical optimization of the response-surface was done.This results in an optimum glutamine concentration (feed) and constant feed rate. The simulation was compared to an implemented process to verify the optimized cultivation parameters.As shown in figure The process was optimized using a model based design instead of performing various experiments in the laboratory. Based on a few shaking flask experiments for kinetic parameter determination,the model was tested for data generation on common fed-batch strategies. Optimized conditions were selected by means of DoE strategies and tested experimentally. In this way, suitable fed-batch strategies for mammalian cell lines were evaluated in silico before bioreactor experiments had to be performed.This results in a significant reduction of required experiments and is therefore an inexpensive and time-saving alternative to entire statistical optimization methods.The cell line CHO-XM-111 (CCOS-837) was obtained from the Culture Collection of Switzerland, Wädenswil.


Staphylococcus aureus and extended-spectrum β-lactamase-producing Klebsiella pneumoniae. The resulting dissimilar fitness should have influenced the clonal dynamics and thereby the rates of resistance for these pathogens. Moreover, a similar mechanism was recently proposed for the emergence of the H30 and H30R lineages of ESBL-producing E. coli and the major international clone (ribotype 027) of Clostridium difficile. Furthermore, several additional international clones of various multiresistant bacteria are suspect to have been selected by an analogous process. An ability to develop favorable mutations in the gyrase and topoisomerase IV genes seems to be a prerequisite for pathogens to retain fitness while showing high-level resistance to fluoroquinolones. Since, the consumption of other “non-fluoroquinolone” groups of antibiotics have also contributed to the rise in resistance rates a more judicious use of antibiotics in general and of fluoroquinolones in particular could ameliorate the international resistance situation.Fitness cost associated with resistance to fluoroquinolones was recently shown to vary across clones of methicillin-resistant  Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing Klebsiellapneumoniae and Clostridium difficile. The question arises what sort of driving force(s) could have reshaped the clonal landscape?Though, clonal spread has always been a hallmark of many serious pathogens it is striking and remains enigmatic why the clonal spectra of several multiresistant bacteria have undergone a reduction at some point during the last three decades. We have witnessed a worldwide clonal shrink among others in methicillin-resistant It is well-established that some clones of MRSA have for some time been on the advance replacing others and disseminating in novel geographic areas. Among the foraying MRSA clones ST22 (EMRSA-15) and CC5 proved particularly adept. In Europe where solid, up-to-date information on the clonal distribution of MRSA is available they are well-established to have become the dominant clones at the expense of multiple others . In addiK. pneumoniae. A few STs of the pathogen mostly genetically related to each other have become internationally dominant during the last decade  and South-German (ST228) clones almost completely supplanted isolates of the then resident Hungarian/Brazilian clone ST239; . MoreoveSince, the observed clonal shifts in MRSA were supposed to have been promoted by varying fitness cost associated with resistance to some antibiotic the impact of fluoroquinolone resistance on the vitality of MRSA has been tested by us in a clonal affiliation .Propagation assays from various clones of MRSA showing similar MIC values for fluoroquinolones established that isolates from the New York–Japan (ST5) and South-German clones (ST228) retained significantly more fitness than strains from the Hungarian/Brazilian clone (ST239) which they replaced in Hungary . In addiFigure 1).The superior fitness shown by the newly invading MRSA clones should well-explain the increase in the rate for MRSA detected transiently subsequent to both clonal replacements . The MRSA rates also displayed a significant association with the consumption of second generation cephalosporins though the relationship proved somewhat weaker . The consumption of third generation cephalosprorins was unrelated to the rate for MRSA 1,2.However, it must have been a rise in the use of fluoroquinolones that could have allowed the widespread dissemination of the highly fluoroquinolone resistant major clones of MRSA in Hungary. Trends in the rate for MRSA indeed proved significantly associated with trends in the consumption of fluoroquinolone type antibiotics in the country  clone its general biofilm-producing capacity is in fact inferior compared with those of the ST228 and ST8 strains it readily replaces in the healthcare setting .Although, resistance markers to “non-fluoroquinolone” antibiotics should also impact the fitness of MRSA the preeminence of fluoroquinolone resistance-associated fitness cost is reflected in the observation that our CA-MRSA strains were usually more susceptible to various additional groups of antibiotics than the major clone HA-MRSA isolates, nevertheless, they suffered more fitness cost upon acquiring resistance to fluroquinolones than them. Interestingly, the CA-MRSA isolate suffering the most fitness cost subsequent to the induction of resistance to ciprofloxacin was the ST30 strain showing resistance exclusively to β-lactam antibiotics apart from fluoroquinolones .K. pneumoniae in Hungary  were originally detected in France3. Interestingly, in contrast to previous isolates, all of the novel strains carried the CTX-M-15 ESBL gene . This rise, however, in contrast to MRSA, was not expected to be significantly associated with the consumption of fluoroquinolones for two reasons.Similarly, to the MRSA situation the observed clonal change was associated with a rise in the rate for ESBL-producing (1)K. pneumoniae clones “invaded Hungary” a couple of years subsequent to the increase in the consumption of fluoroquinolones and, thus, commenced to expand in 2004 after the advent of the widespread use of fluoroquinolones in the country .The new ESBL-producing (2)K. pneumoniae clones did not prove as exclusive as that of the major HA-MRSA clones in Hungary . Despite this strong circumstantial evidence the abscence of an established mechanism for how fluoroquinolones could influence the rate of β-lactam resistance in Staphylococcus aureus might have precluded the acceptence of a causal relationship  in its 2003 guideline  and the tionship .An additional factor may complicate the situation somewhat further. One of the groups investigating a possible link between fluoroquinolone use and the rate for MRSA  observedSince the recognition of novel lineages/sublineages of MRSA seem to be of utmost importance efficient techniques for the identification of newly emerging variants of the pathogen are warranted. Spa typing proved very useful in detecting variations within STs  and the C. difficile is also relevant: the proportion of the “fluoroquinolone-related” clones is substantial Major international clones of several multiresistant pathogens have emerged during the past three decades (see literature above).(2)K. pneumoniae, ESBL-producing E. coli, and multiresistant C. difficile. These findings are based on two lines of evidence.Experimental findings show that diverse fitness cost associated with resistance to fluoroquinolones could have influenced the clonal dynamics in MRSA, ESBL-producing (a)K. pneumoniae, ESBL-producing E. coli, and C. difficile were shown to command favorable fitness when displaying high-level resistance to fluoroquinolones K. pneumoniae, multiresistant C. difficile, and ESBL-producing E. coli when showing high-level resistance to fluoroquinolones (see literature above). Some additional mechanisms of resistance to fluoroquinolones in K. pneumoniae seem to be either expendable (qnr type resistance) or may not provide a viable alternative In vitro observations concerning the fitness of various clones of MRSA K. pneumoniae showing resistance to fluoroquinolones may account for the worldwide dissemination of this enzyme at the expense of SHV type ESBLs in this species (K. pneumoniae and ESBL-producing E. coli.The observations that plasmids harboring the CTX-M-15 gene – in contrast to those with SHV type ESBL genes – proved impossible to eliminate from  species . The issC. difficile is well-established (see literature above).The impact of fluoroquinolone consumption on the prevalence of MRSA and the major international clones of K. pneumoniae and ESBL-producing E. coli could be properly investigated by determining the clonal affiliation of the isolates and appreciating the change exclusively in the rates of those major clone strains which are known to have been selected by fluoroquinolones.The influence of fluoroquinolone use on the incidence of ESBL-producing Staphylococcus aureus and ESBL-producing Klebsiella pneumoniae. He perused the literature related to clonality and fluoroquinolone resistance-associated fitness cost in various pathogens and compiled the manuscript himself. All the inferences presented in the paper have been drawn by him.MF was head of the research team establishing the impact of fluoroquinolone resistance-associated fitness cost on the clonal dynamics of methicillin-resistant The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies have demonstrated that machine learning based molecular cancer classification using gene expression profiling (GEP) data is promising for the clinic diagnosis and treatment of cancer. Novel classification methods with high efficiency and prediction accuracy are still needed to deal with high dimensionality and small sample size of typical GEP data. Recently the sparse representation (SR) method has been successfully applied to the cancer classification. Nevertheless, its efficiency needs to be improved when analyzing large-scale GEP data.l2-norm or l1-norm of the coding residual.In this paper we present the meta-sample-based regularized robust coding classification (MRRCC), a novel effective cancer classification technique that combines the idea of meta-sample-based cluster method with regularized robust coding (RRC) method. It assumes that the coding residual and the coding coefficient are respectively independent and identically distributed. Similar to meta-sample-based SR classification (MSRC), MRRCC extracts a set of meta-samples from the training samples, and then encodes a testing sample as the sparse linear combination of these meta-samples. The representation fidelity is measured by the Extensive experiments on publicly available GEP datasets demonstrate that the proposed method is more efficient while its prediction accuracy is equivalent to existing MSRC-based methods and better than other state-of-the-art dimension reduction based methods. It haia (ALL) ,8 and aria (ALL) ,9 may noDimension reduction methods can be grouped into two categories: feature selection and feature reduction approaches. Feature selection methods , such aset al. proposed a SR-based classification (SRC) method using et al. further presented a new SR-based method for GEP-based cancer classification, termed meta-sample-based SR classification (MSRC), where a set of meta-samples are extracted from training samples, and then a testing sample is represented as the linear combination of these meta-samples by et al. proposed a new classifier, meta-sample-based robust sparse representation classifier (MRSRC) based on the MSRC method, for cancer classification [Machine learning based methods are also often called model-based methods because a predictive model is built for predicting the label of test sample. The model selection is a complex training procedure, which easily leads to over-fitting and decreased prediction performance. Recently, sparse representation (SR), a powerful data processing method that does not require model selection, has been extensively applied to face recognition ,19 and f samples . They ape method . Their efication . Their eet al. proposed a new coding model, namely regularized robust coding (RRC) for face recognition [Previous SR-based model assumes that the coding residual follows Gaussian or Laplacian distribution, which may not be effective for describing the coding residual in practical GEP datasets, and another problem is that the sparsity constraint on coding coefficients leads to the high computational cost of SRC method. To deal with the problem, Yang ognition . Here, wognition . Our extn-dimensional space. Each of the m columns consists of an n-element expression vector for a single sample. Let Let where The analysis flowchart of the meta-sample-based SR method is different from those of traditional model-based and template-based methods Figure . The cla1) The whole sample set is randomly split into two disjoint parts: training set and test set, and then the meta-samples are extracted only from the training set using singular value composition (SVD).2) The weight of each gene is calculated according to a weight function, and the genes with lower weight are removed in a test sample 3) The test sample 4) We can reconstruct the test sample for each subclass by using the meta-samples and the coding coefficient of the original test sample 5) The distance between the processed test sample and each reconstructed test sample et al. used singular value decomposition (SVD) to transform GEP data from a space of genes The meta-sample extracted from GEP data is commonly defined as a linear combination of all training samples. In this paper, a set of meta-sample is extracted from all training samples of one cancer type. We find that meta-sample can capture the structures to the data and offer biological insight. on the other hand, the linear combination of the meta-samples can roughly estimate genetic expression pattern of gene data . Alter, '' space , where twhere matrix The meta-sample set  formula where There are two key issues in solving the RRC model. The first one is how to determine the distribution where where The logistic function has the same properties as the hinge loss function in SVM , so we cwhere For cancer classification, the coding coefficients associated with the dictionary atoms from the same subclass would have big absolute values. However, we do not know which subclass the testing sample will belong to. Therefore, we actually assume that the coding coefficient where The RRC model has two vital cases when c values .When When 3C) algorithm was designed by Yang, et al. to solve the RRC model efficiently [Iteratively reweighted regularized robust coding .3C is actually a set of meta-samples which are extracted by SVD from the original training set, so we call our methods as MRRCC1 (the meta-sample-based regularized robust coding classification 1) and MRRCC2 (the meta-sample-based regularized robust coding classification 2) corresponding to the two cases RRC_L1 and RRC_L2, respectively.In literature  the RRC GEP data can be obtained by two technologies, DNA microarray and next-generation sequencing (NGS) technologies. In our experiments five microarray and four NGS cancer datasets are used to evaluate the proposed method Table . The fivhttp://cancergenome.nih.gov/). They include Breast invasive carcinoma , Kidney renal clear cell carcinoma (KIRCCancer), Lung adenocarcinoma (LUADCancer), and Thyroid carcinoma (THCACancer). All samples are matched cancer and normal tissue samples.The four NGS datasets are downloaded from the web site: The Cancer Genome Atlas (TCGA) , one of simplest classification methods, with correlation distance is used to classify cancer samples (here 5 nearest neighbors are used). For LDA method and the datasets with two subclasses, Euclidean distance is used because only one feature is extracted. To avoid over-fitting, before classification we extract only 5 features using these feature extraction methods except LDA whose number extracted is A two-stage method can be used to reduce the dimensionality of dataset before classification. The first stage is a process of adopting a gene filter method such as KWRST   or RelieExperiments indicate that the different divisions of training sets and test sets can also greatly affect the classification performance. In our experiments, the Balance Division Method (BDM) is used to divide each original dataset into balanced training sets and test sets . For thel1-norm regularization but the former has much lower computational cost [With the development of microarray and NGS technologies, a huge amount of GEP data is rapidly accumulated, demanding more efficient analysis methods to analyze these data. In this paper we present a novel meta-sample-based regularized robust coding for cancer classification (MRRCC) that firstly represents each test sample as a linear combination of all meta-samples which are extracted from the original training set using SVD. The coefficient vector is then obtained by nal cost . The expThe authors declare that they have no competing interests.Shu-Lin Wang designed the framework of analysis, performed the partial experiments, and drafted the manuscript. Liuchao Sun also performed the partial experiments, and Jianwen Fang analyzed the numerical results and revised the manuscript. All authors read and approved the final manuscript.


Under pentobarbital anesthesia, rats were subjected to unilateral nerve injury. Mechanical allodynia was tested for two weeks after nerve injury. After nerve injury, neuropathic pain behaviors developed progressively. The crossed-withdrawal reflex was observed at two weeks postoperatively. Contralateral enhancement of 2-DG uptake in the ventral horn of the spinal cord to electrical stimulation of the uninjured paw was observed. These results suggest that the facilitation of information processing from the uninjured side to the injured side may contribute to the crossed-withdrawal reflex by plastic changes in the spinal cord of nerve-injured rats.We have previously reported the crossed-withdrawal reflex in which the rats with nerve injury developed behavioral pain responses of the injured paw to stimuli applied to the contralateral uninjured paw. This reflex indicates that contralateral plastic changes may occur in the spinal cord after unilateral nerve injury. The present study was performed to elucidate the mechanisms and morphological correlates underlying the crossed-withdrawal reflex by using quantitative  It has been shown that peripheral nerve injury can cause severe chronic pain in humans , 2. HumaOccasionally, neuropathic pain symptoms can be observed on the opposite side of the nerve injury in humans or in experimental animal models of neuropathic pain –4. It inIn our previous study , we obse14C-2-deoxyglucose (2-DG) autoradiography, which is a functional mapping technique established by Sokoloff et al. -methyl methacrylate standards  to X-ray film (Kodak BioMax MR) for 10 days. For quantitative analysis, the spinal cord gray matter was divided into 4 regions: the dorsal horn (corresponding approximately to laminae I-IV), the intermediate horn (laminae V-VI), the ventral horn (laminae VII-IX), and the central gray (lamina X). The selected regions were digitized using computer-generated templates for each spinal lumbar segment from L1 to L6. The digitized images were quantified using the MetaMorph Imaging System .The sections were attached to a cardboard and were exposed with precalibrated /[metabolic activity in the ipsilateral ventral horn] × 100. t-test.Data were expressed as means ± S.E.M. at each level of the lumbar spinal cord. Cross ratios of the nerve-injured rats and those of normal control rats were compared at each spinal level using the independentThe result of mechanical allodynia is shown in P < 0.05, paired t-test) but the reflex was not observed in normal control animals.In To observe any increased crossing input from the uninjured side to the injured side in the spinal cord, the metabolic activities of the lumbar spinal cord in normal controls and nerve-injured rats that expressed cross-withdrawal reflexes were compared. Electrical stimulation of the uninjured paw of neuropathic rats produced significantly increased 2-DG uptake in contralateral ventral horn in the lumbar spinal cord; but, in normal controls, upregulation of 2-DG uptake was produced in the ipsilateral ventral horn where the electrical stimulation was applied .P < 0.05, independent t-test). In the L1-L2 segments, the cross ratios of nerve-injured rats tended to be slightly higher but were not significantly different from controls .Cross ratios from different levels of the spinal cord are compared in After nerve injury, bilateral allodynia including original pain and mirror image pain can be found in experimental animal models as well as human patients. It has been shown that the mirror image pain develops in a minority of human patients but it recurs frequently .Contralateral changes resulting from ipsilateral pain symptoms have also been reported through different experimental paradigms in the absence of lesions of the nervous systems. Subcutaneous injection of dilute formalin solution into a hind paw induced a mirror-image-like pain, and the rats frequently licked the contralateral untreated hind paw . A similIn the present study, the crossed-withdrawal reflex represents information processing from input on the uninjured side to output on the injured side. This phenomenon was morphologically determined by using the 2-DG autoradiography technique. As determined by cross ratio using the 2-DG technique, the magnitude of the contralateral metabolic 2-DG activity over the ipsilateral activity by the stimulation of the uninjured side in the neuropathic rat was greater than that in the normal control animals. This result indicates that information processing from input on the uninjured side to output on the injured side must be facilitated in rats with nerve injury. This result provides a morphological and metabolic evidence for the behavioral cross-withdrawal reflex.Similarly, it was found that spinal cord glucose consumption increased both contralaterally and ipsilaterally after chronic constriction injury of the unilateral sciatic nerve , 12. ConOur previous study  reportedOur present study showed that electrical stimulation on the uninjured side increased metabolic activities on the injured side through the multisegments of the spinal cord. These results suggest that maybe there exist heterotopic transneuronal signaling pathways between the uninjured side and the injured side. There are evidences supporting the existence of the transneuronal signaling pathway. The anatomical study by Fitzgerald  reportedThe detailed mechanisms of the crossed-withdrawal reflex with nerve injury still remain to be determined. However, this result is in good agreement with the report which demonstrates that peripheral nerve injury, a model for neuropathic pain, is associated with a synaptic plasticity in the spinal dorsal horn . The croWe observed that the rats with unilateral nerve injury showed withdrawal responses of the injured paw to stimuli applied to the contralateral uninjured paw. We called this unusual response type “the crossed-withdrawal reflex.” We determined the “crossed-withdrawal reflex” by behavioral and electrophysiological aspects in our previous report.In the present study, electrical stimulation of the uninjured paw of neuropathic rats produced significant increase of 2-DG uptake in contralateral ventral horn in the lumbar spinal cord. This result is in accordance with the behavioral and electrophysiological findings of the crossed-withdrawal reflex and suggests that information processing from input on the uninjured side to output on the injured side may be facilitated in neuropathic rats by neuroplastic changes through the heterotopic transneuronal signaling pathway in the spinal cord after peripheral nerve injury.


Purpose: Some studies suggest that the pattern of glucose hypometabolism relates not only to the ictal-onset zone but also reflects seizure propagation. We investigated metabolic changes in patients with occipital lobe epilepsy (OLE) that may reflect propagation of ictal discharge during seizures with automatisms.Methods: Fifteen patients who had undergone epilepsy surgery for intractable OLE and had undergone interictal Fluorine-18-fluorodeoxyglucose positron-emission tomography (18F-FDG-PET) between 1994 and 2004 were divided into two groups (with and without automatisms during seizure). Significant regions of hypometabolism were identified by comparing 18F-FDG-PET results from each group with 16 healthy controls by using statistical parametric mapping.Key Findings: Significant hypometabolism was confined largely to the epileptogenic occipital lobe in the patient group without automatisms. In patients with automatisms, glucose hypometabolism extended from the epileptogenic occipital lobe into the ipsilateral temporal lobe.Significance: We identified a distinctive hypometabolic pattern that was specific for OLE patients with automatisms during a seizure. This finding supports the postulate that seizure propagation is a cause of glucose hypometabolism beyond the region of seizure onset. However, the interictal 18F-FDG-PET hypometabolism often extends beyond the epileptogenic region into the adjacent and remote cortical and subcortical structures , oral and manual automatisms are common and are often attributed to seizure propagation from the occipital to the temporal lobe , 8. This18F-FDG-PET between 1994 and 2004 at the Westmead and Royal Prince Alfred Hospitals, Australia. The study was approved by the ethics committees in Central Sydney (Protocol no. X03-0161) and Western Sydney Area Health Services [HREC reference no: HS/TG HREC 2003/6/4.13 (1670)]. Patients with resection of the parieto-occipital cortex were not included. The occipital lobe boundaries were established as defined by the Tzourio-Mazoyer atlas  at the voxel-by-voxel level using two-sample t-tests to identify all clusters of voxels exhibiting significant hypometabolism. SPM analysis identifies regions with cluster size (extent threshold) larger than 250 contiguous voxels and with voxel-level significance (height threshold) of p ≥ 0.01. Only regions with clusters of voxels that exceeds this extent and height threshold and achieved corrected cluster-level significance of p < 0.05 were considered significant. Group analysis was performed to eliminate interindividual metabolic variability. The analysis allowed the identification and comparison of hypometabolic patterns for the seizures with automatisms and the seizures without automatisms groups when compared to normal healthy controls.Methods for FDG-PET acquisition and for statistical parametric mapping  and spatial pre-processing of FDG-PET images of patients and 16 healthy controls have been described in detail elsewhere , 13. In ocedures . For staFifteen patients  with age of seizure onset between 2 and 20 years  were studied. The median age of the study population was 25 years old . Ten patients were admitted to hospital for in-patient prolonged video EEG on at least two occasions (range 1–4 video-EEG monitoring). The median number of seizures recorded was 16 seizures . Among the patients, 13 had intracranial video-EEG studies and showed an ictal-onset zone within the occipital lobe. Two patients (patient 6 and 13) did not undergo invasive monitoring but showed a MRI abnormality with concordant scalp video-EEG findings; both became seizure free after surgery. The median follow-up duration following surgery was 10.1 years . Twelve patients achieved Engel class 1 outcome, 2 had significant seizure improvement (Engel 2) and 1 had worthwhile improvement (Engel 3). The histopathology was summarized in Table Nine patients had seizures with automatisms occurring as a component of their habitual seizures. The other six patients, who never had automatisms as a feature of their habitual seizures, were assigned to the group without automatisms for SPM group analysis. Table In the seizure without automatisms group, SPM analysis revealed significant glucose hypometabolism involving primarily the epileptogenic occipital lobe and extending marginally into the posterior temporal region Figure A. In conp < 0.001, median 1223 voxels; IQR 928–4207 voxels) when compared to patients without automatisms during seizures . No significant association was found between the extent of temporal lobe involvement, duration of epilepsy before FDG-PET, age when FDG-PET was performed and seizure outcome.The extent of temporal lobe involvement was significantly associated with the presence of automatisms during seizures (In this study, we determined the interictal metabolic patterns of glucose in patients with OLE with and without automatisms. The major difference in OLE patients with automatisms was the presence of significant glucose hypometabolism in the temporal lobe. We suggest that this interictal metabolic change reflects evidence for the propagation pathway of seizures in patients with automatisms.18F-FDG-PET has been used to localize ictal focus by showing regional glucose hypometabolism in the epileptogenic occipital lobe in OLE (18F-FDG-PET revealed focal areas of relative hypometabolism that was associated with the epileptogenic zone.Significant hypometabolism was present in the occipital lobe in both groups of patients with OLE. e in OLE , 17. OurThe occipital lobe is connected to the mesial and lateral temporal structures by abundant multisynaptic projections , 19. Sevin situ. This suggests involvement of the temporal lobe reflects spread of ictal activity, rather than the temporal lobe being a key part of the epileptogenic network.Several studies support the hypothesis that the topography of glucose hypometabolism relates, at least in part, to brain regions involved in the ictal onset and to pathways of seizure propagation generating the clinical manifestations , 4, 23. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic obstructive pulmonary disease (COPD) has been recently recognized as an inflammatory disease. A recent Swedish hospital-based study found an increased risk of COPD in patients with a number of autoimmune conditions including systemic lupus erythematosus (SLE). We wonder whether the risk is also present in SLE patients from the general population. The objective was to assess the future risk of newly recorded COPD cases among incident SLE cases compared with controls from the general population using physician billing and hospitalization data that cover the entire province of British Columbia (BC), Canada.Our data include all health professionals and hospital visits covered by the comprehensive provincial medical services plan (1990 to 2010) and all dispensed medication (1996 to 2010), for all BC residents. We conducted a retrospective matched cohort (1996 to 2010) study among patients satisfying at least one of the following validated criteria: one diagnostic code for SLE (ICD-9-CM = 710.0) on at least two visits within a 2-year period by a nonrheumatologist physician; one ICD-9 code by a rheumatologist or from hospitalization; and absence of a prior SLE diagnosis between 1990 and 1995. Ten controls matched by birth year, sex and calendar year of exposure were randomly selected from the general population for each case. Outcome: we used a validated criteria to define COPD  from hospitals or death certificates. We estimated relative risks (RRs) by comparing SLE cases with age-matched, sex-matched and entry-time-matched comparison cohorts, adjusting for confounders. Sensitivity analyses were conducted to assess for unmeasured confounders.Among 4,486 individuals with incident SLE, 96 developed COPD  (Table This is the first general population-based study indicating a twofold increased risk of COPD in patients with SLE. The risk of developing COPD was highest within the first year, declining thereafter, suggesting the potential pathogenic role of inflammation in the development of COPD.


Ursus maritimus) have been identified as vulnerable to changes in sea ice conditions. We use sea ice projections for the Canadian Arctic Archipelago from 2006 – 2100 to gain insight into the conservation challenges for polar bears with respect to habitat loss using metrics developed from polar bear energetics modeling.Sea ice across the Arctic is declining and altering physical characteristics of marine ecosystems. Polar bears (st century with projected warming. Each polar bear population in the Archipelago may undergo 2–5 months of ice-free conditions, where no such conditions exist presently. We identify spatially and temporally explicit ice-free periods that extend beyond what polar bears require for nutritional and reproductive demands.Shifts away from multiyear ice to annual ice cover throughout the region, as well as lengthening ice-free periods, may become critical for polar bears before the end of the 21Under business-as-usual climate projections, polar bears may face starvation and reproductive failure across the entire Archipelago by the year 2100. Ursus maritimus) are inextricably linked to Arctic sea ice and are sensitive to sea ice loss Pusa hispida), the primary prey species of polar bears Observed changes in global climate have influenced Arctic sea ice cover more than most models have predicted st century, with significant losses in the Hudson Bay and peripheral Arctic seas Optimal polar bear habitat is predicted to decline in the 21st century The CAA and Greenland were thought to have the greatest likelihood of sustaining polar bears to the end of the 21www.gfdl.noaa.gov/coupled-physical-model-cm3). This simulation includes a realistic spatial distribution of sea ice extent and thickness and simulates the trend in observed minimum sea ice extent during the observational record (1979–2013). This pilot simulation was dynamically downscaled using the ice-ocean Massachusetts Institute of Technology General Circulation Model (MITgcm) simulation in regional mode over the Arctic at a resolution of 18km (available from http://mitgcm.org). The 3-hourly atmospheric forcing fields from GFDL-CM3 were bias-corrected at the monthly scale using differences for variables  or ratios for variables  between the Japanese 25 year Reanalysis (JRA25) st century with large trends in many of the Arctic climate forcing fields. MITgcm parameters were provided by Nguyen Global climate simulations contributed to the Coupled Model Intercomparison Project Phase 5 (CMIP5) are too coarse to effectively resolve the narrow channels of the CAA. Although numerically challenging, one solution is to dynamically downscale a global simulation onto a finer grid using a regional climate model. A comparison between over 30 different CMIP5 models led us to select one simulation from the Geophysical Fluid Dynamics Laboratory Coupled Physical Model (GFDL-CM3) driven by radiative scenario RCP8.5 to pilot the regional model  and central regions  may be entirely ice-free for 5 months, and may no longer reach 100% SIC at maximum ice extent. In the north , the simulation estimates a 2–4 month ice-free season by the end of the 21st century, and maximum concentrations <100% in 2080–2090. Ice thickness and snow depth exhibit similar declines throughout the CAA. Ice thickness in the late 20th century was twice to nearly five times the thickness of the projected thickness in the same month of the late 21st century.While multiyear ice is not good hunting habitat due to its low prey abundance eptember . By the th and late 21st centuries in the western regions . Furthermore, using a conservative estimate of a minimum 20 cm snow depth requirement for seal habitat Snow depth declines in part due to the reduction in sea ice surface and dates of formation, but also due to a predicted shift in precipitation from snow to rain Polar bears fare poorly when sea ice is absent for prolonged periods, losing body mass without the opportunity to hunt st century with ice-free seasons reaching critical duration, and early break-ups occurring in parts of all populations we examined. Similarly, to the east of the CAA, the west coast of Greenland and much of Baffin Bay may no longer be suitable habitat for polar bears before 2050, though ice should persist along the east coast of Baffin Island until much later. Early break-up in the narrow channels of the central CAA may become critical in 2060–70s, whereas the adjacent coastlines of the open Arctic Ocean remain largely non-critical until near 2100.We find that sea ice conditions may become unsupportive of polar bear population persistence in the CAA and its surroundings by the late 21st century.It is important to consider that what we deem a critical point-of-no-return occurs once the ice-free period crosses our critical threshold and remains critical for the remainder of the modeled period. Nevertheless, it is feasible that single seasons, or clusters of seasons, may become critically ice-free before that point, with subsequent seasons being non-critical. As such, we examined the cumulative number of critical events for of the aforementioned critical periods (A–D) by population . We founst century.Without exception, our simulation projects the sea ice habitat in all polar bear populations of the CAA may change from a multiyear to an annual ice system before the end of the century, and the remaining annual ice might not persist sufficiently long each year to allow hunting opportunities for polar bears as we currently understand them. Our model suggests that, by 2070, over 80% of the CAA might experience break-up in July, forcing pregnant females to retreat to land early, with possible negative effects on their reproductive output. Given that our study area comprises approximately one quarter of the world's polar bears, and nearly one-tenth of the total current habitat, our analyses project significant habitat loss and alteration under the business-as-usual model scenario used to estimate sea ice conditions over the coming 21Conservation efforts to protect polar bear habitat in the Canadian Arctic should focus on regions that are slower to experience change in sea-ice concentration and ice-free period. The Queen Elizabeth and Norwegian Bay populations retain multiyear ice the longest, and their northerly fjords and channels consistently exhibit the fewest critical ice-free events. Nevertheless, by 2100 all regions of the study area may cross the critical point-of-no-return, putting the persistence of the CAA polar bear populations in jeopardy.


Inadequate cardiac output is associated with poor outcome following cardiac surgery and is generally modified by use of inotropic agents, volume resuscitation and epicardial pacing. Although temporary epicardial pacing is frequently utilised post-cardiac surgery, the potential impact of determining the optimal R-R interval in the critically ill has largely been ignored. Echocardiography (Echo) has been used to optimise cardiac output by modifying pacing settings.We sought to determine the impact of the R-R interval on cardiac electromechanics, cardiac output and stroke volume in patients following cardiac surgery on a cardiothoracic intensive care unit.24 sequential patients being paced following cardiac surgery were assessed using transthoracic echocardiography within 4 hours of returning to the CICU. Pacing was set to the presumed optimal rate basing on clinical evaluation by the treating anesthesiologist. Echo data included LV/RV systolic & diastolic function including Doppler assessment of stroke volume (SV), Cardiac Output (CO) and total isovolumic contraction/relaxation time derived by ejection time (ET) & filling time (FT). tIVT was calculated as [60-; t-IVT > 14 s/m is associated with left ventricular dyssynchrony [Results are shown in Table 2-0.88 - Figure 2).There was no consistent nor predictable correlation between changes in HR and SV, CO or CI in the overall population studied except in mitral valve surgery patients. A strong linear negative correlation was found between LV t-IVT and SV, CO and CI in all the groups (p < 0.0001 rThis is the first report of the role of HR optimization in post-cardiac surgery patients with epicardial wire, which led to changes in pacemaker baseline setting in 79% of the patients. This underlines that the clinical assessment of the pacemaker setting is not sufficient and that the HR has to be set on the patient's features. The t-IVT showed to be a sensitive index to assess the best hemodynamic and electromechanic profile in our population.


Purpose. To assess the ability of optical coherence tomography-angiography (OCT-A) to show and analyze retinal vascular patterns and the choroidal neovascularization (CNV) in retinal vascular diseases. Methods. Seven eyes of seven consecutive patients with retinal vascular diseases were examined. Two healthy subjects served as controls. All eyes were scanned with the SD-OCT XR Avanti . Split spectrum amplitude decorrelation angiography algorithm was used to identify the blood flow within the tissue. Fluorescein angiography (FA) and indocyanine green angiography (ICGA) with Spectralis HRA + OCT (Heidelberg Engineering GmbH) were performed. Results. In healthy subjects OCT-A visualized major macular vessels and detailed capillary networks around the foveal avascular zone. Patients were affected with myopic CNV (2 eyes), age-related macular degeneration related (2), branch retinal vein occlusion (BRVO) (2), and branch retinal artery occlusion (BRAO) (1). OCT-A images provided distinct vascular patterns, distinguishing perfused and nonperfused areas in BRVO and BRAO and recognizing the presence, location, and size of CNV. Conclusions. OCT-A provides detailed images of retinal vascular plexuses and quantitative data of pathologic structures. Further studies are warranted to define the role of OCT-A in the assessment of retinovascular diseases, with respect to conventional FA and ICG-A. Fifty years have passed since Novotny and Alvis performed the first fluorescein angiography (FA). This invasive method uses intravenous fluorescein to produce fluorescence images of circulating blood in the human retina . For manFrom its commercialization, both time and spectral domain (SD) optical coherence tomography (OCT) modalities have dramatically changed the daily clinical practice in ophthalmology , 4. WithRecently, a novel dyeless method of microvasculature imaging called OCT angiography (OCT-A) was introduced. Several prototypes used the normal movement of the red blood cells in the retinal capillaries as intrinsic contrast medium to generate flow imaging. The phase-based and amplitude-based modalities showed feasibility of rendering deeper structural details of retinal and choroidal microvascular structures when motion-based contrast techniques are used –9. ThreeAmplitude-based OCT signal analysis may be advantageous for ophthalmic imaging since it uses a speckle variance method that does not suffer from phase noise artifacts and does not require complex phase correction methods . Both phμm. Each B-scan contained 216 A-scans. The scanning procedure was described in detail elsewhere [This prospective unmasked study adhered to the tenets of the Declaration of Helsinki, and informed consent was obtained from all patients prior to their enrolment. Our local Ethics Board was notified and stated that their approval was not necessary. Seven eyes of seven consecutive Caucasian patients  ranging in age from 54 to 64 years (mean 59.1 years), referred to the Ophthalmic Clinic of University Chieti-Pescara, Italy, for the presence of retinal vascular diseases from October 1 to 31, 2014, were examined. Two healthy subjects with no significant medical history and no signs or symptoms of retinal vascular disease were included as controls. FA was performed in all subjects with the Spectralis HRA + OCT (Heidelberg Engineering GmbH). Indocyanine green angiography (ICGA) was performed only in patients with suspected choroidal neovascularization (CNV). All eyes were also scanned with the SD-OCT XR Avanti , using the following settings: high speed , 840 nm wavelength (band-width 45 nm), and an axial resolution of 5 lsewhere . The SSAZ position) of the highly reflective RPE was identified through the analysis of the reflectance and reflectance gradient profiles in depth [ en face X-Y projection angiograms were produced by selecting the maximum decorrelation value along the axial (Z) direction in each layer. Segmentation of the retina in specific layers provided a simple en face visualization of the corresponding vascular supply for that layer.Decorrelation of OCT signal amplitude between B-scans taken at the same nominal position could be caused by flow, bulk tissue motion, scanner position error, and background noise. To enhance true flow in the images and improve the signal-to-noise ratio for flow detection, the decorrelation due to bulk motion and background noise were eliminated. The SSADA data was separated into retinal and choroidal regions with the dividing boundary set at the retinal pigment epithelium (RPE). The depth , manual correct of the segmentation was necessary in order to avoid both image processing software and segmentation errors . The allIn healthy subjects OCT-A visualized major macular vessels and detailed capillary networks around the foveal avascular zone. The vascular network was similar to those reported using FA . NotablyPatients 1 and 2 were affected with ischemic branch retinal vein occlusion (BRVO). A composite map composed of three 8 × 8 mm scans from patient 1 and an 8 × 8 mm OCT-A scan from patient 2 are shown in Figures Patient 3 was affected with branch retinal artery occlusion (BRAO). The main finding was the low flow area in OCT-A, which correlated with the area of hypofluorescence in FA due to retinal artery hypoperfusion. Moreover, the en face image adapted to the RPE surface showed the presence of ischemia-induced area of hyporeflectivity and longitudinal B-scan revealed focal thickening and hyperreflectivity within both inner and deep capillary plexuses .In patient 4, the color-mode OCT-A aided in detecting the vascular network of myopic CNV and in defining specific morphological features . The entPatients 6 and 7 were affected with CNV secondary to age-related macular degeneration (AMD). Classic CNV  focused This study aimed at examining the ability of OCT-A, a promising and noninvasive imaging modality, to image the vascular modifications within the inner and outer retinal layers in several retinovascular diseases. Currently, FA and ICGA are considered the gold standard for defining functional and morphological features in patients affected with ocular vascular diseases. However, both techniques are invasive since they require intravenous dye injection and are bothersome for some patients who complain of side effects that range from nausea/vomiting to serious anaphylactic reactions . Moreover, FA can only show vessels in a nearly transparent structure with a thickness that is on the order of only hundreds of micrometers. This allows a good visualization of the superficial capillary network of the retina but not of the deep capillary layer , 16. On In the present study both OCT-A and FA provided clinically useful images of the inner retinal vascular plexus. This was in accordance with results of Imai et al.  who usedOne of the most promising fields for the application of OCT-A is CNV imaging. Other structural OCT imaging modalities cannot directly identify the CNV structure, but only the presence of changes such as abnormal tissue above or below the RPE. Therefore, FA and ICGA are still needed in the initial diagnosis of neovascular AMD.The present study showed that OCT-A was also valuable in imaging and quantifying CNV in myopia and AMD since the neovascularization was successfully identified in all patients presenting this lesion. The SSADA algorithm was crucial in differentiating CNV from the surrounding outer retinal tissue, RPE, and hemorrhages. This confirmed the preliminary data reported by Jja et al. in a swept-source OCT prototype study . NotablyIt is important to keep in mind that subretinal and surrounding CNV fluid appeared as a low flow area in OCT-A (because SSADA identifies only moving flow), whereas it produced a hyperfluorescent pattern in FA; where the fluid outside of the blood vessels generates dye leakage Overall, OCT-A was not completely comparable to FA and ICGA angiograms. In fact, OCT-A provided static images of the vascular flow, without providing dynamic information, which was conversely provided by classical ocular angiographies with dyes. However, the integration of structural and quantitative data obtained with OCT has the potential to be a new useful tool for studying retinal vascular diseases.Although this pilot study has the weaknesses of limited sample size it showed the potential of OCT-A for providing quantitative data for diagnosis and follow-up of different retinochoroidal diseases without the use of intravenous dyes. The first limit of this new technique was that the field of view  was smaller than conventional FA. In the future the FOV should increase as scan speeds increase. The second limit was that a precise fixation was required in order to obtain good images, so this technique was not reproducible in patients with low vision. Further investigations will be needed to validate the clinical application of this novel method.


Right-sided lesions occur in 5-25% of patients after arterial switch operation (ASO) and percutaneous or surgical treatments are available .We present the first reported case of iatrogenic aorto-pulmonary window (APW) due to transcatheter pulmonary valve implantation after ASO and the surgical strategy for repair.A 12-year old boy (d-transposition of the great arteries and ASO (LeCompte maneuver) in the neonatal period) was referred for transcatheter pulmonary valve implantation due to pulmonary regurgitation. A 22-mm stent-mounted valved bovine jugular vein graft  was implanted, but the patient became hemodynamically unstable (pulmonary-to-systemic ratio 1.96). Transesophageal echocardiography (TEE) showed a traumatic APW in the uppermost portion of the ascending aorta. Haemodynamic stability was achieved following closure using a 10-mm Amplatzer-Muscular-VSD-Occluder but he remained symptomatic in the subsequent weeks. TEE showed residual left-to-right shunt in the proximal margin and a covered stent was implanted on the left pulmonary artery (LPA) to deal with the residual shunt, but the Amplatzer was dislocated. He was referred for emergent surgery. He was cooled to 18°C and ventricular fibrillation was achieved. Carbon dioxide field flooding was employed throughout. The circulation was arrested and the right PA was incised and extended toward the LPA. Both percutaneous devices were removed and the APW was closed (Gore-Tex patch). The PA and branches were reconstructed (bypass time 150 and arrest time 23 minutes). TEE showed no residual shunts.He had an uneventful recovery and was discharged on 10th postoperative day.The adherences between the aorta and PA on performing the LeCompte maneuver and the protrusion of the stent-mounted valve into the PA bifurcation may result in an APW. Care should be taken in avoiding Melody valve in patients with ASO and surgical option such as valved conduit is advised when significant pulmonary regurgitation is developed.Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.