Less attention has been given to tests for categorical data, such as the Chi-square. This project takes a first step by developing an open-source software tool, Ptest, that addresses the need to offer public software tools incorporating these and other statistical tests with options for correcting for multiple hypotheses.Genomics and proteomics analyses regularly involve the simultaneous test of hundreds of hypotheses, either on numerical or categorical data. To correct for the occurrence of false positives, validation tests based on multiple testing correction, such as Bonferroni and Benjamini and Hochberg, and re-sampling, such as permutation tests, are frequently used. Despite the known power of permutation-based tests, most available tools offer such tests for either This study developed a public-domain, user-friendly software whose purpose was twofold: first, to estimate test statistics for categorical and numerical data; and second, to validate the significance of the test statistics via Bonferroni, Benjamini and Hochberg, and a permutation test of numerical and categorical data. The tool allows the calculation of Chi-square test for categorical data, and ANOVA test, Bartlett's test and t-test for paired and unpaired data. Once a test statistic is calculated, Bonferroni, Benjamini and Hochberg, and a permutation tests are implemented, independently, to control for Type I errors. An evaluation of the software using different public data sets is reported, which illustrates the power of permutation tests for multiple hypotheses assessment and for controlling the rate of Type I errors.The analytical options offered by the software can be applied to support a significant spectrum of hypothesis testing tasks in functional genomics, using both numerical and categorical data. Current statistical inference problems in areas such as genomics and proteomics regularly involve the simultaneous test of hundreds of null hypotheses. This strategy has allowed scientists to unveil important cues on the mechanisms involved in the development of deadly diseases. For example, Barth et al. 2006)  analysed006  analTo correct for the occurrence of false positives, validation tests based on multiple testing corrections and re-sampling techniques (i.e. permutation-based test) are frequently used. Although both strategies aim to control Type I error, these techniques implement different approaches to estimating errors and rejecting null hypotheses. Traditional multiple-testing corrections, such as Bonferroni and variations, adjust P-values derived from multiple statistical tests to correct for the occurrence of false positives . The Bent-test or ANOVA only. Another example is GeneSpring [t-test or ANOVA test statistics only. These and other tools published do not offer multiple-testing solutions for categorical data, such as the Chi-square. This test is appropriate for the analysis of SNPs (single nucleotide polymorphisms) data to identify significant patterns of genetic variability, i.e. variation-phenotype associations. Another important statistical significance assessment technique not available in well-known open-source tools is the Bartlett test, which may be used for testing equality of variances or the significance of data dispersion differences across groups. Moreover, the Bartlett test should also be used before attempting the calculation of either ANOVA or t-test, as they assume that variances are equal across groups or samples.To date, permutation tests have become widely accepted and recommended in studies that involved multiple statistical testing ,6,7. DesneSpring  that offt-test for paired and unpaired data. Once a test statistic is calculated, Bonferroni, B&H and a permutation tests are implemented, independently, to control for Type I errors. P-values from the permutation test were estimated as follow, using the data encoding format shown in Figure Given the evident need to offer software tools incorporating such statistical tests with options for correcting for multiple tests, this study takes a first step by developing a public-domain, user-friendly software with the following functionality. The tool allows the calculation of Chi-square test for categorical data, ANOVA test, Bartlett's test and B times and test statistics are calculated on each permuted data set. Third, permuted distribution is calculated by: counting the times (K) the statistic value obtained in the original data set was smaller than the statistic value obtained from the permuted data sets, and dividing that value by the number of random permutations i.e. K/B. Results are stored in a text file for subsequent analyses. Table First, test statistic and corresponding P-value are calculated on the original data set. Data are permuted at random The software is a Java-based, command-line tool [see Additional files The tool has at the user's disposal the following statistic tests: Student's test for numerical data, two classes; Bartlett's test for numerical data, two classes; ANOVA test for numerical data, more than three classes; and Chi-square for categorical data, two or more classes. For detailed information about each test, please refer to National Institute of Standards and Technology/Semiconductor Manufacturing Technology e-Handbook of Statistical Methods .N) number of samples, (C) number of classes, (F) number of features, (S) significance level, (B) number of permutations, and (T(obs)) test statistic, validation methods are as described bellow:Given (T(obs); sample's labels are shuffled B times and T(obs)s' are obtained; if T(obs) <T(obs)' a counter T(per) is increased by 1. The probability that T(obs) occurred by chance alone is: T(per)/B.Multiple testing correction: P-values, according to test statistic and degrees of freedom (N-2), were obtained and adjusted under Bonferroni and B&H multiple testing corrections B. Permutat-test is selected, the user should indicate whether samples (i.e. groups being compared) are independent or not (paired). The user is also allowed to specify which type of distribution should be used: one- or two-tailed distribution. Once the required information is provided, the tool performs the analysis and displays those features whose raw P-values are below the significance level, their corrected P-value after Bonferroni correction, their corrected P-value after B&H correction, and their corrected P-values after performing the permutation test.Typical usage involves a user providing the following information: file name containing the data to be analysed, the name of new file where results are to be stored, the selection of test statistic to be calculated, the significance level at which the null hypothesis is to be rejected, and the number permutated data sets to be created for the estimation of the null-hypothesis distribution Figure  see Add. DependiFigure To illustrate some of the advantages of using the permutation-based test for multiple hypotheses validation, this section summarises examples of analyses using publicly available data. This includes a comparison with results obtained when Bonferroni correction was applied Table .Three data sets were used in the analysis:1) A microarray data set generated by a study in dilated cardiomyopathy was obtained from the GEO (Gene Expression Omnibus) , accessi2) A genotype data set  was obtained from the Single Nucleotide Polymorphism database (SNPdb) . This da3) A microarray data set, oligo array, generated by a study in heart failure was obtained from the GEO, accession number GDS1362, was composed of 37 samples: 7, 20 and 10 samples were obtained from non-failing hearts, DCM heart, and Ischemic cardiomyopathy (ICM) patients respectively.Microarray data: probe sets with absent calls in more than 50% of their transcripts were discarded. Transcripts of probe sets corresponding to similar gene symbols were averaged. Data were normalised per chip and then per gene. Values were transformed using the mean and standard deviation of the row (per gene) or column (per chip). Genotype data did not require pre-processing.The first analysis calculated Bartlett's test statistic to determine whether the variances of two experimental groups, from a microarray data set [see Additional file t-test : equal variances and two tailed) to estimate the potential statistical significant difference between the means of two  experimental groups, from the same microarray data set analysed above [see Additional file The second analysis implemented the The third analysis implemented the Chi-square test on categorical data derived from a genetic variation data set (SNPs) [see Additional file t-test is designed to perform pair-wise comparisons, only. The null hypothesis of this ANOVA analysis was that there were no differences between the means of the three groups, and the significance level to reject the null hypothesis was set to 0.05. In this case, the raw P-values of 6371 genes were under the significance level (P < 0.05), 3331 genes were under the significance level after correcting with B&H, and only nine genes were under the significance level after correcting with Bonferroni. After performing the permutation test, 6262 genes were found significantly differentially expressed (P < 0.05). The genes reported as significantly differentially after correcting via Bonferroni were not included in the set of potentially significant genes detected by the permutation test. In addition, we compared our results against those previously reported by Kittleson, et al. (2005) [A fourth analysis implemented the ANOVA test to estimate the potential statistical significant difference between the means of three  experimental groups. Samples in this data set were obtained from heart tissue of healthy donors, as well as from donors suffering from either dilated or ischemic cardiomyopathy [see Additional file . (2005)  and founThe techniques for multiple testing offered here through a platform-independent tool are relevant to a variety of data analysis tasks in biology and medicine. The results also allowed us to illustrate the power of a permutation test for multiple hypotheses assessment procedures and for controlling the rate of Type I errors. We also demonstrated that even when P-values were corrected via B&H, which is considered a less stringent method as opposed to Bonferroni, a number of potentially significant features were dismissed. The software is easy to use and it offers the basis for future extensions. Another key contribution is the implementation of multiple hypotheses statistical testing techniques for both numerical and categorical data. The analytical options offered can be applied to support a significant spectrum of hypothesis testing tasks in functional genomics, e.g. fast detection of significantly differentially expressed genes and genotypes. Moreover, to the best of our knowledge, this is the first open-source software tool freely available for supporting less traditional genomic applications, such as the detection of between-group differences on the basis of SNPs. In this area multiple-testing procedures have traditionally relied on very stringent adjustment approaches (e.g. Bonferroni).Despite its simplicity, in terms of usability, this tool in comparison with others, such as GeneSpring and TIGR MeV, offers the following advantages: Freely-available, as TIGR MeV does, no computational installation cost, easy to use, computationally inexpensive. Moreover it allows the calculation of traditional statistical tests and multiple testing with categorical data, as well as test- and distribution-independent permutation-based tests.We expect to continue expanding the tool with alternative statistical significance measures, such as Fisher's exact test, Z or Wald scores. We will welcome additional user's feedback after the publication of this article.Project name: Permutation-based statistical tests for multiple hypothesesProject home page: Operating system(s): Platform independentProgramming language: JavaOther requirements: Java 1.5.1 or higherLicense: NoneAny restrictions to use by non-academics: NoneANOVA: Analysis of variance; DCM: Dilated CardioMyopathy; SAM: Significant Analysis of Microarray; NSC: Nearest Shrunken Centroid; SNP: Single nucleotide polymorphismsThe authors declare that they have no competing interests.AC co-designed the software, wrote and implemented all source code, co-evaluated their outcomes and co-wrote the manuscript. FA conceived the original study, contributed to the testing and evaluation phases, and co-wrote the manuscript. HW co-designed the software, co-evaluated their outcomes and co-wrote the manuscript. HZ co-designed the software, co-evaluated their outcomes and co-wrote the manuscript. All authors read and approved the final manuscript.Ptest software. Execute from command line like this\java Ptest.Click here for fileEasyInput library. Used by Ptest.Click here for fileREADME. Microsoft word file, it should be open with Microsoft word because it contains mathematical equations.Click here for fileExample data set for Bartlett's test. 2 classes, 12 samples, 5 and 17 respectively, 8068 features. Plain text file can be open with any word processor.Click here for fileExample data set for  t-Test – unpaired samples. 2 classes, 12 samples – 5 and 17 respectively – 8068 features. Plain text file can be open with any word processor.Click here for fileExample data set for Chi-square test – categorical data. 3 classes, 33 samples – 10, 12, 11 samples respectively – 334 features. Plain text file can be open with any word processor.Click here for fileExample data set for ANOVA test. 3 classes, 37 samples – 7, 20 and 10 respectively – 14976 features. Plain text file can be open with any word processor.Click here for file


For advanced prostate cancer, the main hormone treatment against which other treatments are assessed is surgical castration. It is simple, safe and effective, however it is not acceptable to all patients. Medical castration by means of luteinizing hormone-releasing hormone (LH-RH) analogues such as goserelin acetate provides an alternative to surgical castration. Diethylstilboestrol, previously the only non-surgical alternative to orchidectomy, is no longer routinely used. Castration reduces serum testosterone by around 90%, but does not affect androgen biosynthesis in the adrenal glands. Addition of an anti-androgen to medical or surgical castration blocks the effect of remaining testosterone on prostate cells and is termed combined androgen blockade (CAB). CAB has now been compared with castration alone  in numerous clinical trials. Some trials show advantage of CAB over castration, whereas others report no significant difference. The author favours the view that CAB has an advantage over castration. No study has reported that CAB is less effective than castration. Of the anti-androgens which are available for use in CAB, bicalutamide may be associated with a lower incidence of side-effects compared with the other non-steroidal anti-androgens and, in common with nilutamide, has the advantage of once-daily dosing. Only one study has compared anti-androgens within CAB: bicalutamide plus LH-RH analogue and flutamide plus LH-RH analogue. At 160-week follow-up, the groups were equivalent in terms of survival and time to progression. However, bicalutamide caused significantly less diarrhoea than flutamide. Withdrawal and intermittent therapy with anti-androgens extend the range of treatment options. © 1999 Cancer Research Campaign


Nuclear receptors (NRs) and Receptor tyrosine kinases (RTKs) are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention.We constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs.We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved.http://www.bioinfo-cbs.org/NR-RTK/Availability: NR-RTK database is accessible at We infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs.This article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein), Dr. Arcady Mushegian and Dr. Anthony Almudevar. The receptor-ligand binding triggers a series of reactions in a living system. So, detailed knowledge and data about receptors such as nuclear receptors and receptor tyrosine kinases and their ligands are important for understanding living systems and diseases, and for designing new drugs.Nuclear receptors (NRs) are one of the most abundant classes of transcriptional regulators in animals. They regulate diverse functions, such as homeostasis, reproduction, development, or metabolism. They are prominent pharmaceutical targets for diseases such as hypertension, cancer, diabetes, cardiovascular disease and the metabolic syndrome ,2. NucleReceptor tyrosine kinases (RTKs) transmit their activation signal across the plasma membrane, and many studies have demonstrated that the receptors, and not the growth factors, mediate the pleiotropic cellular responses. Growth factors recognize and activate their cognate receptors and stimulate receptor dimerization, tyrosine kinase activation and autophosphorylation. The autophosphorylated RTKs recruit and activate a receptor-specific complement of intracellular signalling pathways that relay information to the nucleus and other intracellular compartments .Beyond this well-established mechanism of RTK and NR signallings, crosstalk between the RTK and NR receptors has been reported in many cases, such asi) Complex interactions between steroid receptor (estrogen receptor (ER) and progesterone receptor) and growth factor receptor signalling governed breast cancer evolution .ii) TrkB-GR interaction plays a critical role in the BDNF-stimulated PLC-γ pathway, which is required for glutamate release and the decrease in TrkB-GR interaction caused by chronic exposure to glucocorticoids results in the suppression of BDNF-mediated neurotransmitter release via a glutamate transporter .iii) Nuclear RTKs regulate a variety of cellular functions, such as cell proliferation, DNA damage repair and signal transduction, both in normal tissues and in human cancer cells .Here, our goal is to get a global understanding of the NR-RTK network using automated methods. We also developed a database which summarizes various data related to RTKs, NRs and their interactions and makes our results available for further discussion and investigation.We integrated results derived from genomic context, high-throughput experiments, coexpression (conserved) and text mining related to human NRs and RTKs to obtain the complete dataset, with a total of 159 protein-protein interactions including 38 RTK-RTK, 42 NR-RTK and 79 NR-NR protein interactions as shown in Additional file We then analysed the RTK-RTK, NR-NR interaction networks separately. The RTK-RTK interaction network is shown in figure Our analysis reveals 42 NR-RTK interactions when the two networks were merged into one figure . Moreovein vivo or in vitro assays: DHTR-Erbb2 [Among the 42 predicted NR-RTK interactions, four interactions had been previously validated by TR-Erbb2 , EGFR-ESTR-Erbb2 , Erbb2-ETR-Erbb2  and IGF1TR-Erbb2 . The intOn the other hand, we should take into account the substantial time lag between the first publication of a finding and at the time at which the PPI is entered in a database. It could therefore be postulated that many of our predicted PPIs predicted today are in fact correct, but have not been entered in a database for several years.k interactors against log(k). For the whole NR-RTK and RTK-RTK networks, we obtained a log-log plot linear regression R2 value of 0.794 and 0.899, respectively. This indicates that the networks have a scale-free topology. Nevertheless, this statistical property was not observed for the NR-NR protein interaction network (log-log plot linear regression R2 = 0.39) , the other vertebrate orthologs were taken from Chimpanzee, Dog, Mouse Rat and Chicken organisms.The premise behind the design of the database was to utilize as many public, continually-updated databases as possible. Thus, the menu items are linked to resources such as the National Center for Biotechnology Information (NCBI), Pfam, MutDB, CGAP, PharmGKB Table . MoreoveNR-RTK database is constructed through three modules. Each module performs specialized functions that provide users with a convenient way to find desired information according to the standard classification of these two kinds of receptors. The modules are described in the following sections:Receptor tyrosine kinases are classified into 20 subfamilies according to their variable extracellular domain as described by .Nuclear receptors are classified into seven groups (denoted from 0 to 6) according to Nuclear Receptor Nomenclature Committee.This module provides protein-protein associations derived from high-throughput experimental data, from the mining of databases and literature, and from predictions based on genomic context analysis.This module contains data on NR-NR, RTK-RTK and NR-RTK interactions inferred as previously described. The data are collected in an Excel file containing interaction method detection, data mining references (PMID) and combined score for every association. From a functional perspective, 'association' can mean direct physical binding, but can also mean indirect interaction such as participation in the same metabolic pathway or cellular process.http://www.bioinfo-cbs.org/NR-RTK/NR-RTK database is accessible at We infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs.We searched Swiss Prot Database for human NR and RTK and retrieved protein identifiers for 48 and 56 of them, respectively.Protein-Protein interaction network was constructed by STRING . The intIn order to assess whether a network is scale-free or not, the distribution of connectivity is plotted. Specifically, we plotted the log of frequency of proteins with K interactions, versus the log (K). A network may resemble a scale-free topology if the distribution of connectivity decays in a power-law fashion. Therefore, the better the trendline (in the log-log plot) fits a linear regression, the more the network resembles a scale-free topology .The topological and statistical significance of network have been calculated using Cytoscape plugins Network Analyzer  and Centhttp://med.bioinf.mpi-inf.mpg.de/netanalyzer/help/2.6.1/index.html.The definitions of calculated parameters are available at http://www.mysql.com. The application runs on an Apache 2.0 HTML server http://www.apache.org.All programming languages and software used were Open source, supplied under a general public license. We used the MySQL database server software DHTR: Dihydrotestosterone Receptor; EGFR: Epidermal Growth Factor Receptor; Erbb2: Receptor tyrosine-protein kinase erbB-2; ESR1: Estrogen Receptor 1; ESR2: Estrogen Receptor 2; IGF1R: Insulin-like growth factor 1 receptor; NR: Nuclear Receptor; NR0B2: SHP: Short heterodimer partner; NR1D1: nuclear receptor subfamily 1, group D, member 1; NR2C1: nuclear receptor subfamily 2, group C, member 1; NR2F1: nuclear receptor subfamily 2, group F, member 1; NR2F6: nuclear receptor subfamily 2, group F, member 6 PGR: Progesterone Receptor; RTK: Receptor Tyrosine Kinase; RXRA: Retinoic acid receptor RXR-alpha.The authors declare that they have no competing interests.MC did the computational analyses, data extraction and curation and database design. She also wrote the manuscript. AR supervised the work and corrected the manuscript. All authors read and approved the final manuscript.Paul Harrison Department of Biology, McGill University, Canada . One should really check other possible equations also (not just a power law equation).Author's response: We agree with the reviewer that we should check other equations. We feel that testing the scale-free topology and biological interpretation is sufficient for publication on this topic. As we are carrying on other works with the data, a more detailed comparison with other power law equation is desirable and we hope to complete that in the near future.* Thirdly, in the database, the Pubmed links in the lists of families do not appear to work.Author's response: Done, all links work.* Fourthly, the authors should make sure that the format of the webpages and download files is adequately explained. Currently, there is not sufficient explanation. For example, there is not a detailed explanation of the columns in the Excel file of downloadable interactions. Help pages with this information should be provided.Author's response: This information is explained in the manuscript in Results and Discussion section ("NR-RTK module"). Furthermore, we are now in the process of generating a new website to house these data.Arcady Mushegian, Department of Bioinformatics, Stowers Institute for Medical Research, Kansas City, Missouri, USA.* This study is suitable for publication as a Discovery Note, not as a Research Article. Targeted reconstruction of a protein-centered interaction subnetwork by extracting and scoring all relationships of that protein may be of interest if this results in novel observations about biological system, and I would suggest to show more of it in this note.Author's response: We don't think that this study can be considered as a Discovery Note. We believe that the significance of this work consists not just in the approach, but in the combination of methodology and biology to get new insights on the important receptors in signal transduction.* For instance, instead of primarily focusing on the node degree of the network, the authors may discuss in more detail the actual gene content of the modules that they discover. How many of the connections and of module composition are well-known and how many are novel/not covered in the literature? Any new hypotheses suggested by the module inference? What type of evidence makes the most significant contribution to the network? Are there types of evidence that do/do not improve the score?Author's response: The sub-networks discovered are RTK-RTK and NR-NR networks. The gene content of each sub-network is quite clearly described in the database such are their potential connections and their citations in literature. The module inference is exactly the main aim of the presented paper. Our approach may serve as predictive tool for indentifying key interactions and providing insight in experimental validation (in vivo and in vitro assays) (table1). The experimental assays and co-citations in literature for a given interaction improve the score.The figures as they stand now are typical of many "systems-biology" papers, but it is not clear what to make of them. Are we supposed to eyeball the list of gene names (in which case the table would suffice)? Is the density of links supposed to be the main message? A position of particular nodes?Author's response: The figures represent the interactions derived from different sources of evidence showing the central role of some proteins so called "hubs". The main point is the identification of these hubs according to their position in the network and other topological parameters .Finally, about the "scale-free" character of the network. First, I am not sure that it is useful to compute the node degree distribution of the local, i.e., protein-centric network: if it is by construction a set of interactors of one protein, it is guaranteed that at least one protein will be very highly connected. More formally, it is known that fitting to the power law is not a right test here: many purported "scale-free" networks have been proven to reject the hypothesis in standard tests , or fit the power law only on an interval, or on different intervals with different values of the gamma parameter . Moreover, suppose that the distribution can be fit to a mixture of functions - what would the biological conclusion be?Author's response: We agree with reviewer. Although the statistical test shows that the network is scale-free, we are also aware that much more tests should be performed especially for such complex system.Regarding your question supposing a mixture of functions, this would be explained by two types of interactions: physical interactions and cross-talking with the two types of receptors that modulate gene expression.Reviewer's report 3: Anthony Almudevar, Department of Biostatistics and Computational Biology University of Rochester Medical Center, Rochester, NYThe authors are concerned with two classes of proteins, nuclear receptors (NR) and receptor tyrosine kinases (RTK). These have broad functionality, with variants implicated in many disease states, including cancer. A database is constructed based on a protein-protein interaction (PPI) network of 48 NRs and 53 RTKs. The PPIs are compiled using various existing knowledge sources.Author's response: We corrected the typing error in manuscript, 56 RTKs instead of 53 RTKs.Three networks are analyzed, with NR proteins only (NR-NR), with RTK proteins only (RTK-RTK), and with all proteins (NR-RTK). It is reported that the RTK-RTK and NR-RTK networks have scale-free topology . This does not hold for the NR-NR network, an observation which conforms to another published finding. A hypothesis for this observation is given, involving the presence of a large number of negative feedback loops.Proteins which form highly connected hubs in the NR-RTK network (11 listed) are conjectured to influence the transmission of information between the NR and RTK networks.The NR-RTK database is available at a URL given in the article. It is fairly basic in structure, but provides a useful way to explore the properties of the network and its components.Overall, the paper is concerned with the construction of a knowledge database, and is not concerned with new methodology. As such, some potentially useful hypotheses concerning signaling pathways, and their role in disease states, are generated. The methods used seem sound. The given biological background, and hence the motivation for the database, is interesting.Some suggestions:(1) Abstract - Results: "We constructed a human signalling network ... that indentified a much more connected network topology than previously thought." Is it possible to provide citations, or to elaborate on this claim?Author's response: To elaborate this result, we rephrased this sentence.(2) In "Results and discussion" and "Methods: sections: The method used by the authors for testing the scale-free property is given in Barabási and Albert (1999) Science, vol 286, p -509-512. The exponent of the power law (the slope of the regression line) might also be reported, since it is sometimes used to characterize network properties, and might allow for a useful comparison to other cellular networks.Author's response: We added this reference.Minor points:Background- paragraph 4 - item iii): "of NR-RTK network" - > "of the NR-RTK network"Results and discussion - NR-RTK signalling network- paragraph 2: enclose "especially EGFR" in commas.- paragraph 4: rephrase sentence starting "The remaining ..."- paragraph 5: "and the PPI" - > "and the time at which the PPI ..."Results and discussion - Topology of the interaction networks- paragraph 2: "few nodes" - > "a few nodes"- paragraph 2: "network" - > "networks"- paragraph 2: "on large" - > "on a large"Author's response: we corrected these points accordingly.RTK-NR interactions. It contains in 4 worksheets 1) the protein-protein interactions, their scores, their source, the interaction detection method and PMIDs 2) NR-RTK distribution 3)RTK-RTK distribution 4) NR-NR distributionClick here for fileTopological coefficients of NR-RTK interaction network. It contains in 6 worksheets 1) degree 2) Clustering coefficient 3)Closeness centrality 4)Eccentricity 5)Neighborhood connectivity 6) Average shortest path length.Click here for file


To ascertain if the polymerization reaction also contributes additionally to the antibacterial effects of two commonly used cyanoacrylate tissue adhesives.Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Pseudomonas aeruginosa. Immediately after the discs were applied, the cultures were incubated at 35° C for 24 h. Bacterial inhibitory halos were measured in the cultures at the end of the incubation period.Fresh liquid ethyl-cyanoacrylate (EC) and N-butyl-cyanoacrylate (BC) adhesives were applied onto 6-mm sterile filter paper discs. In the first group, the adhesive-soaked discs were immediately placed onto confluent monolayer cultures of bacteria, allowing the polymerization reaction to proceed while in culture. In the second group, the adhesive-soaked disc was allowed to first polymerize prior to being placed onto the bacterial cultures. Four types of bacteria were studied: Streptococcus pneumonia, Staphylococcus aureus and Escherichia coli. For BC, it increased the bacterial inhibitory halos in Staphylococcus aureus and Streptococcus pneumoniae. No inhibitory halos were observed in Pseudomonas aeruginosa. The bactericidal effect was higher in actively polymerizing EC, compared to previously polymerized EC in Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli; however, no such differences were observed for BC.For EC, exposure of the bacteria to the cyanoacrylate polymerization reaction increased the bacterial inhibitory halos in The polymerization reaction may also be an important factor in the antibacterial properties of EC and BC. Cyanoacrylate polymers have been used as biological adhesives in the cornea for over 40 years.–3 Their The polymerized adhesive promotes wound healing, vascularization, and epithelialization of the injured corneal stroma. It also The antimicrobial properties of cyanoacrylate tissue adhesives have been reported previously and some authors have even promoted its use in the prophylaxis or treatment of infection in corneal ulcers.–9 It hasThe aim of the study was to establish the role of the polymerization reaction in conferring additional antibacterial properties to cyanoacrylate tissue adhesives.in vitro experiments: Ethyl-cyanoacrylate (EC)  and N-butyl-cyanoacrylate (BC) . Six microliters of adhesive were applied onto standard 6-mm sterile filter-paper discs, using sterile micropipettes  under sterile conditions.Two cyanoacrylate tissue adhesives were studied in the Staphylococcus aureus (ATCC25924), Streptococcus pneumoniae (ATCC49619), Escherichia coli (ATCC25922), and Pseudomonas aeruginosa (ATCC27853). They were primarily incubated in a nutrient broth at a temperature of 35°C until reaching 0.5 on the McFarland scale . The bacteria were then transferred as confluent monolayer cultures to Müller-Hinton media following the Kirby-Bauer modified technique, with the exception of Streptococcus pneumoniae, which was transferred to blood agar media.The following bacterial strains from the American type culture collection (ATCC) were analyzed: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Twenty blood agar plates (90-mm diameter) were used for Streptococcus pneumoniae. A blank disc without adhesive was placed in the center of each bacterial plate as a control.Ten Müller-Hinton agar plates (150-mm diameter) were used for each of the following bacteria: In each Müller-Hinton plate, eight additional discs were placed onto the cultures (in addition to the control disc): two with actively polymerizing (liquid) EC (EC-liq); two with previously polymerized (solidified) EC (EC-poly); two with actively polymerizing (liquid) BC (BC-liq); and two with previously polymerized (solidified) BC (BC-poly). For the blood agar plates, four discs were placed onto the cultures (in addition to the control disc): one with actively polymerizing (liquid) EC (EC-liq); one with previously polymerized (solidified) EC (EC-poly); one with actively polymerizing (liquid) BC (BC-liq); and one with previously polymerized (solidified) BC (BC-poly). To achieve prior polymerization of the cyanoacrylate in EC-poly and BC-poly, the adhesive-soaked discs were exposed to air under a sterile hood for 30 min before being placed on the cultures. All actively polymerized samples were applied directly into the disc placed in the plate. All plates were then incubated at 35°C for 24 h, after which the bacterial inhibitory halos, if present, were measured in millimeters .P < 0.05.In order to evaluate if the bacterial inhibitory halos were the result of mere bacteriostasis or actual bactericidal effects, two samples per halo were collected from the clear agar within the inhibitory halos and re-cultured on new bacterial culture plates. The new plates were re-incubated at 35°C and analyzed after 48 h. Bactericidal activity was measured by calculating the percentage of plates with no bacterial growth. All the microbiological procedures and readings were performed by one author (CBS). The results were statistically analyzed with the Student T-test and significance was defined as Streptococcus pneumonia (P=0.0014), Escherichia coli (P<0.0001) and Staphylococcus aureus (P=0.019), but not in Pseudomonas aeruginosa.Staphylococcus aureus (P<0.0001) and Streptococcus pneumoniae (P=0.0010), but not in Escherichia coli and Pseudomonas aeruginosa.Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, but this bactericidal enhancement by the polymerization reaction was not observed in BC. The bactericidal effect was not analyzed for Pseudomonas aeruginosa, since there was no inhibitory halo.The results of the bactericidal analyses of EC and BC are summarized in Streptococcus pneumonia, Staphylococus aureus and Escherichia coli. In Escherichia coli, however, inhibitory halos were totally absent without exposure to the active polymerization reaction. For BC, inhibitory halos were observed only for gram-positive bacteria. No inhibitory halo was observed for Pseudomonas aeruginosa for either EC or BC, with or without exposure to the active polymerization reaction.Antibacterial effects were enhanced by the polymerization reaction in EC for Staphylococcus aureus and Streptococcus pneumoniae, to be much greater than of the two gram-negative organisms tested, Escherichia coli and Pseudomonas aeruginosa. Eiferman et al. reported the absence of inhibitory halos in Klebsiella pneumoniae[Escherichia coli.The antibacterial effects of cyanoacrylate are greater in gram-positive bacteria than in gram-negative, possibly because the latter are protected by an outer carbohydrate capsule. Our resuneumoniae a gram-nStaphylococcus aureus and Escherichia coli. This difference may be due to the greater tissue toxicity and antimicrobial effects associated with shorter-alkyl chain cyanoacrylates, such as EC, over the longer-alkyl chain cyanoacrylates, such as BC. As a rule, shorter alkyl-chain cyanoacrylates are characterized by greater instability and faster chemical degradation than the longer alkyl-chain counterparts, resulting in higher levels of ambient degradation, products such as cyanoacetate and formaldehyde.[EC appeared to have a greater antibacterial effect than BC for aldehyde.et al. established a standard volume of adhesive of 100 μl, no bactericidal studies were performed.[The bactericidal effect of the adhesives was lower than in previous studies.–912 This9erformed.A polymer of cyanoacrylate is formed as a number of monomers join together under the effect of any catalyst (water). The polymer decomposes to produce cyanoacetate and formaldehyde. Such degradation products can diffuse out. Therefore, there is an inhibition halo even in a polymerized state.5Escherichia coli, the effect of the polymerization reaction in EC was very important.Although the effect of the polymerization reaction was relatively small for gram-positive micro organisms, the values were statistically significant. Nevertheless, as per gram-negative micro organism like in vivo” reproduction is not possible because the material loses its adhesive capacity to mold onto irregular surfaces after polymerization reaction. On top of that, when cyanoacrylate-based tissue adhesive gets into contact with water, it solidifies. Therefore, it is not possible to use different concentrations of the adhesive and to obtain the MIC 90, which is used to evaluate antimicrobial properties in antibiotics and is defined as the antimicrobial concentration that inhibits growth of 90% of the microorganisms.Regarding restrictions of this study, “In conclusion, the polymerization reaction appears to have an important contributory role in the antibacterial activity of cyanoacrylate tissue adhesives and may be exploited in the treatment of corneal ulcers. Therefore, in severe or recalcitrant gram-positive bacterial corneal ulcers, for instance, one may consider the use of shorter alkyl-chain adhesives with their strong antibacterial properties, as a supplement to topical antibiotics. This antibacterial effect may be an advantage when glue is applied to the cornea in patients with melting or perforation.


P. falciparum merozoites is a complex multistep process involving a series of molecular recognition events between parasite ligands and their receptors on the erythrocyte surface. Although many potential P. falciparum invasion ligands are known, the erythrocyte receptors they interact with have been identified only for a handful of cases. Studying extracellular binding events between membrane-embedded proteins in vitro is challenging and beyond the reach of standard approaches such as co-immunoprecipitation and yeast two-hybrid screens. These interactions are generally of very low affinity and often require specific motifs generated via post-translational processing; furthermore, amphipathic membrane-spanning proteins are difficult to manipulate biochemically. The Duffy Antigen Receptor for Chemokines (DARC), a multi-pass G-protein coupled erythrocyte receptor is known to be important for invasion by P. vivax but no multi-pass receptor has been established to play a role in P. falciparum invasion. With the aim of identifying such a receptor, we have developed a flow cytometry based, high-throughput strategy that can detect transient, extracellular protein: protein interactions. We are in the process of screening the soluble, recombinant ectodomains of 39 P. falciparum merozoite surface proteins against an expression library of 45 erythrocyte multi-pass surface proteins. We will present our screening strategy and preliminary results.Invasion of erythrocytes by


Kaposi sarcoma-associated herpesvirus  is a γ-2 herpesvirus that causes Kaposi sarcoma. KSHV seroprevalence rates vary geographically with variable rates recorded in different sub Sahara African countries, suggesting that effects of genetic and/or environmental factors may influence the risk of infection. One study conducted in South Africa, where KSHV seroprevalence is relatively low, found that carriage of human leukocyte antigen (HLA) alleles HLA-A*6801, HLA-A*30, HLA-A*4301, and HLA-DRB1*04 was associated with increased shedding of KSHV DNA in saliva. Confirmation of those results would strengthen the hypothesis that genetic factors may influence KSHV distribution by modulating KSHV shedding in saliva. To explore these associations in another setting, we used high resolution HLA-A, B, and DRB1 typing on residual samples from the Uganda Sickle Cell Anemia KSHV study, conducted in a high KSHV seroprevalence region, to investigate associations between HLA and KSHV shedding in saliva or peripheral blood among 233 children and their mothers. HLA-A and HLA-DRB1 alleles were not associated with KSHV shedding in our study, but our study was small and was not adequately powered to exclude small associations. In exploratory analyses, we found marginal association of KSHV DNA shedding in saliva but not in peripheral blood among children carrying HLA- B*4415 and marginal association of KSHV DNA shedding in peripheral blood but not in saliva among children carrying HLA- B*0801 alleles. The contribution of individual HLA polymorphisms to KSHV shedding is important but it may vary in different populations. Larger population-based studies are needed to estimate the magnitude and direction of association of HLA with KSHV shedding and viral control. KSHV sethropods . The rolthropods . Recentlthropods -11, thisthropods .The Uganda Sickle Cell Anemia KSHV study included 600 children with sickle cell anemia, but without KS, who were enrolled from November 2001 to April 2002 at the Sickle Cell Clinic at Mulago Hospital . The motWe performed high-resolution HLA-A, -B and -DRB1 typing on residual DNA obtained from saliva or buffy coat of the children and their mothers from the KSHV viral study. Alleles were separated by a group-specific amplification approach using multiple amplification primer mixes in parallel  . The preWe calculated the weighted distribution of HLA-A, -B, and -DRB1 allele group frequencies among the children and their mothers separately. The prevalence of allele groups was weighted back to the original study population. Thus, the weighted prevalence of HLA allele group (X) in the children =  + . The prevalence of HLA allele groups for the mothers was weighted back to the original study population based on KSHV status of the children because the mothers were selected when their child was selected.a priori based on previous associations with KSHV shedding in saliva: HLA-A*6801, HLA-A*30 and HLA-A*4301, and HLA-DRB1*04 separately for the children and their mothers. We also performed exploratory analyses to identify new associations. Odds ratios (ORs) for association and corresponding 95% confidence intervals (95% CIs) were computed using logistic regression models. We adjusted for KSHV serostatus by including the posterior probability of KSHV infection estimated using multivariate mixture models as previously described [p-values < 0.05 were considered statistically significant.We assessed associations between HLA allele groups and KSHV DNA detection for the alleles selected escribed . All staAmong 233 children with DNA, HLA was successfully typed for the A locus in 223, for the B locus in 225, for the DRB1 locus in 223 children. Among 233 mothers with DNA, HLA was successfully typed for the A locus in 226, for the B locus in 226, for the DRB1 locus in 227 mothers. The weighted prevalence for HLA loci, HLA-A, -B, and -DRB1 and the number of people carrying the allele group for children and their mothers separately are shown in Table HLA-A*3001, HLA-A*3002, HLA-A*6801, HLA-A*6802, and DRB1*0405 alleles were not associated with detection of KSHV DNA in saliva or in peripheral blood in the children (Table et al., [Our study did not replicate HLA associations with KSHV DNA shedding reported in South Africa . Our nulet al., , risk foet al.,  and, thuet al.,  provide The strengths of our study include assessment of HLA polymorphisms on three loci among children and their mothers. The pre-selected sample of children with sickle cell disease, one time sampling of subjects and small sample size were limitations. Thus, it is possible that play of chance and multiple testing in small studies could explain findings from both ours and Alkharsah's studies. Large and better designed studies are warranted to evaluate the contribution of individual HLA polymorphisms to KSHV shedding in different populations.The authors declare that they have no competing interests.MGO analyzed and interpreted data, and drafted the manuscript. MV, TFS, BEV, CF, and RB used high resolution technique for HLA-A, B, and DRB1 allele typing. RMP contributed to analysis and interpreted data. CMN, AMO, PMB, interpreted data and edited the paper, KB interpreted data. SMM conceived the idea, guided data analysis, interpreted data and edited the paper. All authors had access to data, commented on and contributed to the final draft of the manuscript. All authors read and approved the final paper.


Haemophilus influenzae type B (Hib). To ensure adequate antigen supply, GlaxoSmithKline Biologicals has introduced a new DTPw antigen source and developed a new DTPw-HBV/Hib combination vaccine containing a reduced amount of Hib polyribosylribitol phosphate (PRP). This study was undertaken to compare the immunogenicity and reactogenicity of this new DTPw-HBV/Hib vaccine with a licensed DTPw-HBV/Hib vaccine (Tritanrix™-HBV/Hib).Diphtheria-tetanus-whole-cell pertussis (DTPw)-based combination vaccines are an attractive option to rapidly achieve high coverage and protection against other important pathogens, such as hepatitis B virus (HBV) and This was a randomized, partially-blind, multicenter study in three countries in Latin America . Healthy children received either the new DTPw-HBV/Hib vaccine  or Tritanrix™-HBV/Hib  co-administered with oral poliovirus vaccine (OPV) at 2, 4 and 6 months, with a booster dose at 18-24 months.One month after the end of the 3-dose primary vaccination course, the new DTPw-HBV/Hib vaccine was non-inferior to Tritanrix™-HBV/Hib in terms of seroprotection/vaccine response rates for all component antigens; ≥97.3% and ≥93.9% of subjects in the two groups, respectively, had seroprotective levels of antibodies against diphtheria, tetanus, hepatitis B and Hib and a vaccine response to the pertussis component. Persistence of antibodies against all vaccine antigens was comparable between groups, with marked increases in all antibody concentrations after booster administration in both groups. Both vaccines were generally well-tolerated as primary and booster doses.Results confirm the suitability of this new DTPw-HBV/Hib vaccine comprising antigens from a new source and a reduced PRP content for inclusion into routine childhood vaccination programs.http://www.clinicaltrials.gov NCT00332566 Haemophilus influenzae type b (Hib) infections remain endemic in many parts of the world, causing disease that can readily be prevented by vaccination  and for anti-BPT, the 90% confidence interval for the ratio of geometric mean antibody concentrations (GMCs) between each pair of lots was within  (data not shown). Based on these findings, data for the three vaccine lots used in this study were pooled for comparison against the control vaccine.Tritanrix™-HBV/Hib, with the exception of anti-PRP GMC. However, almost all subjects achieved anti-PRP concentrations ≥1.0 μg/mL (≥97.3%) and post-primary anti-PRP GMCs were high (> 12.5 μg/mL) in both groups, suggesting that intergroup differences are unlikely to be of clinical relevance. A large body of evidence shows reduction of the PRP content of Hib conjugate-based vaccines to have no negative impact on either immune response or induction of immune memory to Hib [Antibody GMCs induced by the new DTPw-HBV/Hib vaccine following primary vaccination were observed similar to those induced by y to Hib -26. Effey to Hib ,18. In oTritanrix™-HBV/Hib group). Indeed, by eliminating the need for multiple injections, such combination vaccines are likely to promote compliance with infant immunization schedules [The new DTPw-HBV/Hib vaccine was found to have an acceptable reactogenicity profile. Some differences in reactogenicity were observed between the two vaccines. However, since very few subjects in either group sought medical advice for solicited symptoms it is likely that any differences had minimal clinical impact. Also, as no correction for multiplicity of endpoints was applied, statistically significant differences in reactogenicity should be interpreted with caution. Very few subjects in this study failed to receive all planned vaccine doses due to adverse events (2 in the DTPw-HBV/Hib group and 1 in the chedules . In bothchedules ,17,27,28In conclusion, the development of new DTPw-based combination vaccines is essential for the success of current and future childhood mass vaccination programs in countries where resources are limited. Combining DTPw vaccines with HBV and Hib performs a key role in increasing vaccine coverage rates in line with WHO targets. Results of this study confirm the suitability of GSK Biologicals' new DTPw-HBV/Hib vaccine containing DTPw antigens from a new source and a reduced quantity of PRP as an alternative to currently licensed DTPw-based combination vaccines for use as a primary or booster dose in routine pediatric vaccination programs.Bordetella pertussis toxin; CI: confidence interval; DTPw: Diphtheria-tetanus-whole-cell pertussis; ELISA: enzyme-linked immunosorbent assay; GMC: geometric mean antibody concentration; GSK: GlaxoSmithKline Biologicals; HBsAg: hepatitis B virus surface antigen; Hib: Haemophilus influenzae type B; HBV: hepatitis B virus; IU: international units; OPV: oral poliovirus vaccine; PRP: polyribosylribitol phosphate; SAE: serious adverse events; SD: standard deviation; WHO: World Health OrganizationATP: according to protocol; BPT: This study was funded by GlaxoSmithKline Biologicals. JC, AC, ILF, J-MJ are employees of GlaxoSmithKline Biologicals or were employed at the time of the study.All authors participated in the design or implementation, analysis and interpretation of the study, the writing of the report and the decision to submit the manuscript for publication.AC participated in the design of the study and performed the statistical analysis. FE, MT, AG, KA, JC, ILF, J-MJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/297/prepub


A wide range of e-learning modalities are widely integrated in medical education. However, some of the key questions related to the role of e-learning remain unanswered, such as (1) what is an effective approach to integrating technology into pre-clinical vs. clinical training?; (2) what evidence exists regarding the type and format of e-learning technology suitable for medical specialties and clinical settings?; (3) which design features are known to be effective in designing on-line patient simulation cases, tutorials, or clinical exams?; and (4) what guidelines exist for determining an appropriate blend of instructional strategies, including on-line learning, face-to-face instruction, and performance-based skill practices? Based on the existing literature and a variety of e-learning examples of synchronous learning tools and simulation technology, this paper addresses the following three questions: (1) what is the current trend of e-learning in medical education?; (2) what do we know about the effective use of e-learning?; and (3) what is the role of e-learning in facilitating newly emerging competency-based training? As e-learning continues to be widely integrated in training future physicians, it is critical that our efforts in conducting evaluative studies should target specific e-learning features that can best mediate intended learning goals and objectives. Without an evolving knowledge base on how best to design e-learning applications, the gap between what we know about technology use and how we deploy e-learning in training settings will continue to widen. The direction of the future of e-learning in medical education is heavily influenced by three major trends: 1) the rapid adoption of emerging communication, simulation, and information technology in undergraduate, graduate, and continuing medical education; 2) a national call for competency-based, patient outcome-oriented training across the continuum of education; and (3) the rapidly changing health care environment including advances in the biomedical sciences as well as in the diagnoses and management of diseases, organization, financing, and delivery of health care services, and changes in the societal expectations [ the rapi a nationhttp://www.ivimeds.org/) and the Virtual Campus of the King's College of University of London (http://gktvc1.kcl.ac.uk/) are examples of e-learning training at the undergraduate, residency, and continuing professional levels [E-learning is defined as learning mediated by technology, such as the World Wide Web, intranet, and multi-media based computer applications , 4. In rl levels . The IVIAs new technology is developed and deployed in learning settings at a rapid pace, the question that begs an answer is: what specific contributions can technology make towards improving the quality of medical education? Even with today's extensive and pervasive use of technology in medical education, some of the basic questions related to the role of e-learning remain unanswered , 6, suchWhat is an effective approach to integrating technology into pre-clinical vs. clinical training?What evidence exists regarding the type and format of e-learning technology suitable for medical specialties and clinical settings?Which design features are known to be effective in designing on-line patient simulation cases, tutorials, or clinical exams?What guidelines exist for determining an appropriate blend of instructional strategies, including on-line learning, face-to-face instruction, and performance-based skill practices?        The central tenet of this paper is three fold. First, technology will continue to dominate the way we train future physicians. Second, there exists an untapped potential for synchronous delivery and simulation technology to help meet training needs in emerging competencies . Third, What is the current trend of e-learning in medical education?What do we know about the effective use of e-learning?What is the role of e-learning in facilitating newly emerging competency-based training?        The Liaison Committee on Medical Education in the U.S. conducts an annual survey of medical schools, in which several questions are asked about the use of technology in the medical curriculum . One itehttp://www.acgme.org/Outcome/).At the CME  level, Curran and Fleet  report thttp://www.aamc.org/mededportal), End of Life/Palliative Education Resource Center by Medical College of Wisconsin (http://www.eperc.mcw.edu/), The Health Education Assets Library , Multimedia Educational Resource for Learning and On-line Teaching , and Family Medicine Digital Resource Library (http://fmdrl.org). These initiatives recognize the need to create a mechanism for sharing quality e-learning resources across institutions and to reward the works of faculty and staff through peer reviewed processes.At the national and international levels, a number of initiatives have emerged with the purpose of creating a digital repository of peer-reviewed electronic resources for public dissemination . Some ofIn understanding the impact of e-learning on learning, Kirkpatrick's work has been cited as a useful model . The modLearner satisfaction mainly encompasses participants' perceptions and satisfaction with learning objectives, content, format, and instructor's effectiveness. Learning outcomes include assessment of learners' knowledge, attitudes, and skills. Performance improvement targets changes in practice behaviors as a direct result of the newly acquired knowledge, attitudes, and skills. Lastly, patient and health outcomes include changes in patients' behaviors and health indicators as a result of trainees' improved practice patterns. For example, in a study by Curran and Fleet , 10 whicA number of systematic reviews have been conducted to examine the effectiveness of e-learning across the continuum of medical training covering medical students, residents, and practicing physicians. http://fammed.washington.edu/predoctoral/clerkship/casedemo.html). The purpose of the Case for Change is to teach third-year medical students how to conduct a patient-centered communication targeting a patient's behavioral change. This Web-based tool is required of all students to complete during their 6-weeks of Family Medicine clerkship rotation. These rotations take place at 24 training sites in the five-state region . The main components of Case for Change include: (1) video cases that simulate a provider-patient interaction using a scenario of reducing risky sexual behavior; (2) free-text input by students what they would say to the patient after viewing the video segments; and (3) multiple choice options selected by students that best match their free-text inputs. Multiple video segments are available to students that are associated with key stages of patient-centered behavioral change discussions, including building rapport, eliciting patient concerns, negotiating agenda, determining risk patterns, etc. Our evaluation involved coding of students' free-text responses and scoring of their multiple choice options using the expert's approach as the gold standard. Four categories of coding were used in analyzing students' free-text responses:One example of a novel approach to incorporating Kirkpatrick's model in assessing an e-learning application involves the Case for Change tool developed at the University of Washington  mandate required of all U.S. residency training programs, residents have to demonstrate their competencies in six core areas, including: (1) patient care, (2) medical knowledge, (3) practice-based learning and improvement, (4) interpersonal and communication skills, (5) professionalism, and (6) systems-based practice. There is an increasing call at the national level to (1) restructure the undergraduate and continuing medical education using the residency competencies ; and (2)So the question is: what is the role of technology in teaching and assessing competencies across undergraduate, graduate, and continuing medical education. How do we adapt the current e-learning applications for targeting the following competency skills in future physicians:Patient-centered communication skillsCompetency in providing culturally sensitive careExhibiting professionalism in all aspects of a physician's lifeExercising evidence-based decision makingPatient safety/medical error reductionInter-professional team careLife-long learningContinuous self-assessmentImproving practice performanceEvidence-based critical thinking and clinical reasoning        There are three e-learning modalities that promise a great potential for innovative training in the future. These modalities include: (1) simulation technology; (2) synchronous learning delivery; and (3) Web-based or videoconferencing for standardized patient-based training.The state of the art simulation centers exist in many U.S. medical institutions. These centers not only support performance- based training of surgery and anesthesiology trainees, but also internists and family physicians who are required to demonstrate a wide range of procedural skills. These skills encompass difficult airway management, cardiac life support, bronchoscopy, endoscopy, and suturing, etc. Goals of the simulation centers are to provide trainees with multiple opportunities to practice a wide range of procedural skills and to reduce preventable medical errors, thereby ensuring patient safety.http://simcen.usuhs.mil/home.html); (2) Harvard Center for Medical Simulation (http://www.harvardmedsim.org/); (3) Peter Winter Institute for Simulation, Education, Research at University of Pittsburgh (http://www.wiser.pitt.edu/); (4) Bristol Simulation Center (http://www.bris.ac.uk/Depts/BMSC/); (5) The Simulation Group at Massachusetts General Hospital (http://www.thesimgroup.org/); and (6) SimSuite from Medical Simulation Corporation (http://www.medsimulation.com/education_system/centers.asp). Typically, these centers use full mannequins or models connected to various display units that guide the trainees' performance during simulation sessions. Evaluating trainees' skills are usually conducted via observation by faculty members, who complete checklist forms for assessing trainees' psychomotor and technical skills. Depending on the scenarios developed for trainees' interactions with simulators, evaluation can also include trainees' skills in patient assessment, management of critical events, communication, and interpersonal relationship. Although expensive and required of highly skilled personnel to operate, simulation technologies will certainly be on the leading edge of e-learning applications in medical education. These technologies can serve as effective modalities for delivering competency-training in future physicians, which is difficult to deliver using conventional teaching methods.Some of the notable centers include: (1) National Capital Area Medical Simulation Center (http://www.adobe.com/products/breeze/for demonstration of system features). Breeze integrates a wide range of features for providing both instructors and learners a high level of flexibility. Instructors and learners can view each other via video streaming during live lectures, instructors can create on the fly on-line quizzes or surveys for learners to complete, and learning resources, such as Powerpoint slides and lecture notes, can be archived for students' self-paced learning. The entire Breeze session can be video or audio taped and archived for later access by learners as well. At University of Washington, third year students complete their clerkship at remote training sites in a five-state region that encompasses three-time zones. Breeze can be used to connect students or faculty members at clerkship sites to receive didactic sessions on commonly seen conditions in outpatient settings and to discuss interesting or rare cases that may not be frequently encountered at other sites.The advancement in technology and access to broadband connectivity from remote training sites have made a synchronous learning delivery a possibility. The synchronous learning modality, such as Webcast, consists of a live video/audio broadcast of training sessions and archival of training materials for later access by participants. Many benefits include: (a) connecting learners from distant sites to live training sessions; (b) creating opportunities for trainers and participants to interact in real time; (c) fostering peer-to-peer feedback; (d) interacting with learning resources such as lecture notes or simulated cases; and (e) accessing training materials for self-paced review. The product developed by Macromedia, Breeze, offers one example of how a synchronous e-learning technology may be used in medical education  and video technology have been piloted to test whether performance-based skills, such as decision making or error disclosure skills, can be taught and evaluated. One study by Clever, et al.  examinedThis paper presented a general overview of the current trend in e-learning with a focus on the U.S. medical education, main findings from the recent systematic reviews of studies involving e-learning that show many gaps in the way the effectiveness of e-learning is being examined, and a review of emerging technologies that have potentials for meeting new requirements for competency-based training. The paper also provided examples of emerging consortiums of institutions that have created common e-learning experiences for their medical trainees. As e-learning continues to be widely integrated in the training of future physicians, it is critical that our efforts in conducting evaluative studies should target specific e-learning features that can best mediate intended learning goals and objectives. Without an evolving knowledge base on how best to design e-learning applications, the gap between what we know about technology use and how we deploy e-learning in training settings will continue to widen.


Prognostic factors are defined as biological or clinical measurement associated with overall survival and/or disease-free survival. Previous studies have shown that patients with estrogen receptor (ER) positive cancers have a better prognosis than patients whose cancers do not have these receptors.This study investigated the assessment of variables in defining prognosis of 742 breast cancer women with pathological stage (pTNM) I-III diagnosed between 1980 and 2005 at the Kyoto University Hospital in Japan, by age, clinical stage (cTNM), pTNM, the numbers of positive lymph nodes (pN), and ER status.Multivariate analysis demonstrated that pTNM and ER status were the independent prognostic factors for overall survival, and that pTNM and pN were the independent prognostic factors for disease-free survival. For the 0- to 2-year interval, the hazard of recurrence was higher for the ER-negative patients than the ER-positive patients, and beyond 3 years the hazard was higher for ER-positive patients.The present study confirmed the previous reports which showed favorable prognosis of the patients with lesser pTNM or positive ER status. A reversal of recurrence hazard rate between ER positive and negative breast cancer patients beyond 3 years after operation was detected. The fact may indicate the importance of long term adjuvant hormone therapy for ER positive cancer patients. A prognostic factor is defined as a biological or clinical measurement that is associated with overall survival and/or disease-free survival . The knohttp://www.niph.go.jp/english2/english ver/ethical-gl/guidelines.htm, this study was exempt from ethical approval under Japanese law and guidelines. Moreover, all treatments for breast cancer were undertaken with informed consent and consents were also taken to confirm cancer diagnosis. These patients underwent surgery with axillary lymph node dissection. The operation methods were classified into three groups: breast conserving surgery, modified radical mastectomy, and standard radical mastectomy. All the patients with breast conserving surgery received radiation therapy. Staging of cTNM and pTNM was evaluated according to UICC stage [per os only) with or without hormone therapy (tamoxifen or tremifene) was included. The patients received adjuvant chemotherapy with LH-RH agonist after 2001, cyclophosphamide, epirubicin and 5-fluorouracil (CEF) or Cyclophosphamide, methotrexate and 5-fluouracil (CMF) regimen after 2002, and rational developers such as taxane, trastuzumab, or aromatase inhibitor therapy after 2004.742 female beast cancer patients aged between 21 and 80 with stage I-III of pTNM were selected from the patients treated at Kyoto University Hospital in Japan from 1980 to 2005. Based on the section 2 in chapter 1 of Japanese ethical guidelines for epidemiological research CC stage . Accordip <0.05 was regarded as statistically significant. The statistical analysis was conducted with SPSS version 11.0 statistical software.Disease-free survival was defined from the operation day to the identification date of recurrence of cancer or death from any cause, and overall survival was defined from the operation day to death from any cause. Survival curves were estimated with the Kaplan-Meier method. To identify prognostic factors independently associated with the overall survival or disease-free survival and to estimate the hazard ratios, the Cox proportional hazard model was applied. Two-sided Patient characteristics are summarized in Table The 10-year overall survival rates classified by age, cTNM, pTNM, pN, ER status and types of breast surgery are shown in Table The 10-year disease-free survival rates classified by age, cTNM, pTNM, pN, ER status and types of breast surgery are shown in Table p = 0.009). Moreover, among 452 ER-positive cases, at 1 year after surgery, the hazard of recurrence was higher for the patients with adjuvant hormone therapy than the patients without adjuvant hormone therapy, but between 2 and 4 years, the hazard was higher for the patients without adjuvant hormone therapy , cTNM (stage I-III), pTNM (stage I-III), pN , ER status , and types of breast surgery  were analyzed as potential prognostic factors by the Cox proportional hazard model. Both univariate analyses to determine prognostic factors associated with overall survival and disease-free survival that the features with ; ≥ 55, cTumor staging systems provide information about extent of disease that can be used to guide treatment recommendations and provide estimates of patient prognosis. It is well known that pathological stage is the most significant independent prognostic factor for determining survival in breast cancer . Our stuMany studies have shown that women with ER positive cancers have a better prognosis than patients whose cancers do not have this receptor ,10. In tHortobagyi et al.  previousTraditional prognostic factors, i.e., those that predict the risk of recurrence or death from breast cancer, include number of positive axillary nodes . It has The univariate Cox regression analysis for overall survival and disease-free survival demonstrated that the hazard ratio of patients with breast conserving surgery was lower than that of patients with standard radical mastectomy Table  &3. ThisIn conclusion, the present study presented the data of the long term survival of pathological stage I-III patients with breast cancers at our institution. For the 0- to 2-year interval, the hazard of recurrence was higher for the ER-negative patients than the ER-positive patients, and beyond 3 years the hazard was higher for ER-positive patients. Additionally, disease free survival 10 years after operation was reversed between ER-positive and negative patients. Therefore, the fact may indicate the importance of long term adjuvant hormone therapy for ER positive cancer patients.cTNM: clinical stage; ER: estrogen receptor; pTNM: pathological stage; pN: positive lymph nodes.The authors declare that they have no competing interests.TI and MF designed this study. MU, KY, HK, TI collected and assembled the data. TI organized the data. TK, TN, ST, TI and MF contributed to the statistical analyses and interpretations. TK, TI, MT and MF contributed to writing and finalizing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/8/323/prepub


The relationship of cholesterol homeostasis and carotid intima-media thickness (cIMT) is unknown. To address this, we assessed markers of cholesterol homeostasis  and cIMT in a middle-aged, statin-naive population.In this prospective study of primary prevention cIMT was measured by ultrasound in 583 hospital employees aged 25–60 years without prevalent cardiovascular disease or lipid-modifying medication. The serum concentrations of plant sterols (as markers of cholesterol absorption) were measured by gas-liquid chromatography. Lathosterol serum concentrations were quantitated to assess hepatic cholesterol synthesis.P<0.0005) and lathosterol-to-cholesterol . In contrast, plant sterols, as markers of cholesterol absorption, showed a weak negative correlation to cIMT measurements . Stratifying subjects by serum sterol levels, we found that cIMT increased continuously over quintiles of serum cholesterol (P<0.0005) and was positively associated to serum lathosterol-to-cholesterol levels (P = 0.007), on the other hand, plant sterol levels showed a weak negative association to cIMT (P<0.001 for campesterol-to-cholesterol).cIMT correlated positively with serum cholesterol (r = 0.22, In this population without prevalent cardiovascular diseases or lipid-modifying medication, markers of increased endogenous cholesterol synthesis correlated positively with cIMT, while markers of cholesterol absorption showed a weakly negative correlation. These data suggest that not only total serum cholesterol levels but also differences in cholesterol homeostasis are associated with cIMT. Plasma cholesterol is derived from enteral cholesterol absorption and hepatic cholesterol synthesis,Ultrasound of the carotid arteries allows clinicians to detect the development of atherosclerosis at an early stage. Measurements of carotid intima-media-thickness (cIMT), which is a marker of future cardiovascular events In the prospective I LIKE HOMe study , we evaluated cholesterol absorption and synthesis in relation to cIMT in 583 middle-aged hospital employees. Participants had no known cardiovascular disease (CVD), and were not taking lipid-modifying medication Healthcare workers at the Saarland University Hospital in Homburg/Saar, Germany, aged 25–60 years who were scheduled to undergo routine medical checkups were invited to have an ultrasonographic examination of their carotid arteries. Participants were excluded if they had prevalent CVD, diabetes mellitus, active tumor disease, inflammatory/autoimmune disease requiring systemic immunosuppressive treatment, stage 4 or stage 5 chronic kidney disease, were strict vegetarians or were on any lipid-modifying medications. Prevalent CVD was diagnosed in subjects who had a history of myocardial infarction, coronary artery angioplasty, stenting, bypass surgery, major stroke, carotid endarterectomy, non-traumatic lower extremity amputation, lower limb artery bypass surgery, or angioplasty. Subjects on antihypertensive medication, hormone replacement therapy, or oral contraceptives were allowed to enter the I LIKE HOMe study.A total of 583 participants met these inclusion criteria and were included in the analysis. Written informed consent was obtained from all study participants. The study protocol was approved by the Ethics Committee of the University of Saarland. A standardized questionnaire was used to record patient histories of smoking, diabetes, current drug intake, cardiovascular comorbidity, and premature onset of CVD in the family . Anthropomorphometric measurements and resting blood pressure were recorded. Body mass index (BMI) was calculated as the individual's body weight divided by their height squared. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate were measured after 5 min of rest. Mean blood pressure was calculated as DBP+ [(SBP-DBP)/3].Participants were categorized as active smokers if they were current smokers or had stopped smoking less than 1 month before entry into the study. Individuals who had self-reported diabetes mellitus, a non-fasting blood sugar level of >200 mg/dl, a fasting blood sugar level of >126 mg/dl, or were currently using hypoglycemic medication were categorized as diabetic and excluded from the study.Blood samples were taken from all subjects under standardized conditions. Plasma glucose, creatinine, total cholesterol, and high-density lipoprotein (HDL) cholesterol were obtained using standard techniques. Gas chromatography-flame ionization analysis was performed for quantification of cholesterol and mass spectrometry was performed for lathosterol and plant sterols as described previously The cIMT of the common carotid artery was measured from high-resolution, two-dimensional ultrasound images obtained using a linear-array 7.5 MHz transducer . With the subject in a supine position and the head slightly extended and turned in the opposite direction, longitudinal B-mode images of the distal common carotid artery and the carotid bulb were acquired and digitally stored for offline reading by a single blinded investigator. The cIMT was defined as the distance between the leading edge of the lumen interface and the media-adventitia interface of the far wall. Three representative measurements were calculated for the far wall of both common carotid arteries at predefined positions , and these six IMT readings were averaged to give the mean common cIMT. The cIMT was not measured at the site of a carotid plaque, which was defined as a focal thickening of the intima-media complex ≥1.1 mm irrespective of echogenicity.U test. Categorical variables are presented as the percentage of participants. Correlation coefficients were calculated by the Spearman test. A value of P<0.05 was considered statistically significant. Subjects were stratified into quintiles of serum cholesterol and non-cholesterol sterol levels. We recorded data on cIMT measurements, age, BMI, and lipid metabolism parameter trends across increasing quintiles of cholesterol, lathosterol, campesterol, and sitosterol using one-way analysis of variance (ANOVA) and partitioning the between-group sums of squares into trend components. Multivariable regression analyses were performed to further analyze markers of cholesterol metabolism in relation to cIMT. The Sobel test was used to assess whether the association between BMI and cIMT was partially mediated by lathosterol.Data management and statistical analyses were performed using SPSS 13.0. Unless otherwise indicated, continuous data are expressed as the mean ± standard deviation (SD) and compared by the Mann-Whitney The baseline characteristics of the 583 subjects enrolled in the study are shown in P<0.0005), age , mean blood pressure , plasma low density lipoprotein (LDL) cholesterol  and triglycerides . The plasma level of HDL cholesterol was negatively correlated to cIMT . As a consequence, we found a significant association between cIMT and Framingham risk score-based categories of risk for coronary heart disease (CHD)  .P<0.0005), the sitosterol-to-cholesterol ratio , campesterol , and the campesterol-to-cholesterol ratio  were negatively correlated with BMI, markers of endogenous cholesterol synthesis, lathosterol, and the lathosterol-to-cholesterol ratio, showed a positive correlation with BMI . Furthermore, blood pressure was positively correlated with lathosterol  and lathosterol-to-cholesterol ratio , but correlated only weakly, if at all, with cholesterol absorption . Age was weakly correlated with lathosterol  and lathosterol-to-cholesterol ratio , but not with sitosterol or campesterol. As a result, the Framingham risk score correlated with lathosterol  and the lathosterol-to-cholesterol ratio , but correlated only weakly, if at all, with campesterol  and the campesterol-to-cholesterol ratio  and sitosterol  and the sitosterol-to-cholesterol ratio .BMI was associated with non-cholesterol sterols and their ratios to cholesterol. While sitosterol . Stratifying subjects into quintiles based on serum cholesterol level, we found that cIMT measurements increased continuously over quintiles of serum cholesterol  with subjects in quintiles 5 and 4 having higher cIMT measurements than those in quintile 1  and lathosterol-to-cholesterol ratio  as a surrogate for endogenous cholesterol synthesis. The latter association was found in both men  and women . After stratification into quintiles by serum lathosterol levels, cIMT measurements increased continuously over quintiles of serum lathosterol  with significantly higher values in quintiles 5 and 4 than in quintile 1 . The absolute concentration of sitosterol did not correlate with cIMT ; the sitosterol-to-cholesterol ratio showed a weakly negative correlation with cIMT . Again, the association of cIMT measurements with the campesterol-to-cholesterol ratio and sitosterol-to-cholesterol ratio were found in both men and women (data not shown).In contrast to serum cholesterol and the cholesterol precursor lathosterol, serum levels of the plant sterol campesterol and the campesterol-to-cholesterol ratio showed a weakly negative correlation with cIMT  is associated with increased cardiovascular risk.