Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects.The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV ( Clinical RNA-seq datasets can predict clinical outcomes. Here, Shen et al. report a statistical method for survival analysis of mRNA isoform variation using clinical RNA-seq datasets, and the identified isoform based survival predictors outperform gene expression based survival predictors using TCGA data on six cancer types. Production of mRNA isoforms through alternative processing and modification of RNA is essential for generating this complexity. A prevalent mechanism for producing mRNA isoforms is the alternative splicing of precursor mRNA2457RNA sequencing (RNA-seq) has become a popular and cost-effective technology to study transcriptome regulation and mRNA isoform variation13P-value cutoff of 0.05 , a statistical model for identifying mRNA isoform ratios associated with patient survival times in large-scale cancer RNA-seq data sets. SURVIV models the estimation uncertainty of mRNA isoform ratios in RNA-seq data and tests the survival effects of isoform variation in both censored and uncensored survival data. In simulation studies, SURVIV consistently outperforms the conventional Cox regression survival analysis that ignores the measurement uncertainty of mRNA isoform ratio. We used SURVIV to identify alternatively spliced exons whose exon-inclusion levels significantly correlated with the survival times of invasive ductal carcinoma (IDC) patients from the TCGA breast cancer cohort. Survival-associated alternative splicing events are identified in gene pathways associated with apoptosis, oxidative stress and DNA damage repair. Importantly, we show that alternative splicing-based survival predictors outperform gene expression-based survival predictors in the TCGA IDC RNA-seq data set, as well as in TCGA data of five additional cancer types. Moreover, the integration of clinical information, gene expression and alternative splicing profiles leads to the best prediction of survival time.Here we introduce SURVIV .Briefly, for any individual exon-skipping event, the first component of SURVIV uses a proportional hazards model to establish the relationship between patient β. A positive β means increased exon inclusion is associated with higher hazard rate and poorer survival, while a negative β means increased exon inclusion is associated with lower hazard rate and better survival. λ0(t) is the baseline hazard rate estimated from the survival data of all patients (see Sk(t) can be calculated from the patient-specific hazard rate λk(t) as β=−1 and a constant baseline hazard rate λ0(t)=1, where higher exon-inclusion levels are associated with lower hazard rates and higher survival probabilities.For each exon, the association between the exon-inclusion level and patient survival time is reflected by the survival coefficient ψk of a given exon in a particular sample can be estimated by the RNA-seq read count specific to the exon inclusion isoform (ICk) and the exon-skipping isoform (SCk). Other types of alternative splicing and mRNA isoform variation can be similarly modelled by this frameworklI) and the exon-skipping isoform (lS), the exon-inclusion level ψk can be estimated as ICk follows a binomial distribution with the total read count nk=ICk+SCk, we have:The second component of SURVIV models the exon-inclusion level and its estimation uncertainty in individual patient samples. As illustrated in ψk as influenced by the total read count nk, in which the parameter pk represents the proportion of reads from the exon-inclusion isoform, given the exon-inclusion level ψk adjusted by a length normalization function f(ψk) based on the effective lengths of the isoforms. The definitions of effective lengths for all basic types of alternative splicing patterns are described in ref. The binomial distribution models the estimation uncertainty of ψk estimated from RNA-seq count data, and the confidence of ψk estimate for a given exon in a particular sample depends on the RNA-seq read coverage. We use the statistical framework of survival measurement error modelH0:β=0. The false discovery rate (FDR) is estimated using the Benjamini and Hochberg approachDistinct from conventional survival analyses in which predictors do not have estimation uncertainty, the predictors in SURVIV are exon-inclusion levels β=0), and the remaining 10% of the exons from the alternative hypothesis that the exons were associated with survival time. The survival coefficient β of exons representing the alternative hypothesis was randomly sampled from the top 100 significant exons based on SURVIV analysis of all IDC patients (see n) with mean value , with 90% of the exons from the null hypothesis that the exons were not associated with survival time . Using the exon-inclusion levels of these 176 exons, we clustered IDC patients into three major subgroups, with 95, 194 and 389 patients, respectively. As illustrated by the Kaplan–Meier survival plots, the three subgroups had significantly different survival times  RNA-seq data set (see Methods for details of the data source and processing pipeline). We chose to analyse IDC because it is the most frequent type of breast canceral times .STAT5A  was found to be significantly associated with IDC patient survival  had significantly longer survival times as compared with the second group of 262 patients with lower exon 5 inclusion levels .A total of 165 significant correlations existed among the 84 survival-associated alternative exons and the three splicing factors in this network . As the Our combined survival and co-expression network analysis identified splicing factors known to be dysregulated in breast cancer. For example, TRA2B is known to have elevated expression in breast cancers compared with normal breast tissues, and it regulates the alternative splicing of a number of genes with key functions in cancer development404142P-value threshold of ≤0.01. Then, a penalized Cox regression of L1 penalty was used to build the predictive model combining the individual alternative splicing events identified by SURVIV. We then applied the predictive model built from the training group to predict the survival outcome in the testing group. The cross-validation was repeated for 100 times. The discriminatory power of the survival predictors was measured by the C-indexP=1.5e−3; splicing versus clinical P=4.7e−4; two-sided Wilcoxon test). We also observed that alternative splicing predictors alone performed better than gene expression predictors . Adding clinical parameters to the predictive model collectively improved the performance of both gene expression and alternative splicing-based predictors, while the combined model of alternative splicing plus clinical parameters outperformed the combined model of gene expression plus clinical parameters . We also evaluated the combined effect of incorporating clinical, gene expression and alternative splicing data. A predictive model incorporating all three types of information performed significantly better than any one type of predictors alone . The improved prediction accuracy was not caused by over-fitting since the training and testing data were completely separated during cross-validation. This result illustrates the potential value of utilizing alternative splicing information for improved prognosis of cancer patients.Finally, we investigated the prognostic value of alternative splicing as predictors of IDC patient survival. Recent work by the TCGA consortium evaluated the clinical utilities of diverse molecular data  as well as clinical parameters in predicting cancer patient survivalP-value significance cutoffs varied across cancer types, with LGG having the strongest survival-associated alternative splicing signals with 660 significant exon-skipping events at FDR≤5% , KIRC , LGG (lower grade glioma), LUSC (lung squamous cell carcinoma) and OV (ovarian serous cystadenocarcinoma). As expected, the number of significant events at different FDR or t FDR≤5%  and 4. St FDR≤5% , consistAlternative processing and modification of mRNA, such as alternative splicing, allow cells to generate a large number of mRNA and protein isoforms with diverse regulatory and functional properties. The plasticity of alternative splicing is often exploited by cancer cells to produce isoform switches that promote cancer cell survival, proliferation and metastasis715471821We have developed SURVIV, a novel statistical model for survival analysis of alternative isoform variation using cancer RNA-seq data. SURVIV uses a survival measurement error model to simultaneously model the estimation uncertainty of mRNA isoform ratio in individual patients and the association of mRNA isoform ratio with survival time across patients. Compared with the conventional Cox regression model that uses each patient's mRNA isoform ratio as a point estimate, SURVIV achieves a higher accuracy as indicated by simulation studies under a variety of settings. Of note, we observed a particularly marked improvement of SURVIV over Cox regression for low- and moderate-depth RNA-seq data . This haP-values≤0.01 in multiple clinical subgroups. While the statistical threshold seemed loose, several lines of evidence suggest the functional and clinical relevance of these survival-associated alternative splicing events. These alternative splicing events were frequently identified and enriched in the gene functional groups important for cancer development and progression, including apoptosis, DNA damage response and oxidative stress. While some of these events may simply reflect correlation but not causal effect on cancer patient survival, other events may play an active role in regulating cancer cell phenotypes. For example, a survival-associated alternative splicing event involving exon 5 of STAT5A is known to regulate the activity of this transcription factor with important roles in epithelial cell growth and apoptosis4041424344Using the TCGA IDC breast cancer RNA-seq data of 682 patients, SURVIV identified 229 alternative splicing events associated with patient survival time, which met the criteria of SURVIV We performed cross-validation analyses to evaluate and compare the prognostic value of alternative splicing, gene expression and clinical information for predicting patient survival, either independently or in combination. As expected, the combined use of all three types of information led to the best prediction accuracy. Because we used penalized regression to build the prediction model, combining information from multiple layers of data did not necessarily increase the number of predictors in the model. The perhaps more surprising and intriguing result is that alternative splicing-based predictors appear to outperform gene expression-based predictors when used alone and when either type of data was combined with clinical information . We obsehttps://github.com/Xinglab/SURVIV. SURVIV is a general statistical model for survival analysis of mRNA isoform ratio using RNA-seq data. The current statistical framework of SURVIV is applicable to RNA-seq based count data for all basic types of alternative splicing patterns involving two isoform choices from an alternatively spliced region, such as exon-skipping, alternative 5′ splice sites, alternative 3′ splice sites, mutually exclusive exons and retained introns, as well as other forms of alternative isoform variation such as RNA editing. With the rapid accumulation of clinical RNA-seq data sets, SURVIV will be a useful tool for elucidating the clinical relevance and potential functional significance of alternative isoform variation in cancer and other diseases.The SURVIV source code is available for download at n) with the mean value .We designed simulation studies to compare the performance of SURVIV with the conventional Cox regression survival analysis using point estimates of mRNA isoform ratios. We mimicked the characteristics of the TCGA IDC breast cancer RNA-seq data wherever possible in the simulations. For each simulation, we simulated a data set of 600 individuals, which was similar to the sample size of the TCGA IDC data. To study the effect of RNA-seq depth, for individual alternative splicing events we simulated the total number of splice junction reads  for each individual k as: β=0.05, which produced a read count variance similar to the variance in the TCGA IDC data. Then, we generated the total number of splice junction reads (nk) for each individual k as: ψ) according to a distribution Beta that fitted the observed distribution of all alternative exons from the TCGA IDC data set. Similarly, the variance of the exon-inclusion levels (σ) was simulated according to a distribution Gamma that fitted the observed variance of all alternative exons from the TCGA IDC data set. Each individual's exon-inclusion level (ψk) was simulated as: For each exon, we then simulated the total number of splice junction reads and exon-inclusion levels across the 600 individuals. To model the variation in the total splice junction counts across individuals, we used a Gamma–Poisson model to simulate the total number of splice junction reads of each individual ψk was used as the covariate. We used the Nelson–Aalen estimatorWe simulated the patient survival time based on the proportional hazards model, in which the individual exon-inclusion level https://tcga-data.nci.nih.gov/tcga/). For each exon-skipping event defined in the Ensembl annotations (release #75), we identified the read counts for both exon-inclusion and -skipping splice junctions from the Level 3 data. The IDC patient list is provided in We obtained the RNA-seq read counts and clinical parameters of 682 IDC patients from TCGA breast cancer (BRCA) cohort (TCGA dbGaP Accession ID phs000178). The RNA-seq reads were mapped to the human genome and transcriptome by the TCGA consortium using Burrows-Wheeler Aligner (BWA) with default settingsP≤0.01) to select potential survival predictors associated with patient survival time. We used Cox regression for the univariate analysis of clinical information and gene expression. We used SURVIV for the univariate analysis of alternative splicing. Then, starting from the predictors selected from univariate regression, we used a L1-penalized Cox regression modelz-score normalization was used to scale the gene expression and alternative splicing predictors in the penalized regression. After the model was built using the training group, we used the remaining patients from the testing group to test the model. The prediction accuracy of the model was measured by the C-indexWe evaluated and compared the survival prediction using clinical information, gene expression and alternative splicing. We used two-fold cross-validation to split the IDC patients into separate training and testing groups. The even split of the IDC patients ensured that there were enough death events in both training and testing groups for accurate model training and testing. The cross-validation was repeated for 100 times. Each time, half of the IDC patients were randomly selected as the training group and the other half were selected as the testing group. Using the training group, we built a series of survival prediction models based on clinical information, gene expression or alternative splicing predictors as well as the combination of these predictors. The clinical parameters used in the Monte Carlo cross-validation were identical to those used for the initial SURVIV analysis of 26 clinical subgroups , which iHow to cite this article: Shen, S. et al. SURVIV for survival analysis of mRNA isoform variation. Nat. Commun. 7:11548 doi: 10.1038/ncomms11548 (2016).Supplementary Figures 1-3, Supplementary Methods and Supplementary ReferencesList of 229 survival associated alternative splicing events in the TCGA IDC data. From column G, the table contains the log10 SURVIV P-values for each clinical subgroup. The positive or negative signs in front of the SURVIV P-values indicate the direction of the association. Positive signs indicate that higher exon inclusion levels are associated with better survival probabilities. Negative signs indicate that lower exon inclusion levels are associated with better survival probabilities.List of 6 splicing factors whose gene expression levels are significantly associated with IDC patient survival. In the enrichment analysis, all other alternatively spliced exons from genes containing survival associated alternative splicing events were used as the background control, so that the background exons and the survival associated exons were from the same set of genes and did not have systematic differences in overall gene expression levels and RNA-seq coverage.Numbers of significant survival associated alternative exons in six TCGA cancer types at various FDR/P-value significance cutoffs.Full lists of survival associated alternative splicing events at nominal P-value cutoff of 0.01 for six cancer types.List of IDC patients from TCGA.


Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are characterized by a promptly release of proinflammatory mediators that downregulate natural anticoagulant mechanisms, initiate the coagulation system, impair fibrinolysis and produce the rupture of the endothelial and epithelial monolayer . Inflammin-vitro ALI.To evaluate the effect of the treatment with heparin in human alveolar cells and alveolar macrophages after induce an injury, mimicking an Escherichia coli 055 : B5; 1000 ng/ml). The effect of heparin was assessed by the analysis of proinflammatory markers  and anti-inflammatory markers (IL10 and IL13), cell proliferation and permeability (measuring transmembrane resistance). Data are expressed as mean ± SEM (units are relative to the expression of control group). Statistical analysis was performed using One-Way-ANOVA and post-hoc (Newman Keuls) test. Statistical significance p ≤ 0.05 is considered.Human alveolar primary cells and alveolar macrophages from pulmonary biopsies were isolated and seeded. Sodium non-fractioned heparin (100 UI/ml) was administered to the cells after the induction of an injury with a pro-inflammatory stimulus  in the case of macrophages . The permeability of the monolayer and cell proliferation of alveolar cells did not show changes.Heparin has an immunomodulatory effect in alveolar cells and reduces inflammation in macrophages. This mechanism produced by heparin could have a beneficial effect in ALI.


Abundance of immune cells has been shown to have prognostic and predictive significance in many tumor types. Beyond abundance, the spatial organization of immune cells in relation to cancer cells may also have significant functional and clinical implications. However there is a lack of systematic methods to quantify spatial associations between immune and cancer cells.We applied ecological measures of species interactions to digital pathology images for investigating the spatial associations of immune and cancer cells in breast cancer. We used the Morisita-Horn similarity index, an ecological measure of community structure and predator–prey interactions, to quantify the extent to which cancer cells and immune cells colocalize in whole-tumor histology sections. We related this index to disease-specific survival of 486 women with breast cancer and validated our findings in a set of 516 patients from different hospitals.Colocalization of immune cells with cancer cells was significantly associated with a disease-specific survival benefit for all breast cancers combined. In HER2-positive subtypes, the prognostic value of immune-cancer cell colocalization was highly significant and exceeded those of known clinical variables. Furthermore, colocalization was a significant predictive factor for long-term outcome following chemotherapy and radiotherapy in HER2 and Luminal A subtypes, independent of and stronger than all known clinical variables.Our study demonstrates how ecological methods applied to the tumor microenvironment using routine histology can provide reproducible, quantitative biomarkers for identifying high-risk breast cancer patients. We found that the clinical value of immune-cancer interaction patterns is highly subtype-specific but substantial and independent to known clinicopathologic variables that mostly focused on cancer itself. Our approach can be developed into computer-assisted prediction based on histology samples that are already routinely collected.The online version of this article (doi:10.1186/s13058-015-0638-4) contains supplementary material, which is available to authorized users. In recent years, the recognition that cancer is an evolutionary process has penetrated much of cancer biology and evolutionary biology , 2, and Immune cell infiltration is one of the most important aspects of the ecology of neoplastic cells –11. An aEvolution and ecology can provide a framework for understanding these complex dynamics and predicting clinical outcomes . The Morp <0.0001); 2) 10-fold cross-validation within the training set (90.1 % accuracy) . R code and the dataset for performing the proposed methodology and reproducing reported results are provided as Additional file


An effective approach termed Recursive Gaussian Maximum Likelihood Estimation (RGMLE) is developed in this paper to suppress 2-D impulse noise. And two algorithms termed RGMLE-C and RGMLE-CS are derived by using spatially-adaptive variances, which are respectively estimated based on certainty and joint certainty & similarity information. To give reliable implementation of RGMLE-C and RGMLE-CS algorithms, a novel recursion stopping strategy is proposed by evaluating the estimation error of uncorrupted pixels. Numerical experiments on different noise densities show that the proposed two algorithms can lead to significantly better results than some typical median type filters. Efficient implementation is also realized via GPU (Graphic Processing Unit)-based parallelization techniques. Median type filters are known as the effective methods to suppress impulse noise which often arises from sensor damage, malfunctioning or timing errors in signal acquisition Several methods have been developed to overcome the drawback of SMF. Adaptive Median Filter (AMF) was proposed in In suppressing impulse noise, median-type methods suffer from the image degradation of detail blurring and staircase artifacts. This problem might be more serious for high noise density cases The structure of this paper is organized as follows: In section II, we explain the RGMLE approach together with the two derived algorithms RGMLE-C and RGMLE-CS. Experimental results are given and discussed in section III. Section IV concludes this paper with a brief description of its contributions and some open questions for future work.N i.i.d. Laplacian distributed observations with unknown location parameter Based on the theory of statistical estimation, the sample median corresponds to the maximum likelihood estimators of locations for independent and identically distributed (i.i.d.) observations obeying the Laplacian distributions with probability density function (pdf) shown in Likewise, with the Gaussian distribution shown in y the noisy image and i, the weight However, in 2-D impulse noise suppression, assuming an identical variance for all samples in (3), the averaging filtering often leads to a non-discriminative averaging operation and might tend to produce oversmoothing in the restored images(we would illustrate this in the following section on experiment). Improved performance can be expected if a discriminative estimation of variance is applied based on the different reliabilities of different observations. Furthermore, considering the fact that the estimation error (or variance) decreases as the available uncorrupted observations increase, the information from corrected intensities should be exploited in a recursive way to refine the final restoration. In this work, we develop a Recursive Gaussian Maximum Likelihood Estimation (RGMLE) for impulse noise suppression. In this 2-D noise suppression case, with y, and let T is set when the noise density is larger than 70%. We judge the PSNR increase for the estimation of the originally uncorrupted pixels by evaluating the error improvement The recursive update of image y. In estimating each In RGMLE-C algorithm, we consider the certainty measure as whether the pixels are corrupted in the original image h in (13) should be set to modulate the attenuation of the exponent function with respect to distance Within a filter window for one specific point, two pixels belonging to the same structure tend to come from the same class, and should be assigned low variances (or large weights) in estimating each other. We borrow the patch similarity idea in where, two binary patch masks http://links.uwaterloo.ca/Repository.html. The intensities of Lena and Peppers images are 8 bit and within the interval .The MRI image is in DICOM  format and was collected from a head scanning of a 57 years old patient in the first hospital of Nanjing. The intensity range of the MRI image is . The patient in this manuscript has given the written informed consent of using the MRI image data. A wide range of salt-and-pepper noise with density from 10% to 90% (10% increments) is considered. The same simulation as the analysis in above Section II is used. The AMF, DBA and MDBUTMF methods are implemented based on N in the AMF, DBA, MDBUTMF and MDBUTAF methods. For the SKR method, the global smoothing parameter and the kernel size were manually tuned to give the results with the highest PSNR. For the RGMLE-C and RGMLE-CS algorithms, several parameters need to be set, namely the size of filtering window W, the certainty parameter T. Here h and the size of patch h is set to 3.5 for Lena and Peppers images, and to 10 for the MRI image with a larger intensity range. Specially, for RGMLE-CS algorithm, we set T is set to 50 to include a long recursion in restoration using RGMLE-C and RGMLE-CS algorithms.Three The pixel-wise operation (7) in RGMLE-C and RGMLE-CS algorithms allows for easy parallelization using GPU technique Restoration performances are quantitatively measured by PSNR and the structural similarity index comparisons (SSIM) proposed in Obviously, the proposed RGMLE-CS algorithm gives good performance in terms of both noise suppression and detail preservation(see images (i) in In T = 50) for the whole restored image  and the PSNR_U (via above (9)) of the re-estimated uncorrupted pixels (green lines) under different noise densities. Lena image with the same parameter setting as above is used in this validation. This paper proposed a Recursive Gaussian Maximum Likelihood Estimation (RGMLE) for 2-D impulse noise removal. Assuming Gaussian distribution for neighboring intensities, the proposed RGMLE approach solves the maximum likelihood estimation (MLE) of corrupted points through recursive variance-weighted averaging. Simulation in Section II reveals that Gaussian distribution assumption is theoretically preferable to Laplacian distribution assumption in estimating corrupted points under high noise densities. In validation we use salt-and-pepper type impulse noise with pre-known intensities. Two noise removal algorithms RGMLE-C and RGMLE-CS are respectively derived by using certainty based or certainty&similarity based variance estimations. An effective stopping strategy for recursion is also developed based on the estimation error of uncorrupted pixels using restored image information. Extensive experiments have been performed to show the RGMLE-C and RGMLE-CS algorithm can obtain significantly better results than the comparative median type filters. Among all the methods, the RGMLE-CS algorithm achieves good restoration performance through the incorporation of patch similarity information. The application of the proposed RGMLE methods can be easily extended to suppress general impulse noise with random values when combined with the detection methods proposed in h is currently used in the RGMLE-CS algorithm, and we can improve the algorithm by optimizing the estimation of parameter hWith GPU parallelization, the RGMLE-CS algorithm can be efficiently applied and should be suggested for impulse noise suppression. A constant smoothing parameter


Yet there is a significant lack of user-friendly and powerful tools geared towards co-localization analysis based exploratory research. Most tools currently used for co-localization analysis are command line only and require extensive installation procedures and Linux expertise. Online tools partially address the usability issues of command line tools, but slow response times and few customization features make them unsuitable for rapid data-driven interactive exploratory research. We have developed PAPST: Peak Assignment and Profile Search Tool, a user-friendly yet powerful platform with a unique design, which integrates both gene-centric and peak-centric co-localization analysis into a single package. Most of PAPST’s functions can be completed in less than five seconds, allowing quick cycles of data-driven hypothesis generation and testing. With PAPST, a researcher with or without computational expertise can perform sophisticated co-localization pattern analysis of multiple TFs and EMs, either against all known genes or a set of genomic regions obtained from public repositories or prior analysis. PAPST is a versatile, efficient, and customizable tool for genome-wide data-driven exploratory research. Creatively used, PAPST can be quickly applied to any genomic data analysis that involves a comparison of two or more sets of genomic coordinate intervals, making it a powerful tool for a wide range of exploratory genomic research. We first present PAPST’s general purpose features then apply it to several public ChIP-Seq data sets to demonstrate its rapid execution and potential for cutting-edge research with a case study in enhancer analysis. To our knowledge, PAPST is the first software of its kind to provide efficient and sophisticated post peak-calling ChIP-Seq data analysis as an easy-to-use interactive application. PAPST is available at  ChIP-Seq, one of the most powerful research applications of Next Generation Sequencing (NGS) technology, is fundamental to the investigation of TF-to-DNA interactions and histone modifications. A general ChIP-Seq data analysis pipeline includes a) mapping sequence reads to a genome, b) calling statistically significant peaks, and c) broad down-stream analysis of called peaks including co-localization analysis and assignment of peaks to genes and other annotated genomic regions. There are well-established mapping programs –3 and a https://github.com/paulbible/papst, which provides the application jar, a collection of ready to use TF and EM ChIP-Seq data from [We have developed PAPST (peak assignment and profile search tool), with bench scientists in mind, as an easy-to-use yet powerful stand-alone platform for post peak-calling data analysis, focusing on co-localization analysis of transcription factors and histone modifications, a critical component of integrated ChIP-Seq data analysis . PAPST hata from , a comprCertain fundamental tasks are common in nature to all post peak-calling downstream ChIP-Seq analysis. Fundamentally, called ChIP-Seq peaks can be represented as a set of genomic coordinates and nearly all common ChIP-Seq analysis methods rely on determining whether peaks overlap or are near to known genomic features (gene-centric) or user-defined genomic features such as peaks from a specific TF experiment (peak-centric). At the heart of PAPST are efficient algorithms see  section At the global genomic level, the distribution of protein binding with respect to known features provides a fundamental description of binding behavior. PAPST analyzes peak binding in the promoter, gene body, exon, intron, and Intergenic regions quickly with a single mouse click. The summary distribution information is reported as a percentage of total peaks as well as the number of significant peaks that fall into the genomic region. PAPST was used to calculate the binding distribution for four TFs. The result window of PAPST in Post peak-calling ChIP-Seq analysis of TFs invariably begins by assigning peaks to the nearest gene transcription start site (TSS) to dissect their behavior. Many TFs drive gene regulation by interacting with the upstream promoter of target genes. Although the current understanding of gene regulation has evolved to include distal TF-to-target gene regulation, assignment to the nearest TSS provides the baseline preliminary analysis that offers insight to TF behavior . This feLabs studying specific TFs are typically interested in discovering which genes could be direct regulatory targets. PAPST offers customizable search capabilities that allow users to quickly determine which genes have particular TF binding patterns. The powerful filters feature allows users to create sets of criteria for returning genes of interest. A simple but common task would be to find all genes where a specific TF binds in the promoter. To accomplish this, a PAPST user would simply create a filter, select the promoter option (modify the length if desired), and press the search button. The The above example represents the most basic application of the search features. Much more interesting questions are addressed by combining multiple transcription factors and/or histone marks into the search criteria. A natural extension to the promoter search example described above would be to include the activating histone mark H3K4me3 and filter out lower confidence peaks by tag count. To accomplish this a second filter is added to the search that operates on significant peaks derived from an H3K4me3 ChIP-Seq experiment, and the threshold of the Oct4 filter is adjusted to filter out peaks with fewer than 20 reads. PAPST will process all filters and return only genes that meet all the specified criteria (those with both Oct4 and H3K4me3 binding at the promoter and at least 20 reads for the Oct4 peaks). https://github.com/paulbible/papst.This powerful feature, unique to PAPST, generalizes the multiple criteria based search function to any set of genomic regions enabling peak-centric co-localization analysis. In the previous example, a search was constructed to find genes that met multiple criteria across different experiments. Setting the search mode to “Track as Base” enables the selection of a peak set to serve as the genomic locations that are subjected to multiple user-defined filters. This feature allows complex search criteria to be applied to any set of arbitrary genomic regions of interest. As the current understanding of gene regulation evolves, gene-distal regulatory regions will become increasingly important. PAPST provides easy to use tools to explore protein co-localization patterns at any user-defined set of genomic regions. The Example Applications section below describes a case study that utilizes this feature to detect enhancers. Another example of peak-based search features is presented in the tutorial at Efficient performance and usability are the two main design principles of PAPST. All PAPST’s functions can be selected and executed through a graphical user interface with a few mouse clicks, and the results will return within seconds. In addition, we have also implemented specific features to increase its usability and enhance its ability for efficient data handling. A few examples of PAPST’s usability features are: 1) imbedded customizable parsers simplify importing data from a wide range of file formats, 2) an analysis session of any number of peak sets can be saved into a single file to revisit later or share with a colleague without reloading individual data sets, 3) analysis results can be saved as a csv file for further analysis by spreadsheet programs, 4) specific entries may be easily copied and pasted into the UCSC genome browser for viewing, and 5) analysis results may be quickly reformatted within PAPST and used as a new input for the next round of co-localization analysis, greatly simplifying iterative analyses.To demonstrate its efficient performance and ease of use, we have timed a complete analysis session in which we loaded Oct4, Sox2, and Nanog peaks, determined regions of co-localized binding, loaded the literature derived normal and super enhancer regions , and calThe following sections describe case applications of PAPST, with pubic ChIP-Seq datasets, to demonstrate its typical uses and its great potential in cutting-edge research. The results as compared to those published also validate the algorithms and approaches specifically implemented in PAPST.Efficient co-localization analysis, both peak-centric and gene-centric, is the key feature of PAPST. ESC enhancers are characterized by the co-occupancy of master transcription factors Oct4, Sox2, and Nanog . Young’sPAPST can also perform customizable  gene-centric co-localization analysis to screen for genes with a specific occupancy profile of TFs and/or EMs. For example, we have applied PAPST to identifying 1182 non-redundant ESC genes with co-promoter-occupancy of Oct4, Sox2, Nanog, and H3K27Ac . The genPAPST can generate quantitative data for extended co-localization analysis. ESC super enhancers have been shown to have higher levels of active enhancer epigenetic mark H3K27Ac and binding of key TFs as compared to typical enhancers . We usedPAPST can quickly assign a set of genomic regions such as ChIP-Seq peaks to genes by proximity to their transcription start site (TSS). Using PAPST, we assigned ESC super enhancers to their closest genes. The complete assignment results are presented in the supplemental materials . ImportaPAPST has implemented a novel algorithm for symmetric peak overlap calculation, determining the total number of overlapping peaks for all possible pairwise comparisons of TFs and EMs included in an analysis, with one single mouse click see , using aWe have demonstrated PAPST’s main features by applying it to two publicly available ChIP-Seq data sets from embryonic stem cell research , 17. OurPAPST has been developed based on our extensive experience in ChIP-Seq data analysis –21, whicTwo of the most powerful and unique features of PAPST are: 1) simultaneous co-localization analysis of a large number of TFs and EMs (limited only by a computer’s memory), and 2) the extremely fast return of the analysis results on the scale of seconds. A co-localization calculation of 50 factors took only 1.39 seconds and 1.54 seconds for peak-centric analysis and gene-centric analysis, respectively. As most functions of PAPST may be completed basically as fast as one can click the mouse , and thePAPST offers both gene centric and peak centric features in a single package, capable of analyzing ChIP-Seq peaks both on gene-defined genomic regions  and on peak-defined genomic regions, making it a true genome-wide data analysis platform. Its peak-centric feature can be creatively and easily extended to include the co-localization analysis of TFs and EMs on any set of user-defined genomic regions of interest, such as the ENCODE determined locations of regulatory sequences , regionsFinally, PAPST can also generate quantitative data matrices in the form of normalized peak reads within regions defined by genes or peaks, making it possible to study epigenetic and regulatory data with other quantitative statistical methods such as PCA, K-means and hierarchical clustering, and statistical learning based modeling.The raw data was collected from NCBI GEO using accessions GSE44288  and GSE1http://java.com/en/download/). It operates on the statistically significant peaks called from ChIP-Seq experiments. Any BED-like file is accepted by PAPST. An in-app input wizard allows users to create parsers for custom formatted files as long as the chromosome, start and end location, and tag count or score value are present. Gene and exon information is loaded into PAPST using RefGene files, and PAPST supports all genomes available from the UCSC Genome Browser (GB). The specific RefGene data used in this study were downloaded from GB site on October 13, 2014.PAPST is built on Java 1.7  . Given aMost ChIP-Seq analysis programs calculate the overlap of peak regions in a non-symmetric way. Problems arise when a single peak region from one experiment overlaps multiple peaks in another experiment. For applications such as clustering, having a symmetric overlap relationship is desirable. PAPST uses a novel overlap algorithm that ensures the binary relationship between two peaks sets is symmetric  = Overlap). The algorithm accomplishes this by counting the number of edges that would be drawn to connect a peak with its overlapping peaks in the opposite set . When apGene-centric: ChIP-Seq data analysis on genomic regions defined by genes, such as promoters, exons, introns, and gene bodies. The analysis result usually is a set of genes with shared ChIP-Seq features, such as genes with their promoters co-occupied by two transcription factorsPeak-centric: ChIP-Seq data analysis on genomic regions defined by a set of ChIP-Seq peaks. The analysis result usually is a set of genomic regions with shared ChIP-Seq features, such as those regions co-occupied by a transcription factor and a histone modificationTypical enhancer: normal enhancerSuper enhancer: a cluster of adjacent typical enhancersS1 Appendix(DOC)Click here for additional data file.S1 TableThe data presented in this table provides an example of the gene assignment function of PAPST.(CSV)Click here for additional data file.S2 TableThis file contains the table for all unique genes (isoforms excluded) that have a significant peak located in the promoter regions 2000 up stream of the TSS.(CSV)Click here for additional data file.S3 TableThis file contains the table for all unique genes (isoforms excluded) that have a significant peak for both Oct4 and K4me3 located in the promoter regions 2000 up stream of the TSS.(CSV)Click here for additional data file.S4 TablePAPST searched for gene promoters (+/- 5000 bp from TSS) that contained a binding profile that included all 3 transcription factors and the H3K27ac epigenetic mark. This table contains the exhaustive list of identified target genes.(XLSX)Click here for additional data file.S5 TableUsing PAPST’s gene assignment feature, all super enhancer regions were assigned to the nearest TSS. This table contains all the assigned genes’ accessions, both the enhancer and gene coordinates and the distance from the enhancer to the TSS.(XLSX)Click here for additional data file.S6 TablePAPST was used to generate the all-pairs overlap data. Data generated from overlap comparisons in PAPST is symmetric making it suitable for clustering as well as other multi-dimensional analyses.(CSV)Click here for additional data file.


The eastern part of India has been affected by an ongoing low-intensity conflict between government forces and armed Maoist groups, known as Naxalites. Since 2006, Médecins Sans Frontières (MSF) has been providing primary health care services in the conflict-affected region along the Andhra Pradesh-Chhattisgarh border. In 2011, treatment for drug-resistant tuberculosis (DR-TB) was included in the services provided. This report aims to describe MSF experiences of providing treatment to DR-TB patients in a mobile primary health care outpatient clinic, in a low-intensity conflict setting in India.A total of thirteen patients were diagnosed with drug-resistant TB (DR-TB) between January 2011 and October 2013. An innovative treatment model was developed which delegated responsibility to non-TB clinicians, including primary-care nurses and nurse-aids who were remotely supported by a TB-specialist from the MSF DR-TB project in Mumbai. Individualised regimens were designed for each patient based on WHO guidelines. Of these 13 patients, 10 patients had an outcome, of whom seven (70%) patients were cured. One patient became lost to follow-up prior to treatment initiation, one patient died prior to starting treatment and one patient refused treatment. Three patients were on-treatment, were clinically improving and were culture-negative at the end of their intensive phase of treatment.Drug-resistant tuberculosis diagnosis and treatment is a highly specialised and technical subject which requires continued patient follow-up. However, our study demonstrates that it is feasible to manage DR-TB patients in a conflict setting, using a primary-care model with remote expert support. Long-term commitment and sustainability are essential for continued care, even more so in similar conflict settings. Loss to follow-up in patients remains a programmatic challenge and community involvement may play a key role.Managing DR-TB in a primary health care programme is feasible in a low-conflict setting with an appropriate treatment model. Ambulatory strategies and standardised treatment regimens should be considered to further simplify treatment delivery and allow for scale-up when needed. The eastern part of India has been affected by an ongoing low-intensity conflict between government forces and armed Maoist groups, known as Naxalites . The regThe limited security and almost non-functional healthcare services in these conflict zones have hindered universal coverage of all national health programmes including tuberculosis (TB), and in particular drug-resistant tuberculosis (DR-TB). Multidrug-resistant tuberculosis (MDR-TB) is estimated to account for 2.1% of new cases and 15% of previously treated TB cases –10. The In 2006, in response to the conflict and the resultant significant civilian displacement to temporary relief camps Médecins Sans Frontières (MSF) started a primary health care mobile clinic service in the border area between the states of Chhattisgarh and Andhra Pradesh. This conflict may have contributed to the spread of DR-TB in the area by disrupting health care services, inhibiting both access of directly observed treatment (DOT) providers to their patients and also patient’s access to health services, interrupting drug supplies and the inevitable close habitation of DR-TB patients in temporary housing due to forced displacements. Thus, along with primary healthcare services, the MSF programme provided treatment to patients suffering from drug-sensitive TB and DR-TB.Treatment of DR-TB is highly specialised, complex and expensive, and needs appropriate tools to monitor the long and painful regimen. Availability of these pre-requisites for DR-TB treatment and care in remote rural settings is uncertain. To date, little is known about DR-TB management in a primary health care setting in areas affected by conflict . To addrSince 2006, MSF has been operational in the border region between Andhra Pradesh and Chhattisgarh (AP-CG). The project runs from the town of Bhadrachalam and has sub-bases in the small border towns of Konta and Cherla. The MSF mobile clinics provide care free of charge to patients and can help to facilitate referral to secondary care for patients requiring emergency care. The great majority of patients are from rural forested tribal areas often lacking in basic water or sanitation services or access to functioning healthcare facilities. In general, about one-third of patients attending the MSF clinics walk more than 10 km to reach the clinic location. MSF started providing DR-TB treatment to patients from January 2011. DR-TB care and treatment is offered in an outpatient primary healthcare setting.The study population consisted of all DR-TB patients registered for care within the MSF programme between January 2011 and October 2013.▪ Category 1 and Category 2 treatment failure▪ All retreatment cases and all cases starting Category 2 treatment▪ Close contacts of known DR-TB patients▪ HIV-infected TB patientsAll the patients presenting with presumed TB undergo sputum smear evaluation  in the BSputum collection and transfer to specialist laboratory for culture and Drug Susceptibility Testing (CDST): Two sputum samples, usually a spot and a morning sample, are collected and transferred via courier to the MSF HIV MDR-TB project in Mumbai, India. Delivery of these samples takes 2 working days and the sample is delivered to an RNTCP accredited private laboratory for culture and DST. Information regarding the possible DR-TB patient is made anonymous , entered into a database and sent via email to the MSF Mumbai MDR-TB technical referent. Since July 2013, the samples for line probe assay (LPA) are sent to Hyderabad RNTCP accredited laboratory, Andhra Pradesh.A shared database (electronic- excel based) between the project staff and Mumbai TB technical referent is used to record the patients’ treatment progress. The database specifically monitors patients’ follow up sputum sample outcomes, blood test results and adverse events.Pre-treatment evaluation: Following confirmation of a positive DR-TB result, a pre-treatment evaluation is carried out including basic hematological and biochemical investigations (including creatinine clearance) and an HIV test. Adherence counseling and health education sessions are given. A treatment consent form including a commitment to complete treatment is received from all the patients.Individualised treatment regimen: The treatment protocols follow WHO guidelines . HoweverTreatment initiation and follow up: After treatment initiation, sputum smear and culture is repeated monthly until the end of the intensive treatment phase and every other month during the continuation phase. Chest X-ray is not routinely used for treatment monitoring. A directly-observed treatment (DOT) approach is followed depending on the level of insecurity in the area, using MSF community health workers, Furthermore, an MSF nurse, nurse aid or patient support officer would review the patient on a weekly basis to confirm compliance and report adverse events. If the patient remains stable, medical reviews by the MSF doctor occurs on a monthly basis. All treatment is provided free of charge. Additional supportive counseling is provided to patients who interrupt treatment. The tracing of lost-to-follow-up patients is carried out by a local team of MSF health workers.Patients who fall into the above categories are presumed to have a higher likelihood of DR-TB and their samples are then sent for sputum culture and drug susceptibility testing (DST). The following diagnostic and treatment model is being used:Demographic and clinical information were recorded in patient files. The clinical data being routinely collected for each patient, including treatment and laboratory data, were entered into an electronic shared database. Data from all TB patients initiated on treatment between January 2011 and October 2013 were included in the analysis.This case-study satisfied the criteria for reports using routinely collected programmatic data set by the Médecins Sans Frontières Ethics Review Board (ERB), Geneva, Switzerland. Patient identifying information was removed prior to analysis. As this was a study of routinely collected monitoring data, patient consent was not required.Between Jan 2011 and October 2013, 13 patients were diagnosed with DR-TB  of ten patients with treatment outcome were cured. Adverse events were common (31%) among this cohort of patients. Among them, a patient had baseline renal dysfunction with a creatinine clearance of 21 ml/min, thus the treatment doses were adjusted in line with published recommendations. Another patient developed psychological disturbances after three months of treatment and required referral to a psychiatrist. The patients suffering from ototoxicity or gastrointestinal disturbances were managed symptomatically.Our experiences demonstrate that it is feasible to provide treatment to DR-TB patients within this particular conflict setting. The MSF position as a neutral actor, has allowed it to build trust and ensure continued access to the people it treats, in this chronic low-intensity conflict zone. On a day to day basis there is normally no immediate threat to the health workers which is vital for being able to deliver DR-TB medication on a daily basis. The patient cohort, rather than being direct victims of warfare find themselves as indirect victims. They are affected by intimidation, difficulties in travel and access to healthcare services due to the background security situation, as well as seasonal problems including monsoon rains and flooding. Our study shows that despite these challenges it is possible to deliver care and medications for the duration of the treatment period for DR-TB.Our treatment model shows it is possible to manage and follow-up DR-TB patients in a primary care setting with non-specialists, rather than requiring a vertical DR-TB programme, if there is a system in place for supportive guidance from DR-TB experts, as previously documented in other MSF programmes . It may Our DR-TB patient cohort included similar number of patients from Chhattisgarh and Andhra Pradesh. This accurately reflects the border locations of the MSF clinics. About 23% (3/13) of the patients were women. Further, 7.7% (1/13) patients were children (<16 years old), which may reflect the ongoing challenges in diagnosing and managing children with TB . HoweverThere is little published evidence of the non-governmental organization (NGO) sector managing DR-TB in conflict settings. There is justifiable concern regarding trying to make a diagnosis if the expensive medications and any follow up system is not in place or is unrealistic given the context. Indeed, a sporadic or non-sustainable approach to DR-TB management in conflict settings could do more harm than good including leading to the amplification of resistance. Previous studies from Peru  and MumbA limitation to our treatment model is the varied treatment regimens used to treat our mono-resistant and poly-resistant DR-TB cohort, which in the current era may seem inappropriate. However, these regimens were prepared based on available literature and international guidelines then , 17. TheA second limitation was the prolonged duration of treatment for several of the patients. These were mostly due to delays in receiving patient laboratory reports or due to the patient’s severe or non-improving clinical condition. Another reason behind prolonged treatment was treatment interruptions by the patients. These patients missed appointments ranging from one week-one month during their treatment duration. The MSF staff had to trace back these patients by visiting their residence or sending confidential messages via residents from the same villages.A few months after the MSF project in AP-CG region had been operational; the national TB programme (RNTCP) acknowledged the DR-TB burden in India and initiated the upscaling and investment in services for DR-TB. This includes the investment in molecular resistance testing including line probe assay (LPA) techniques and the availability of free DR-TB medications through the RNTCP. This enabled MSF to adapt its strategy in 2013 from one of being the provider of the entire DR-TB package of care for its patients and thus requiring a minimum commitment of two years in order to ensure the availability of medication for the duration of treatment and taking on more of a facilitator role. This has meant that new suspected DR-TB patients attending the MSF clinics may now have their diagnosis performed in one of the RNTCP accredited laboratories, their medications provided by the national programme but their clinical follow up and delivery of medications to the patients still conducted by the MSF mobile clinic staff. In this way, there is the reassurance that patients can in future be handed over to the RNTCP programme should the need arise albeit within the context of the challenging security situation.Due to sufficient resources and small patient numbers, the DR-TB patients received an individualised treatment regimen based on standard treatment guidelines , 17 suggOne problem encountered is regarding the patients who are lost to follow up either on a temporary basis or permanent, as was the case for one study patient. Significant difficulties exist regarding tracing patients who do not attend appointments or who are not available when their DOT provider goes to observe treatment. To try and limit missed doses, attempts are made to communicate in advance when patients needed to come to clinic, including sending reminders through respected members of the relevant villages. Furthermore, as a considerable number of patient villages are impossible to visit due to limited security, patients are strongly encouraged to come together with their close family members in order to encourage family support of the patient, thereby improving the adherence of the patients during treatment duration. The role of incentives  such as The drug-resistant tuberculosis diagnosis and treatment remains a highly specialised and technical subject which requires continued patient follow-up. However, in areas affected by low-intensity conflicts, our study has shown that with an appropriate treatment model and remotely given expert support, it is possible to implement a DR-TB programme within a primary health care project. The implementation of any such project requires a sustained commitment and an understanding of the implementation challenges.


Parus minor) that bred in the nest-box varied with cavity conditions associated with nest predation risk during the nestling period. The entrance of experimental nest-boxes was enlarged to create a long-term risk soon after clutch competition. A short-term risk, using simulated playbacks with a coexisting control bird and avian nest predator sound, was simultaneously applied to the nest-boxes whether or not the long-term risk existed. We found that the parents reduced their hourly feeding trips, and the nestlings fledged early with the long-term risk, although the nest mortality of the two nest-box types was low and did not differ. While this study presents a portion of prey–predator interactions with the associated uncertainties, our results highlight that the entrance size of cavities for small hole-nesting birds may play an important role in determining their fitness-related traits depending upon the degree of perceived risk of nest predation.Some nest predators visually assess parental activities to locate a prey nest, whereas parents modify fitness-related traits to reduce the probability of nest predation, and/or nestlings fledge early to escape the risky nest environment. Here, we experimentally tested if the parental and fledging behaviours of oriental tits ( Nest predation is one of key drivers of selection for small breeding passerines, in which the parents, offspring, and nest predators may interact to escape the risky nest environment that can impair the reproductive success of prey birds2378Cavity nesters experience relatively lower risks of nest predation due to limited predator access, compared to open-cup nesters111213151617819Parental activity at the nest can attract nest predators, resulting in reduced parental nest activity during the incubation and nestling periods232425Parus minor) to manipulated risks of nest predation during the post-hatching period. Here, we simultaneously applied two types of nest predation risk: “short-term” (simulation of predator presence) versus “long-term” . The long-term risk affected the number of parental feeding trips per hour , but no effect of short-term risk was observed . The varWe examined the influence of cavity condition of oriental tits’ nest-boxes on parental and fledging behaviour , and nesPoecile palustris, versus oriental tits) breeding in nest-boxes with control versus enlarged entrances during the incubation period. Marsh tits with a high frequency of nest trips  and lacked plasticity in their incubation behaviour independent of perceived predation risks experienced relatively high nest mortality during the incubation period. Moreover, few nests of marsh tits survived to fledging in the nest-box with an enlarged entrance; oriental tits, however, tended to experience a low nest predation rate throughout the breeding season. Thus, their anti-predator plasticity (i.e. a steeper slope of reaction norms)The long-term risk of nest predation increased with reduced parental feeding rates in oriental tits; therefore, this long-term risk did not influence fledging success directly when nest predation was minimal in both types of nest-boxes. In general, the degree of nest predation increases with the progress of breeding stages because the number of nest trips made by parents is likely to increase from the incubation period to the nestling period at the expense of nestling growth and survival, after controlling for nest-site effectsDiscrepancies between nestling development and fledging time may depend on the relative degrees of nest versus fledgling predation risks even along the altricial-precocial spectrum in birds333510263439In the present study, the behavioural plasticity in parental activities at the nest improved the rate of nest survival in non-excavating cavity nesting oriental tits, but reduced the duration of the nestling period in the presence of a long-term risk of nest predation. Prior to nesting, nest-site selection may be more adaptive than behavioural plasticity for non-excavating nesters as the cavity conditions for nesting vary in nature. Available nesting cavities are more abundant than previously thought, and cavity nesters may not face a shortage of holes42Periparus ater) shared this entrance size in our study site . We captured adults using mist nets and banded them with a unique combination of three coloured leg bands and a numbered aluminium band  to identify individuals. Sex was determined based on the presence of the cloacal protuberance for males and the brood patch for incubating femalesWe studied the interactions between oriental tits (24 nests) and corvids as avian nest predators inhabiting the campus of the Korea National University of Education, Cheongju, Republic of Korea  during March-June of 2014. The breeding habitat (85 ha) was composed of patchy forest with mixed deciduous and coniferous trees interspersed with buildings. Previously, we monitored about 25–30 pairs of oriental tits each year in our study site. We installed 50 nest boxes (2 m off the ground) during December 2010. Nest boxes (11 cm long × 16 cm deep × 21 cm high), with an initial 2.6 cm diameter entrance hole, were apart from each other by at least 100 m . The prePica pica), Eurasian jays (Garrulus glandarius), Azure-winged magpies (Cyanopica cyanus), and gray-headed woodpeckers (Picus canus), as well as the red squirrel (Sciurus vulgaris) as a mammalian predator. However, grey-headed woodpeckers and red squirrels were rarely detected at the study site, where Eurasian magpies (88%) were detected most frequently, followed by Eurasian jays (8%) and Azure-winged magpies (4%). Our preliminary results indicated that the activity of these avian nest predators also appeared to respond to cavity condition and the parental activity level of cavity-nesting parids during the incubation period.Corvids as generalist omnivores are efficient nest predators that affect many bird species and use parental nest activities as a visual cue to locate prey nests47484950We manipulated the entrance size of the nest-boxes soon after the clutch was completed 2031. The20Streptopelia orientalis) and the most common nest predator (Eurasian magpie) near the nest-box. Oriental turtle-doves were abundant and less likely to compete with the parid species for foraging and nesting at the study siteOriental tits that nested in the control or enlarged nest-boxes with different entrance-hole sizes were simultaneously exposed to two sessions of simulated sound playbacks to examine the effect of short-term risk of nest predation on parental behaviour. We performed sound playback sessions using the sounds of a non-nest predator . We videotaped each nest box using a digital camcorder  without human disturbance (5 m from the nest box) to collect behavioural data during the nestling period in the presence of a mixture of short-term and/or long-term risks of nest predation. Video recording was conducted for 3 h on day 9 and 10 during the nestling period (09:00–12:00 KST) during exposure to the long-term risk (see above). We transcribed the recorded files using media player software . We analysed parental behaviours including the proportion of female’s time spent at the nest  and the number of nest trips by the parents during the total observation time. We also scored the proportion of parental vigilance time (%) from each video. This was calculated as the time spent scanning the surrounding area while perched either outside or inside the nest entrance hole)t test was used to examine the effect of the long-term risk on the length of nestling period. All statistical analyses were performed using SPSS ver. 16.0 . Data are presented as means ± 1·SE.We analyzed female nest attentiveness (%), hourly feeding trips, and vigilance time (%) of the parents in relation to playback stimulus , nest-box type , and the two-way interaction as fixed effects (playback × nest), using a linear mixed model with restricted maximum likelihood (REML). We also included ambient temperature (°C) and brood size (the number of nestlings per nest) as covariates in the model, which were likely to influence parental behaviours such as feeding and brooding nestlings All experimental protocols were approved by the Animal Care and Use Committees at the Korea National University of Education and performed in accordance with the relevant guidelines and regulations of the Ecological Institute for Oriental Stork in the Korea National University of Education.How to cite this article: Yoon, J. et al. Nest predation risk influences a cavity-nesting passerine during the post-hatching care period. Sci. Rep.6, 31989; doi: 10.1038/srep31989 (2016).


Transcatheter aortic valves have benefitted from device miniaturization and in-situ assembly to reduce delivery system caliber and enable trans-vascular delivery. In contrast, investigational transcatheter mitral prostheses are bulky devices that require large caliber access ports. Trans-apical delivery is undesirable because of increased morbidity associated with rib spreading, closure site bleeding and impact on left ventricle (LV) function. Trans-septal delivery is challenging because of the acute angle required to reach the mitral valve. A ‘straight shot' to the mitral valve Fig  that doe2. A direct trajectory to the LA was planned . 3D and cine MRI confirmed that the 18Fr sheath trajectory relative to the plane of the mitral valve was favorable to perform a mitral intervention Fig . The LA Percutaneous Fig  MR guideThis work was supported by the Division of Intramural Research, National Heart Lung and Blood Institute, National Institutes of Health (Z01-HL005062).


Amorphous calcium phosphate (ACP), a postulated precursor in the formation of biological hydroxyapatite, has been evaluated as a filler phase in bioactive polymeric composites that utilize dental monomers to form the matrix phase on polymerization. In addition to excellent biocompatibility, these composites provided sustained release of calcium and phosphate ions into simulated saliva milieus. In an effort to enhance the physicochemical and mechanical properties and extend the utility of remineralizing ACP composites to a greater variety of dental applications, we have focused on: a) hybridizing ACP by introducing silica and/or zirconia, b) assessing the efficacy of potential coupling agents, c) investigating the effects of chemical structure and compositional variation of the resin matrices on the mechanical strength and ion-releasing properties of the composites, and d) improving the intrinsic adhesiveness of composites by using bifunctional monomers with an affinity for tooth structure in resin formulations. Si- and Zr-modified ACPs along with several monomer systems are found useful in formulating composites with improved mechanical and remineralizing properties. Structure-property studies have proven helpful in advancing our understanding of the remineralizing behavior of these bioactive composites. It is expected that this knowledge base will direct future research and lead to clinically valuable products, especially therapeutic materials appropriate for the healing or even regeneration of defective teeth and bone structures. Calcium phosphates (CaPs) are of special interest to oral biology, dentistry and medicine since they occur in normal skeletal tissues  and in pathological  calcifications . Systemain vitro [We have recently developed unique bioactive composites based on filler phases consisting of pyrophosphate-stabilized ACP embedded in certain types of polymer matrix phases derived from the ambient polymerization of acrylic monomers , 8. Signin vitro . HoweverThe matrix resins were formulated from the commercially available dental monomers, coupling agents and components of the photoinitiator systems  and 4. AThe types of ACPs employed in the study are given in d-spacing values was 0.0013, and the measured d-values were within 0.05 % of the reported values of NIST SRM 640 . The wavelength accuracy of FTIR measurements was ≤ 0.01 cm−1 at 2000 cm−1. The particle size distribution (PSD) of the solids dispersed in isopropanol was determined by gravitational and centrifugal sedimentation analysis  following 10 min ultrasonication. The Ca/PO4 ratio of the solids after dissolution in HCl was calculated from solution Ca2+ (atomic adsorption spectroscopy (AAS), Perkin Elmer Mo. 603 spectrophotometer  and PO4  values.The methodologies and techniques utilized to characterize and evaluate the methacrylate resins, the ACP fillers, and their composites are summarized in Volumetric contraction upon polymerization or polymerization shrinkage (PS) of the composites was measured by a computer-controlled mercury dilatometry (DC) attained after polymerization of the composites, a recently developed, non-destructive near infrared (NIR) spectroscopic technique for measuring the methacrylate conversion in dental resins was employed [−1 in the overtone region was used to assess the DC in paired unpolymerized and polymer samples of known thickness. DC was calculated from the decrease in integrated peak area/sample thickness values in going from the unpolymerized to polymerized composites using the following expression:To determine the degree of methacrylate conversion  of each composite disk specimen was determined by using a computer-controlled Universal Testing Machine  operated by Instron Merlin Software Series 9. Detailed description of experimental protocols and calculations used in BFS screening are given in Ref. .2+ and PO4 levels were determined by AAS and UV/VIS spectroscopy, respectively. Ion-release data were corrected for variations in the total area of disk surface exposed to the immersion solution using the simple relation for a given surface area, A: normalized value =  × (500/A).Mineral ion release from each individual composite disk specimen in a continuously stirred, HEPES-buffered (pH = 7.40) 240 mOsm/kg saline solution, was examined at 37 °C. CaWS) profiles, a minimum of five replicate disks in each experimental group were initially dried over CaSO4 until a constant mass was achieved (± 0.1 mg). Specimens were then exposed to 75 % relative humidity (RH) at room temperature (23 °C) by keeping them over aqueous NaCl slurry in a closed system. Gravimetric mass changes were recorded at predetermined time intervals. Degree of water sorption (WS) of any individual specimen at any given time interval (t), expressed as a % mass fraction, was calculated using a simple equation:tW represents sample mass at the time t, and W0 is the initial mass of a dry sample.To determine the water sorption . However, all pair-wise multiple comparisons (Tukey test) revealed that only the differences in dm and SSA between the control HAP powder and Si-hybridized ACP are of statistical significance . The observed increase in the dm, and consequently the lower SSA of Si-ACP compared to HAP, may be explained by different degree (extent) of agglomeration of the Si-hybridized filler. While the Ca/PO4 molar ratio of u-ACP and Si-treated ACPs was practically unchanged, it was significantly higher (Tukey test) for all Zr-treated fillers . In conclusion, /TEGDMA/HEMA formulations have the lowest potential for leaching out unreacted monomeric species. The significantly lower DC obtained with PT composites  indicates a greater probability that the ACP-PT composites will release un-reacted monomers into the oral environment and consequently more likely will have a lower biocompatibility than Bis-GMA-, EBPADMA- and/or UDMA-based ACP composites.The results of the omonomer , a monom EBPADMA . No cleaPS 1.9 % to 4.1 % [PS fell into the category of either flowable composites or adhesive resins ((3.6 to 6.0) % and (6.7 to 13.5) %, respectively [PS and optimal DMC.The PS results showed a very complex dependence on both the resin composition and the filler type . The majto 4.1 % ,19), mosectively ). Reformectively ,21 might4 concentrations were sustained in all but PT composites, which with increased time failed to maintain a favorable remineralizing potential due to the matrix uptake of released Ca via ion binding by the high concentration of carboxylic acid groups of PMGDMA [Ion release from composites was affected by both the chemical structure and the composition of the monomer system as well as by the type of ACP filler . Elevatef PMGDMA . GeneralBFS testing of dry (before immersion) and wet  composites are presented in BFS than unfilled specimens regardless of the type of filler or resin matrix. The strength of all BisGMA-based (except the BTHZr resin), UDMA- based and PT composites deteriorated further upon soaking. Comparison of the BFS values of hybrid vs u- ACP/BTHZr composites showed modest but significant increase in the mechanical strength of Si- and Zr-ACP specimens. However, surface-treated Zr/Zr ACP composites failed to maintain their strength upon immersion. Recent m-FTIR mapping of ACP composites [Results of the mposites  indicatemposites ) decreasBFS usually decreases upon soaking), water sorption/diffusion influences the mineral ion release kinetics and consequently the remineralizing ability of these bioactive materials. Kinetic WS data for unfilled and ACP-filled  XT, XHm, XTH and XHmH  resins (data not shown) indicate that plateau values are reached within a week for XTH and within 2 weeks for XT, XHm and XHmH systems. Generally, XTH composites exhibited the highest and XHm composites exhibited the lowest WS  or hydrophobic (HmDMA) monomers are the dominant components of the resin matrices. No clear-cut conclusion could be made on the effect of the filler type on WS, although certain trends were evident: more water was absorbed by u-ACP composites compared to hybrid ACP composites in Bis-GMA and EBPADMA systems, Zr-ACP composites seemingly have the lowest WS regardless of the resin composition.Studies conducted on the water sorption of dental materials indicate that excessive water uptake may cause a decrease in mechanical strength, distortion, and depression of the glass transition temperature  due to pWS of ACP composites [WS. The possible relevance of this finding is that increased ACP levels increased WS which, in turn, increased ion release and faster ACP to HAP conversion. The resulting disruption in the integrity of the filler/resin interface decreased BFS.It has also been demonstrated that the filler's loading level has a significant effect on the mposites . As seenThese results exemplify the fact that water plays a very significant if not a major role in filler-matrix interactions. It may leach out filler elements, induce filler failures, cause filler-matrix de-bonding and reduce the strength of matrix material. Since the effect of water on most dental composites is irreversible , a true DC of the composites. Utilizing EBPADMA in addition to Bis-GMA as a base monomer and adding moderate amounts of hydrophilic HEMA may be the best route to maximize remineralizing ability of the filler while maintaining low leachability of unreacted monomeric species, i.e., high DC. However, additional adjustments in resin formulations will be necessary to improve the PS of current experimental ACP composites. Finally, Bis-GMA and EBPADMA have proven to be essential in maintaining the mechanical integrity of the composites. As currently formulated, resins that included surface-active adhesive monomers PMGDMA or GDMA did not meet basic physicochemical requirements for ACP composites.In conclusion, results of this study demonstrate that it is possible to improve the remineralizing potential of ACP composites by introducing Si or Zr elements during the low-temperature synthesis of the filler. Si- and Zr- ACPs enhanced the duration of mineral ion release through their ability to slow down the intra-composite ACP to HAP conversion. Additionally, when compounded with BTHZr resins, hybrid ACPs showed improved mechanical properties compared to composites that utilized unmodified ACP. Also, the chemical structure and composition of the monomer system used to form the matrix phase significantly affected ion release, water sorption and the


Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants.PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis.We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.The online version of this article (doi:10.1186/s12864-015-1528-y) contains supplementary material, which is available to authorized users. The extensive use of elite sires exacerbated by the large-scale exploitation of artificial insemination in cattle breeding causes important reductions in effective population size and the common spread of loss-of-function variants. This in turn is responsible for the periodic outburst of genetic defects that cause considerable economic loss and welfare issues. With the development of genome-wide SNP arrays for all livestock species, it has become possible to rapidly map the underlying locus by means of autozygosity mapping to intervals that typically span 2 to 5 megabases thereby proving the inherited nature and mode of inheritance of the corresponding condition (f.i. ). With tWe recently established an “heredo-surveillance platform” to effectively identify and control inherited defects that recurrently emerge as a result of intensive use of elite sires in Belgian Blue and other cattle breeds (f.i. ). TwentyKalimine du Barsy Fontaine, suggesting autosomal recessive inheritance. We therefore genotyped 15 cases with the bovine SNP50 beadchip  to perform a genome-wide association study (GWAS). We used the genotypes from 275 Belgian Blue AI sires, obtained with the bovine HD (700 K) beadchip , as controls. The analysis was restricted to 34,368 SNPs shared by both arrays, and conducted with GLASCOW as previously described TC TCT TC-3′  and 5′-TCT GGC T[G]T CTC TTC -3′  as probes . Reactions were carried out on an ABI7900HT instrument  using standard procedures.A 5′ exonuclease assay was developed to genotype the http://www.ncbi.nlm.nih.gov/SNP/). They will be publicly available with the next dbSNP Build (B145), planned for fall, 2015. Additional files Bos taurus assembly: BosTau6/UMD3) and the list of corresponding sequence variations.All the variations of the IBD disease haplotype have been submitted to dbSNP (


IDH1 mutational status, and MGMT promoter methylation status. We aimed to integrate biological and clinical prognostic factors into a nomogram intended to predict the survival time of an individual GBM patient treated with a standard regimen. In a previous study we showed that the methylation status of the DGKI promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen. We further evaluated the potential prognostic value of DGKI methylation status.Consistently reported prognostic factors for glioblastoma (GBM) are age, extent of surgery, performance status, MGMT-methylated patients (population 2). Cox proportional hazard models were fitted to identify the main prognostic factors. A nomogram was developed for population 1. The prognostic value of DGKI promoter methylation status was evaluated on population 1 and population 2.399 patients with newly diagnosed GBM and treated with a standard regimen were retrospectively included in this study. Survival modelling was performed on two patient populations: intention-to-treat population of all included patients (population 1) and DGKI methylation status for MGMT-methylated patients. We also demonstrated that the DGKI methylation status identified 22% of poorly responding patients in the low-risk group defined by the nomogram.The nomogram-based stratification of the cohort identified two risk groups (high/low) with significantly different median survival. We validated the prognostic value of MGMT stratification of GBM patients receiving standard treatment. These results could help the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.Our results improve the conventional  IDH1) is also associated with a better clinical outcome in gliomas, including glioblastoma. However, this mutation is rare in primary GBMs (approximately 6%) 6-methylguanine DNA methyltransferase gene (MGMT) promoter is currently the strongest predictive biomarker of outcome and benefit from temozolomide-based treatment of GBM et al. integrated biological and clinical prognostic factors and their independent and combined predictive powers into nomograms for GBM patients treated with the standard regimen MGMT promoter methylation status (n = 103).Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Its prognosis remains extremely poor, despite multimodal treatment by surgery, radiotherapy, and temozolomide-based chemotherapy (standard regimen) DGKI) promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen DGKI and the functional consequences of its methylation status have never been investigated in gliomas but DGKI regulates Ras signalling, an oncogenic pathway frequently altered in GBM DGKI methylation status in the context of both MGMT-methylated and intention-to-treat populations.In this study, we retrospectively analysed 399 GBM patients treated with the standard regimen (intention-to-treat). We identified the main clinical prognostic factors in this cohort and compared our results with those of the EORTC (European Organisation for Research and Treatment of Cancer) and NCIC  trial 26981-22981/CE.3. We propose an updated nomogram intended to predict the survival time of an individual GBM patient. In a previous study on 50 GBM patients treated with the standard regimen, we showed that the methylation status of the diacylglycerol kinase iota gene (This multi-center retrospective cohort included 399 patients treated in the Departments of Neurosurgery/Neuro-oncology of Angers (n = 28), Marseille (n = 52), Paris-Salpêtrière (n = 227), Rennes (n = 50), and Poitiers (n = 42) between 2006 and 2011. The inclusion criteria were as follows: 1) patients aged 18 years or more, 2) diagnosis of a primary GBM (WHO grade IV), 3) detailed clinical information at diagnosis and during follow-up, 4) treatment with radiotherapy and concurrent/adjuvant temozolomide (standard regimen), and 5) availability of tumor tissue with informed consent in accordance with French regulations and the Helsinki Declaration. All patients included in this study fulfilled the inclusion criteria. Particularly, all patients received a radio-chemotherapy regimen in accordance with the standard of care. Follow-up for included patients ranged from 24 days to 5.2 years . Tumor samples were snap-frozen immediately after resection and stored in tumor banks under the following authorization numbers: , 2008/70 , DC 2009-957 , DHOS/2004/04056 , and AC-2010-77 . The extent of surgery was evaluated with an enhanced magnetic resonance imaging (MRI) performed within 24 hours after the resection. All samples presented at least 70% of tumor cells. For each tumor sample, DNA was extracted using the NucleoSpin Tissue Kit (Macherey Nagel) according to the manufacturer's instructions. The quality of the DNA samples was assessed by electrophoresis on a 1% agarose gel. Only high quality genomic DNAs were selected for further analyses.MGMT-methylated patients (population 2). Population 1 was used to identify the main clinical prognostic factors and to compare our results with those of the EORTC and NCIC trial 26981-22981/CE.3 MGMT stratification. The effect of DGKI promoter methylation status on the prognosis of GBM patients assigned to standard treatment was also evaluated in MGMT-methylated patients. Patients from population 2 were randomly assigned to a training cohort and a validation cohort of equal sizes in a randomized block design stratified by the hospital center. Two patient populations were considered in this study: the population of all included patients (population 1) and the subgroup of IDH1 codon 132 via exon 4 PCR amplification and direct sequencing as previously described IDH1 mutation is sufficient to establish the “glioma-CpG island methylator phenotype” (G-CIMP), we did not take this phenotype into account in our analysis Tumor DNA was screened for somatic mutations in MGMT promoter was measured using the PyroMark Q96 CpG MGMT kit (Qiagen) (average percentage of the five tested CpG sites). The DGKI promoter's %met was measured using VeraCode GoldenGate Methylation technology (Illumina Inc.) or by pyrosequencing. PCR and pyrosequencing primers were designed using the Pyrosequencing Assay Design Software (Qiagen). The primers and PCR conditions are given in MGMT  and of 26 patients for DGKI . The consistency between VeraCode GoldenGate Methylation technology and pyrosequencing was assessed on 24 patients for DGKI . The assessment of MGMT and DGKI %met was conducted at the Rennes hospital. For MGMT, we used the 8% methylation threshold defined on an independent data set by Quillien et al.risksetROC R package (AUC = 0.61).DNA was bisulfite-modified using the EZ DNA Methylation Kit (Zymo Research). The methylation percentage (%met) of the p-values less than 5% were candidates for the multivariate analyses. In population 1, the Cox proportional hazards model was then used with forward stepwise model selection. We have checked that no evidence of violation of the proportional hazards assumption was found. The probability of inclusion of a factor in the multivariate model was estimated by using the bootstrap resampling technique as described in Gorlia et al. survival R package.Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Comparisons between survival groups were performed using a log-rank test for binary variables, and a log-rank trend test for ordered categories. Cox proportional hazard models provided estimates of the hazard ratios (HRs). From these tests, variables with rms R package.A nomogram was developed for population 1 to predict each patient's median survival and probability of survival at two years, taking into account their clinical characteristics. Variables with a probability of inclusion higher than 90% based on 1000 bootstrap samples were included in the final model. The definition of two risk groups (high/low) was based on the value of the linear predictor underlying the nomogram; values greater than or equal to zero were assigned to the high-risk group, and negative values were assigned to the low-risk group . The accuracy of predictions was assessed by estimating the model's calibration and discrimination measured by the Concordance index corrected for optimism (C-index). The nomogram was built using the IDH1 mutational status, MGMT promoter methylation status, and DGKI promoter methylation status were significantly associated with OS , extent of surgery,  with OS . These v with OS . The finIDH1 wild-type/MGMT-methylated tumor has a total prognostic score of 129 and is predicted to have a median survival of approximately 30 months and a 60% probability of surviving two years. Because the cut-off between high and low risk is 165 points, this patient is assigned to the low-risk group. Patients in the low-risk group had a median OS of 29.6 months , which was significantly longer than 14.9 months  for patients in the high-risk group (p<1e-08). A similar stratification was observed for the PFS  between the DGKI-methylated and DGKI-unmethylated patients (p = 0.23).In population 2, univariate Cox analyses showed that age, KPS, and  cohorts . In the MGMT-methylated and DGKI-methylated patients were not significantly different from the OS and PFS of the MGMT-unmethylated patients . The DGKI methylation status did not stratify the subgroup of high-risk patients in the intention-to-treat population.The DGKI promoter methylation status identified poorly responding patients.We studied a retrospective cohort of 399 GBM patients homogeneously treated with the standard regimen. The higher OS at 6 and 12 months observed in our cohort in comparison to the reference cohort of the EORTC trial IDH1 mutational status, and MGMT promoter methylation status. This result is in agreement with the most consistently reported prognostic factors for GBM The prognostic factors identified in our study were age, KPS, extent of surgery, et al. proposed a nomogram for GBM patients who underwent either a partial or complete resection and were assigned to temozolomide and radiotherapy in the presence of a MGMT promoter methylation assessment . Their nomogram includes MGMT promoter methylation status, Folstein Mini-Mental State Examination (MMSE) score, and WHO performance status MGMT promoter methylation status and Karnofsky performance score but also age, IDH1 mutational status, and extent of surgery. In the EORTC trial, elderly patients (>70 years) were excluded, and the prognostic value of IDH1 was not evident when the trial was designed. Furthermore, the authors discussed the restricted reliability of the extent of surgery in their study. Age and extent of surgery were nonetheless identified by Gorlia et al. in the population of GBM patients who underwent partial or complete resection and were assigned to temozolomide and radiotherapy without the knowledge of the MGMT promoter methylation status . Because it was not routine to collect MMSE score in the neurosurgical units involved, we were unable to evaluate the prognostic value of these nomograms on our cohort. However, the nomogram proposed in the present study showed better performance evaluation (AUC) than MGMT status alone . The potential skewing effect of treatment at recurrence on patient survival was not controlled; however, the validity of our PFS results indicated that our findings could be independent of these treatments. A recent study showed that the nomogram designed by Gorlia et al. for the intention-to-treat population (radiotherapy only or temozolomide and radiotherapy) was a poor predictor of an individual patient's survival because the standard of care has evolved since the EORTC trial et al. for GBM patients who received the standard treatment  were not evaluated in the study by Parks et al. However, our study confirmed the appropriateness of Gorlia et al. findings as we also identified MGMT methylation status, performance status, age and extent of surgery in the nomogram for the population of GBM patients assigned to standard treatment.In 2008, Gorlia MGMT-methylated patients. Interestingly, our previous study showed that the methylation status of the DGKI promoter identified GBM patients with MGMT-methylated tumors who responded poorly to the standard regimen DGKI methylation status for MGMT-methylated patients (population 2). However, this finding could be restricted by the limited size of the training and validation cohorts. We further evaluated the potential predictive value of DGKI methylation status on the intention-to-treat population (population 1). The methylation status of the DGKI promoter identified 22% of poorly responding patients in the low-risk group but had no prognostic value for high-risk patients. The role of DGKI and the functional consequences of its methylation status have never been investigated in gliomas but DGKI regulates Ras signalling, an oncogenic pathway frequently altered in GBM DGKI was hypermethylated in primary hepatocellular carcinoma and was re-expressed in liver cancer cell lines after exposure to reagents reversing DNA methylation DGKI expression is regulated by its promoter methylation. In GBM, we observed an anti-correlation between DGKI expression and methylation levels, in a private cohort and in the TCGA cohort (data not shown). Further functional studies on DGKI are clearly required.A nomogram-based stratification of our cohort of primary GBM patients treated with the standard regimen identified two risk groups (high/low) with significantly different median survival. The low-risk group was almost exclusively composed of MGMT stratification of GBM patients receiving standard treatment. In particular, the DGKI methylation status identified poorly responding patients in the group of low-risk or MGMT-methylated patients. A retrospective study precluding the establishment of firm conclusions, these results need to be validated in a prospectively recruited cohort. They could however be of help in the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.Our results improve the conventional Figure S1Primers and cycling conditions for the pyrosequencing analysis of DGKI_7.(TIFF)Click here for additional data file.Figure S2Prognostic value of DGKI methylation status for GBM patients assigned to standard treatment (population 1). Kaplan-Meier estimation of OS and PFS. M: methylated patients, UM: unmethylated patients, mo: month. The difference in survival between groups is reported . The size and the median survival of each group are also specified.(TIFF)Click here for additional data file.Figure S3Nomogram including the interaction between MGMT and DGKI (population 1).(TIFF)Click here for additional data file.Table S1OS ans PFS - % (95% IC).(DOC)Click here for additional data file.


MR reconstruction of virtual 3 dimensional volumes are routinely used to demonstrate cardiac malformations, particularly the complex geometric relationships between the great arteries and cardiac chambers. However, because of the need to signal average over multiple heart beats, rendering fast moving structures such as atrioventricular (AV) and semilunar valves has been limited. Echocardiography has sufficient frame rates to acquire images of moving valves in real-time 3d volumes.We used state-of-the-art commercially-available computer software  to develop an efficient work flow that permits importation of 3d echo data and co-registration with MR cardiac angiograms. The AV valve plane is localized using single-shot BFFE pulse sequences prior to MR angiography. After the MR acquisition, an echocardiographic 3d volume of the moving AV valve is acquired  and exported to Mimics as DICOM files, 30 phases and 208 slices/phase. Visual inspection of thumbnail images allows phase selection of the fully open valve. Point-to-point registration between the echo-derived AV valve anulus and the MR-derived AV valve plane permits co-registration with the whole heart angiogram. STL files are exported for 3d printing.We have successfully combined 3d echo and MR data to create a physical model in order to plan the operative approach for a 2.5 year old child with single ventricle and single AV valve.Echocardiographic and cardiac MR imaging technologies each have unique advantages that, until now, have been relegated to separate spheres of diagnostic imaging. Integrating these two modalities to create physical models of complex cardiac malformations will give cardiothoracic surgeons and cardiologists a powerful new tool to plan interventions.Friends of Doernbecher Children's Hospital.