Application examples of these workflows carried out on selected cases of drug safety signals are discussed. The methodology and workflows presented offer a novel approach to explore the molecular mechanisms underlying adverse drug reactions.Drug safety issues pose serious health threats to the population and constitute a major cause of mortality worldwide. Due to the prominent implications to both public health and the pharmaceutical industry, it is of great importance to unravel the molecular mechanisms by which an adverse drug reaction can be potentially elicited. These mechanisms can be investigated by placing the pharmaco-epidemiologically detected adverse drug reaction in an information-rich context and by exploiting all currently available biomedical knowledge to substantiate it. We present a computational framework for the biological annotation of potential adverse drug reactions. First, the proposed framework investigates previous evidences on the drug-event association in the context of biomedical literature . Then, it seeks to provide a biological explanation  by exploring mechanistic connections that might explain why a drug produces a specific adverse reaction. The mechanistic connections include the activity of the drug, related compounds and drug metabolites on protein targets, the association of protein targets to clinical events, and the annotation of proteins  to biological pathways. Hence, the workflows for signal filtering and substantiation integrate modules for literature and database mining,  in silico experiments will facilitate research on the mechanisms that underlie ADR, contributing to the development of safer drugs.Adverse drug reactions (ADRs) constitute a major cause of morbidity and mortality worldwide. Due to the relevance of ADRs for both public health and pharmaceutical industry, it is important to develop efficient ways to monitor ADRs in the population. In addition, it is also essential to comprehend why a drug produces an adverse effect. To unravel the molecular mechanisms of ADRs, it is necessary to consider the ADR in the context of current biomedical knowledge that might explain it. Nowadays there are plenty of information sources that can be exploited in order to accomplish this goal. Nevertheless, the fragmentation of information and, more importantly, the diverse knowledge domains that need to be traversed, pose challenges to the task of exploring the molecular mechanisms of ADRs. We present a novel computational framework to aid in the collection and exploration of evidences that support the causal inference of ADRs detected by mining clinical records. This framework was implemented as publicly available tools integrating state-of-the-art bioinformatics methods for the analysis of drugs, targets, biological processes and clinical events. The availability of such tools for  Drug safety issues can arise during pre-clinical screening, clinical trials and, more importantly, after the drug is marketed and tested for the first time on the population In 1998, Lazarou et al estimated that yearly about 2 million patients in the US are affected by a serious adverse drug reactions (ADRs) resulting in approximately 100 000 fatalities, ranking ADRs between the fourth and sixth cause of death in the US, not far behind cancer and heart diseases Due to the important implications of an ADR in both public health and the pharmaceutical industry, unraveling the molecular mechanisms by which the ADR is elicited is of great relevance. Understanding the molecular mechanisms of ADRs can be achieved by placing the drug adverse reaction in the context of current biomedical knowledge that might explain it. Due to the huge amounts of data generated by the “omics” experiments, and the ever-increasing volume of data and knowledge stored in databases related with ADRs, the application of bioinformatics analysis tools is essential in order to study and analyze the molecular and biological basis of ADRs.Although the factors that determine the susceptibility to ADRs are not completely well understood, accumulating evidence over the years indicate an important role of genetic factors Other cases of ADRs may arise as a consequence of drug-drug interactions, or the interplay between the effect of the drug and environmental factors From the above paragraphs, it is clear that the study of the molecular and biological mechanisms underlying ADRs requires achieving a synthesis of information across multiple disciplines. In particular, it requires the integration of information from a variety of knowledge domains, ranging from the chemical to the biological up to the clinical. Different resources cover information about these different knowledge domains, and many of them are freely available on the web, such as biological and chemical databases and the biomedical literature. On the other side, new data is produced continuously, and the list of resources and published papers that a researcher interested in ADRs needs to cope with is turning more into a problem than into a solution. It has been recognized that the adequate management of knowledge is becoming a key factor for biomedical research, especially in the areas that require traversing different disciplines and/or the integration of diverse and heterogeneous pieces of information in silico experiments http://uima.apache.org/) or for combining data mining methods (http://www.knime.org/).On the other hand, approaching current biomedical research questions by computational analysis requires a combination of different methods. An attractive approach that emerged in the last years is the combination of different bioinformatics analysis modules by means of pipelines or workflows signal filtering, we search for previous reports of the potential signal in specialized databases and in the biomedical literature. In the process of signal substantiation, we seek to provide a plausible biological explanation to the potential signal. This framework was implemented by means of software modules accessible through web services and integrated into workflows ready to be used for automatic filtering and substantiation of drug-event associations. Finally, we present a detailed analysis of antipsychotic drugs and their association with the prolongation of the QT interval, as well as a large scale analysis of drug-side effect pairs from SIDER In this article we present a general framework developed in the context of the EU-ADR project for a systematic analysis of adverse drug reactions. The entry point of the system is a potential drug safety signal, which is composed of the drug and its associated adverse reaction. In the process of The here presented framework for the filtering and substantiation of drug safety signals consists of placing the potential signal in the context of current knowledge of biological mechanisms that might explain it. Essentially, we are searching for evidence that supports causal inference of the signal, i.e. feasible paths that connect the drug with the clinical event of the adverse reaction. The signal filtering analysis looks for evidence reporting the drug-event association in the biomedical literature and biomedical databases. The signal substantiation process considers two scenarios able to provide a causal inference of the signal see . First, signal filtering and signal substantiation were implemented using dedicated bioinformatics methods that are accessed through web services and integrated into processing pipelines by means of Taverna workflows. The substantiation workflow results can be visualized and analyzed by means of other bioinformatics tools such as Cytoscape http://dailymed.nlm.nih.gov/) and Medline®. A third Taverna workflow, (ADR-S), performs the signal substantiation process and was implemented by combining in silico target profiling, text mining and pathway analysis, among other bioinformatics approaches. More details about the implementation of web services and workflows can be found in the Our approaches for In the following section we describe the results of the analysis of potential drug safety signals as a proof of concept of the here proposed framework and tools.http://www.qtdrugs.org)   and 2 anugs.org)  indicateugs.org) . This dii in the 0.1 µM range, We furthermore explored the mechanisms underlying the association between QTPROL and the selected antipsychotics using the substantiation workflow. The results are summarized in Interestingly, our analysis also indicates that the antipsychotics in our study have an important activity on adrenergic receptors .i 0.2 µM, Moreover, haloperidol shows activity on the drug transporter encoded by the gene ABCB1 , which connect the drug and the event, and where the involved proteins are expressed in cardiac tissues. It is likely that the effect of a drug on its target proteins will affect proteins in their direct neighborhood in the biological pathway. Hence, we computed the average shortest path length between pairs of drug and event associated proteins in the Reactome pathways and compared them to the average shortest path length between randomly selected drug and event proteins. Interestingly, for all five antipsychotic drugs, the drug and event proteins are in close proximity in the Reactome pathways with average shortest path lengths between 2 and 3, which are significantly shorter than the average shortest path length of 5 of randomly selected drug and event proteins .In summary, the ADR-S workflow provides different hypotheses explaining the antipsychotics-induced QTPROL, including the direct action of the drug on proteins associated with the clinical event (e.g. HERG), the cross-talk between different biological processes , and the differential distribution of drugs among tissues (due to inhibition of transporters exerted by the drug). Moreover, it also highlights several interesting evidences that might explain the differences between low and high-risk antipsychotics.In addition to the example case presented above, the ADR-S workflow was evaluated on a large-scale data set. The SIDER database was used to extract drug-event pairs see . Here, aRecent studies highlight the use of disparate data sets in the study of ADRs, enabled by bioinformatics methodologies. Combining the study of protein–drug interactions on a structural proteome-wide scale with protein functional site similarity search, small molecule screening, and protein–ligand binding affinity profile analysis, Xie and colleagues vide infra). For a drug-event association, we collect information about the drug-targets by querying publicly available databases and by applying in silico drug-target profiling methods phenocopy, a term originally coined by Goldschmidt in 1935 All these examples illustrate how computational approaches are paving the way toward elucidating the molecular mechanisms of ADRs. The here presented framework follows this direction, by traversing and integrating information from the chemical domain, through genes and proteins, molecular and cellular networks, and finally to the clinical domain. The filtering workflows interrogate specialized databases and literature repositories in order to determine the novelty of a drug-event association. On the other hand, the substantiation framework seeks to find hypotheses that might explain drug-induced clinical events by looking for evidences supporting causative connections between the drug, its targets, and their direct or indirect  association to the clinical event. The signal substantiation process can be framed as a closed knowledge discovery process, analogous to the Swanson model based on hidden literature relationships We illustrate our approach by analyzing a clinically relevant drug safety signal: prolongation of the QT interval (QTPROL) leading to cardiac arrhythmias produced by a set of antipsychotic drugs. The results of the filtering workflows show that the association of QTPROL with the antipsychotic drugs has been extensively discussed in the literature and is documented in specialized databases. On the other hand, the substantiation workflow provides different hypotheses explaining the antipsychotics-induced QTPROL. First, we were able to confirm the widely accepted mechanism proposed for drug-induced QTPROL, in which the drug blocks the potassium channel HERG (encoded by the KCNH2 gene) and this blockade leads to a prolongation of the QT interval i 0.2 µM, We furthermore found that activities of haloperidol and pimozide on the drug transporter encoded by the gene ABCB1 , the cross-talk between different biological processes , and the differential distribution of drugs among tissues (due to inhibition of transporters exerted by the drug). Moreover, the analysis also highlights several interesting evidences that might explain the differences between low and high-risk antipsychotics. In addition, we provide the results of a large-scale analysis of drug-side effect pairs from SIDER and show that about 22% of the known side effects of drugs might involve direct effects of drugs on proteins being associated with the events. This relatively small number is not surprising because not all drug side effects can be attributed to the direct action of the drug onto its targets, such as on-target and off-target pharmacological effects. Other mechanisms of drug toxicity have been discussed. For example, metabolites can react with nucleophiles including DNA, which can trigger regulatory processes leading to inflammation, apoptosis and necrosis in silico experimentation, research on the mechanism that underlies drug-induced adverse reactions will be facilitated, which will have great impact in the development of safer drugs.Both filtering and substantiation workflows are available to the community and allow a systematic and automatic analysis of drug safety signals detected by mining clinical records, providing a user-friendly framework for the analysis of drug-event combinations. We believe that with the availability of such tools for http://bioinformatics.ua.pt/euadr/common_types.xsd) and the second one defines the specific types needed for signal filtering and substantiation in the context of the EU-ADR project (http://bioinformatics.ua.pt/euadr/euadr_types.xsd). Both schemas allow a smooth integration of the different modules in Taverna workflows, by enabling content and structure validation for the workflow input and output XML files. Moreover, the use of schemas facilitates further data transformations, for example, by applying XSL transformation to XML files of the signal substantiation workflow to create XGMML file graphs that can be visualized with Cytoscape. The workflows and web services are described in the following sections. All workflows have been implemented and tested using Taverna Workflow Management system version 2.2.The signal filtering and substantiation framework has been implemented by means of software modules that perform specific tasks of the processes. To allow access and integration of the modules in high-level analysis pipelines, the modules were implemented as web services and combined into data processing workflows to achieve the aforementioned signal filtering and signal substantiation. To standardize data exchanges between the different web services, we have developed two complementary schemas using XSD to define a common XML interoperability structure. The first one describes general data types  code of the drug at the 7 digits level (e.g. M01AH02 for rofecoxib) and the event represented by a string as defined in the EU-ADR project .The workflow returns an XML file and an HTML page summarizing the results, showing the PubMed identifiers of the retrieved citations grouped by publication type. A chart of the number of retrieved citations per year is generated using Google Charts Tools (get FilteredRelations (This workflow looks for associations between drugs and side effects that have been recorded in literature (Medline®) or in databases (DailyMed® and Drugbank). These resources have been indexed, and co-occurrences of drugs (corresponding to ATC codes) and side effects as defined in the EU-ADR project were captured and stored in a database. Briefly, all abstracts in the Medline database were split into sentences, and all sentences were indexed by the concept-recognition tool Peregrine elations , which pThe ADR-FD workflow accepts three inputs: the ATC code of the drug at the 7-digit level , the event as defined in the EU-ADR project , and theThe output of the workflow consists of a list of links to entries in the input data sources  in which the input drug-event association is mentioned. The output is generated in XML format and in HTML format.adrPathService). Two SOAP web services (cglService and adrPathService) allowing access to databases and bioinformatics modules relevant for the signal substantiation have been implemented (http://ibi.imim.es/ADR_Substantiation.html.The ADR substantiation (ADR-S) workflow seeks to establish a connection between the clinical event and the drug through (i) proteins targeted by the drug (or by its metabolites) and associated with the clinical event and (ii) biological pathways. In the first connecting path, the link between the drug and the event is established through the set of proteins in common between the Drug-Target-Profile and the Event-Protein-Profile . In the lemented . A tutorlemented  and at hThis method accepts as input a drug encoded by the ATC code at the 7-digits level and provides as output the chemical structure by means of SMILE (Simplified Molecular Input Line Entry Specification).in silico target profiling methods in silico target profiling. The evidences that support each drug-target relationship, such as the binding affinity of the compound to the protein or the source database, are provided.This method accepts as input a drug or metabolite encoded by a SMILE and returns a list of proteins that are related to the drug (Drug-Target-Profile). We use known drug-target associations  and exteThis method accepts as input a clinical event  and the event, which is defined by the three input ports event, eventName and eventType. We allow two different types of event definitions: events as defined in the EU-ADR project ; it is optional and only required for the visualization of the signal substantiation results , the protein profile of the drug or its metabolites (drugTargets), and the protein profile of the event (diseaseProteins) are provided. The signal substantiation workflow combines two ways of connecting drug and event, through proteins or through biological pathways. The outcome of these results is shown to the user during workflow execution by pop-up windows. The list of connecting proteins, that is, the protein annotated to both the drug and the event is provided (connectingProteins). For a user-friendly visualization and analysis of the results, a Cytoscape graph (CytoscapeResultGraph) is generated. The graph is composed of three types of nodes: drug, event, and proteins, and two types of edges: drug-protein, protein-event. The attributes of the edges contain supporting information for each association, such as source databases, association type, binding value for the drug, etc. . For the signal substantiation through pathways, the two protein profiles are subjected to the module getPathways, which returns a list of pathways to which at least one drug and one event protein that are expressed in the same tissue are annotated to. The output is further processed by module ConvertToHTML, which generates an HTML file listing the pathways that connect the drug and the event.A dataset of drug-side effects was downloaded from SIDER (December 2011) http://www.reactome.org/download/current/homo_sapiens.interactions.txt.gz, January 2012) to calculate the shortest path between any pair of antipsychotic drug and QTPROL associated proteins. For this purpose, we used the implementation of the Dijkstra algorithm in the Perl package Graph (http://search.cpan.org/~jhi/Graph-0.94/lib/Graph.pod). We then computed the average shortest path length for randomly chosen combinations of drug and event proteins and used a one-sided t-test to assess if the shortest path between the drug and event proteins as observed in our analysis was shorter than compared to random.We used the protein-protein interaction representation of the Reactome pathways Click here for additional data file.Dataset S2Results of the large-scale analysis of drug-side effects from SIDER using the module ADR-S through pathways.(TXT)Click here for additional data file.Protocol S1Tutorial for the ADR-S workflow.(PDF)Click here for additional data file.


We demonstrate the use of the NetSI family by measuring network stability on four datasets against alternative network reconstruction methods. First, the effect of sample size on stability of inferred networks is studied in a gold standard framework on yeast-like data from the Gene Net Weaver simulator. We also consider the impact of varying modularity on a set of structurally different networks , and then of complex feature covariance structure, showing the different behaviours of standard reconstruction methods based on Pearson correlation, Maximum Information Coefficient (MIC) and False Discovery Rate (FDR) strategy. Finally, we demonstrate a strong combined effect of different reconstruction methods and phenotype subgroups on a hepatocellular carcinoma miRNA microarray dataset (240 subjects), and we validate the analysis on a second dataset (166 subjects) with good reproducibility.The number of available algorithms to infer a biological network from a dataset of high-throughput measurements is overwhelming and keeps growing. However, evaluating their performance is unfeasible unless a ‘gold standard’ is available to measure how close the reconstructed network is to the ground truth. One measure of this is the stability of these predictions to data resampling approaches. We introduce NetSI, a family of Network Stability Indicators, to assess quantitatively the stability of a reconstructed network in terms of inference variability due to data subsampling. In order to evaluate network stability, the main NetSI methods use a global/local network metric in combination with a resampling  procedure. In addition, we provide two normalized variability scores over data resampling to measure edge weight stability and node degree stability, and then introduce a stability ranking for edges and nodes. A complete implementation of the NetSI indicators, including the Hamming-Ipsen-Mikhailov (HIM) network distance adopted in this paper is available with the R package  If a portion of data is randomly removed before inferring the network, the resulting graph is likely to be different from the one reconstructed from the whole dataset and, in general, different subsets of data would give rise to different networks. Thus, in the spirit of applying reproducibility principles to this field, one has to accept the compromise that the inferred/non inferred links are just an estimation, lying within a reasonable probability interval. Here we introduce the Network Stability Indicators (NetSI) family, a set of four indicators allowing the researcher to quantitatively evaluate the reproducibility of the reconstruction process. We propose to quantitatively assess, for a given ratio of removed data and for a given amount of  filter. Finally, we show the use of NetSI measures in a typical application, comparing the stability of relevance networks inferred on a miRNA microarray dataset with paired tissues extracted from a cohort of 241 hepatocellular carcinoma patients We first show the effect of network modularity and the dataset sample size on both the stability and the accuracy of the network inference process. For this purpose, we create two synthetic datasets with a known gold standard. The results are demonstrated for several inference algorithm, such as the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), developed for the reconstruction of gene regulatory networks nettools for the CRAN archives, as well as on GitHub at the address https://github.com/MPBA/nettools.git. For computing efficiency, the software can be used on multicore workstations and on high performance computing (HPC) clusters. Further technical details and preliminary experiments with nettools are available in All the methods (HIM distance and NetSI indicators) have been implemented in the open source R package Before defining the NetSI family we briefly summarize the main definitions and properties of the HIM network distance. Moreover, at the end of this section, we provide a short description of the network inference approaches used in the following experiments.i.e. they focus only on the portions of the network interested by the differences in the presence/absence of matching links. Spectral distances evaluate instead the global structure of the compared topologies, but they cannot distinguish isomorphic or isospectral graphs, which can correspond to quite different conditions within the biological context. Their combination into the HIM distance represents an effective solution to the quantitative evaluation of network differences.The HIM distance Let The definition of the Hamming distance is the following:To guarantee independence from the network dimension (number of nodes), we normalize the above function by the factor When Among spectral distances, we consider the Ipsen-Mikhailov distance IM which has been proven to be the most robust in a wide range of situations We summarize here the mathematical details of the IM definition i.e. the spectra of the associated graph, provide the natural vibrational frequencies where where so that The highest value of e unique  solutionwe can now define the normalized Ipsen-Mikahilov distance IM asso that Finally, the generalized HIM distance is defined by the one-parameter family of product metrics linearly combining with a factor e.g. on brain networks).Obviously, In what follows we will mostly deal with the case The HIM distance can be represented in the Full mathematical details about the HIM distance and its two components H and IM can be found in The mathematical and operational definition of the four NetSI indicators are introduced in We adopt As a first application, we test the difference in stability of the reconstruction process for a set of alternative network co-expression inference algorithms.i.e., the similarity is defined as a power of the absolute correlation , or (iii) the biweight midcorrelation (“bicor” for short) The most famous representative of the correlation-based approaches is surely the Weighted Gene Correlation Network Analysis (WGCNA) The Topological Overlap Measure (TOM) replaces the original co-expression similarity with a measure of interconnectedness (between pairs of nodes) based on shared neighbors The Context Likelihood of Relatedness (CLR) approach The Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE) is another approach relying on mutual information, which was originally developed for inferring regulatory networks of mammalian cells nettools package, based on their Bioconductor and CRAN implementations: minet for ARACNE and CLR, WGCNA for WGCNA, TOM and bicor, and minerva for MIC.A unique interface to all the mentioned algorithms is integrated in the stability analysis tools in the We demonstrate the invariance of the NetSI family with respect to network modularity in a controlled situation. We show that the proposed stability evaluation framework is not affected by various network structures for nine reconstruction algorithms. Moreover, we demonstrate that this property is maintained both if we adopt the HIM metric for the We created a set of networks The simulated gene expression values corresponding to the networks We inferred networks from the 10 datasets with nine algorithms: ARACNE, CLR, cor, TOM, bicor, WGCNA and MIC, where the last two were also used with a permutation-based FDR filter .The stability analysis with three possible network metrics  on networks inferred with the nine mentioned approaches is reported in We investigated the behavior of the NetSI stability indicators for different sample sizes on a yeast-like dataset, again simulated by GNW.InSilicoSize100-Yeast2 dataset with 100 nodes, originally a DREAM3 benchmark, generating 100 samples with default parameter configuration, including noise level, for wild-type steady state  control.We aim to assess differences in the stability of correlation networks inferred by an For an introduction to FDR methods, see for instance In this example, the correlation networks are inferred from a dataset where Starting from the dataset The three networks displayed in Note that although both WGCNA and CORFDR.The  network  C does nSimilar considerations hold for the ranking of the most stable nodes: for WGCNA, the top-ranked nodes are the The nodes The weight and degree stability analysis for the other subsampling cases  and how they evolve in cancer is a key issue for researchers in biology http://www.ncbi.nlm.nih.gov/geo with accession number GSE6857. The dataset collects 482 tissue samples from 241 patients affected by HCC. For each patient, a sample from cancerous hepatic tissue and a sample from surrounding non-cancerous hepatic tissue are available, hybridized on the Ohio State University CCC MicroRNA Microarray Version 2.0 platform consisting of 11,520 probes measuring the expression of 250 non-redundant human and 200 mouse miRNAs. After imputation of missing values The HCC dataset (HCC-B) For validation, we considered a second dataset (HCC-W) recently used to derive a signature of 30 miRNAs for hepatocellular carcinoma Using the CLR algorithm we first generated the four networks inferred from the whole set of datasets and corresponding to the combinations of the two binary phenotypes, discarding links with weight smaller than 0.1. Two different representations of the resulting graphs are shown in In order to explore network reconstruction reliability, we then computed the NetSI indicators and compared the results for CLR with other six reconstruction algorithms ARACNE, cor, TOM, bicor, WGCNA and MIC. The corresponding statistics for the four subsets and different subsampling  are associated with cancer Link (g) connects hsa-mir-092.prec.13.092.1No2 and hsa-mir-092.prec.X.092.2: it is the most stable for FT. The two miRNAs are to known to be associated with chronic lymphocitic leukemia Finally, we compared a disease network inferred from HCC-B Within the first 10 top ranked links for We introduced the NetSI family of indicators for assessing the variability of network reconstruction algorithms as functions of a data subsampling procedure. Our aim here is to provide the researchers with an effective tool to compare either the inference algorithms or properties of the investigated dataset. The first two indicators are global, giving a confidence measure over the whole inferred dataset and are based on a measure of distance between networks. In particular, we demonstrated the proposed framework with the HIM distance, although it is independent from the chosen metric. The other two indicators are local, associating a reliability score to the network nodes and the detected links. They are of particular interest when coupled with algorithms of proven performance, being able to capture the effect of data perturbation on the reconstruction process. We demonstrated their consistency on two synthetic datasets, testing their dependency on different inference methods, and also in comparison with the gold standards. Finally we showed an application for disease networks on miRNA hepatocellular carcinoma data; we found biological or technical evidence for the most stable links in the networks, and good reproducibility on a second dataset having relevant differences in terms of microrray technology and annotation platform.


The responsiveness of the human central nervous system can change profoundly with exercise, injury, disuse, or disease. Changes occur at both cortical and spinal levels but in most cases excitability of the motoneuron pool must be assessed to localize accurately the site of adaptation. Hence, it is critical to understand, and employ correctly, the methods to test motoneuron excitability in humans. Several techniques exist and each has its advantages and disadvantages. This review examines the most common techniques that use evoked compound muscle action potentials to test the excitability of the motoneuron pool and describes the merits and limitations of each. The techniques discussed are the H-reflex, F-wave, tendon jerk, V-wave, cervicomedullary motor evoked potential (CMEP), and motor evoked potential (MEP). A number of limitations with these techniques are presented. The motoneuron was described as the “final common path” of the nervous system , do more or fewer motoneurons generate action potentials after the intervention? A bigger or smaller output would represent a net increase or decrease in motoneuron excitability, respectively. The change in excitability will reflect the balance of inhibition and facilitation but it is difficult to determine the mechanism in a given situation; e.g., a decrease in excitability could be due to either an increase in inhibition or a decrease in facilitation. Further, any change in “excitability” may not apply uniformly across the whole motoneuron pool.Regardless of the methodology or the intervention studied , the goal of testing human motoneuron excitability is the same: to know more about the status of the motoneuron pool. The aim of this review is to discuss briefly the existing methodologies which test motoneuron excitability by evoking compound muscle action potentials with particular focus on the benefits and limitations of each. Discussion of the H-reflex, F-wave, tendon jerk, V-wave, cervicomedullary motor evoked potential (CMEP) and motor evoked potential (MEP) is included below and summarized in Table Hmax. Comparison of H-reflex size to the maximal M-wave enables an estimate of the motoneuron pool involved in the H-reflex. For the soleus muscle in most subjects the percentage of involved motoneurons is ~50% . However, this list of muscles expands greatly if stimulation is given while the subject performs a weak voluntary contraction. This ensures some motoneurons are discharging repetitively, brings others closer to threshold and thus increases motoneuron excitability. Burke and colleagues  identifiThus, far, this section has described homonymous connections, that is from the stimulated Ia afferents to the motoneuron pool of the same muscle. However, there are also heteronymous links between Ia afferents of one nerve to the motoneuron pools of muscles supplied by a different nerve. For example, stimulation of the femoral nerve delivers monosynaptic Ia excitation not only to the quadriceps motoneurons but also to the soleus motoneuron pool  and the consequent difficulty in interpreting a change to the response raises questions about its usefulness as an independent measure of motoneuron excitability. During a maximal voluntary contraction, the size of the V-wave is proposed to indicate the level of descending voluntary drive conveyed by the motoneurons  will be activated by the stimulus, but there is strong evidence that the CMEP is primarily the result of motoneuron activation by a single descending volley elicited by excitation of corticospinal axons  if a given stimulus is delivered during a weak voluntary contraction.Corticospinal (transmastoid) stimulation has recently been paired with transcranial magnetic stimulation (TMS) as a novel means to test motoneuron responsiveness during ongoing muscle activity and fatigue without the confounding influence of unknown levels of descending voluntary drive , is the same change evident when other methods  are applied? We recently compared the effects of fatigue on CMEPs (TMEPs) and F-waves (Giesebrecht et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A set of mediation analyses were carried out in this study using data from It's Your Game. . .Keep It Real (IYG), a successful HIV/STI/pregnancy prevention program. The IYG study evaluated a skill and normbased. HIV/STI/pregnancy prevention program that was implemented from 2004 to 2007 among 907 urban low-income middle school youth in Houston, TX, USA. Analyses were carried out to investigate the degree to which a set of proposed psychosocial measures of behavioral knowledge, perceived self-efficacy, behavioral, and normative beliefs, and perceived risky situations, all targeted by the intervention, mediated the intervention's effectiveness in reducing initiation of sex. The mediation process was assessed by examining the significance and size of the estimated effects from the mediating pathways. The findings from this study provide evidence that the majority of the psychosocial mediators targeted by the IYG intervention are indeed related to the desired behavior and provide evidence that the conceptual theory underlying the targeted psychosocial mediators in the intervention is appropriate. Two of the psychosocial mediators significantly mediated the intervention effect, knowledge of STI signs and symptoms and refusal self-efficacy. This study suggests that the underlying causal mechanisms of action of these interventions are complex and warrant further analyses. The majority of teen HIV/STI/pregnancy prevention programs are theory-based, targeting psychosocial variables to produce changes in sexual risk-taking behaviors. Guided by established psychosocial theories such as social cognitive theory  and sociAdolescents are engaging in sexual risk-taking behaviors at an earlier age, often before they are developmentally ready to deal with potential outcomes. National data illustrate the need to implement intervention programs as early as possible: 11% of 6th graders, 15% of 7th graders, 18% of 8th graders, and 33% of 9th graders have reported lifetime sexual activity . This raIYG was developed using a systematic design process, Intervention Mapping , and wasX and a hypothesized mediator variable M, and (2) the strength of the relationship between the mediator variable M and the dependent variable Y, adjusting for the effects of X; combining these estimates provides a measure of the strength of the mediated or indirect effect of X on Y through mediator variable M. A key assumption of mediation hypotheses is that there is a causal chain of events in which X occurs and produces a subsequent change in M, which in turn causes a subsequent change in Y [Y caused by X indirectly transmitting change through M, that is, the indirect or mediated effect. This indirect effect may be whole or partial, accounting for a proportion of the total effect of X on Y. A mediation analysis involves performing a set of regression analyses to obtain estimates of (1) the strength of the relationship between an independent variable nge in Y , 14. Of n = 907, 588 control, 349 treatment) was followed over a period of two years. Over the study period students were surveyed at baseline, 6, 18, and 24 months using laptop computers via an audio-computer assisted self-interview (ACASI) [The It's Your Game (IYG) study evaluated a skill and norm-based HIV, STI, and pregnancy prevention program that was implemented from 2004 to 2007 among urban low-income middle school youth in Houston, TX, USA. Ten predominately African American and Hispanic middle schools were randomly assigned to either the intervention or comparison condition (five schools to each) using a multiattribute randomization protocol, taking into account the size and racial/ethnic composition of the student body (African American and Hispanic) and the geographic location of the school . Partici (ACASI) . Upon coY) in the mediation models. The primary behavioral outcome for IYG was delay of sexual initiation for those students who reported no lifetime sexual activity at baseline. This was used as the primary behavior . These mediators were included because condom skills training is a recommended element of sexual health education at the middle school level [The analyses include some constructs that may not initially be expected to be associated with delayed initiation  tailored by gender and sexual experience, and “real world”-style teen serials with on-line student feedback. IYG also included six homeworks to facilitate parent-child communication.Specific topics covered in the IYG intervention included characteristics of healthy friendships, setting personal limits and practicing refusal skills in a general context , information about puberty, reproduction, and STIs, and setting personal limits and practicing refusal skills related to sexual behavior, characteristics of healthy dating relationships, the importance of HIV, STI, and pregnancy testing if a person is sexually active, and skills training regarding condom and contraceptive use .X on Y through M. For the present study, X is the intervention indicator variable, taking a value of 0 for those students in the control arm and a value of 1 for those in the intervention arm of the IYG study. The outcome of interest is represented by the variable Y , measured at the 24-month followup. The hypothesized psychosocial mediators are represented by the variable M  and were also measured at the 24-month followup:c is the total effect of X on Y, and this can be decomposed into a direct effect, c′, and an indirect effect, ab, which is carried through the mediator M. The e terms represent the random error component of each model. In the case of single-level ordinary least squares (OLS) regression with an underlying continuous outcome variable, ab is equal to c − c′, which is the difference between the total effect and the direct effect of X on Y [ab does not equal c − c′, although the difference is generally not large, especially for large samples [ab and c − c′ and allows for estimation of standard errors of the mediated effect. In this study ab rather than c − c′ was used to estimate mediated (indirect) effects [f X on Y . For mul samples . In logi effects . a and b effect estimates using the Wald tests and by examining the significance and size of the ab effect estimate using the Sobel test [a and b effects is not necessary steps prior to conducting a test of whether ab is significant and therefore whether a variable is a significant mediator [how an intervention X may produce change in an outcome Y [b and not a suggests either that the mediator being targeted is correct but that the intervention needs to be strengthened in its strategies for influencing the mediator or that the mediator needs to be measured more accurately. Significance of a but not b suggests that although the intervention is successful in influencing the targeted mediator, the psychosocial theory dictating which mediator to target needs some refinement [a and b individually as well as the significance of the mediated effect ab. The mediation process was assessed by examining the significance and size of the bel test , 18, 19.utcome Y . Equatiofinement . Therefoab is divided by its Sobel SE and compared to a critical value from a standard normal distribution and was used to assess mediation effects in the present study [One of the most common methods for estimating the standard error (SE) of a mediated effect is to use the formula first derived by Sobel based on the multivariate delta method , 18, 19:dardized . This ten = 817), in order to look at the main behavioral outcome of initiation of sexual intercourse in 9th grade. The psychosocial mediators were also analyzed on this same subset in order to carry out the mediational analyses. The IYG study design consisted of students nested within schools. In keeping with the analysis of the original study, multilevel regression models were used to estimate the a, b, and c′ effects in order to adjust the standard errors for the presence of any intra-class correlation among students attending the same school. Models were adjusted for age, gender, race/ethnicity, and the baseline measure of the psychosocial mediator. The main outcome, initiation of sexual intercourse, was analyzed using a multilevel logistic regression model; therefore all estimates reported have been standardized in order to carry out the Sobel test [The sample was restricted to those students reporting no sexual experience at the 7th-grade baseline measure . While not statistically significant at the 0.05 level, perceived friend's behavior (P = 0.06), condom knowledge (P = 0.09), and HIV/STI knowledge (P = 0.07) were also most likely influenced by the intervention. The b path provides an estimate of the relationship of the proposed psychosocial mediator to the behavior outcome (initiation of sex). Of the 13 psychosocial mediators under investigation, all but 2 (perceived norms about condoms and HIV/STI knowledge) were significantly related to the behavior (initiation of sex).The c′, estimates how much of this significant intervention effect was direct and not transmitted through any of the measured psychosocial mediators. This direct effect would indicate total mediation if it was estimated at or near zero when the psychosocial mediator was introduced into the model. However, the direct intervention effect remains significant for the mediator sexual beliefs (P = 0.048), but for the other twelve outcomes the intervention effect is no longer significant suggesting some degree of partial mediation. The ab path provides an estimate of the indirect effect or measure of the amount of mediation. While the proportion of intervention effect mediated by the measured psychosocial variables  ranged from <1% to 14.1%, only refusal self-efficacy (P = 0.03) and STI signs and symptoms (P = 0.001) were significant mediators of the intervention effect according to the Sobel test. An increase in the refusal self-efficacy scale was associated with a reduction in the likelihood of initiation of sex. However, an increase in knowledge of STI signs and symptoms was associated with an increase in initiation. These two mediators were estimated to account for 6.7 and 3.6 percent of the total intervention effect, respectively.The IYG study found a significant difference in the proportion of students initiating sex when compared to the control condition . By the b effects, indicate that eleven of the thirteen mediators are related to initiation of sex. This provides the strongest evidence that the mediators currently being targeted by the intervention do have the potential to influence the outcome behaviors. Only two of the psychosocial mediators, HIV/STI knowledge and perceived norms about condoms, were not significantly related to the behavior.Glassman et al. (in submission)found similar b path results in their mediation analysis of a similar theory-based HIV/STI pregnancy prevention program, further supporting the theoretical underpinnings of these types of interventions.The findings from this study provide evidence that the majority of the psychosocial mediators targeted by the IYG intervention are indeed related to the desired behavior and provide evidence that the conceptual theory underlying the targeted psychosocial mediators in the intervention is appropriate. The relationships between the psychosocial mediators and the behavioral outcome, the b effect was in the anticipated direction for most of the mediators, two, STI signs and symptoms knowledge and condom knowledge, had an unexpected result. The results suggest that, as knowledge increases, a student has a higher likelihood of initiation of sex. One possible explanation for this inverse result is a reversal of the causal pathway, meaning that students who already are sexually active may have more knowledge about condoms and STIs. While the theoretical model assumes that the change in the mediator precedes the behavioral change, the timing of the measures in this particular study did not allow us to confirm this temporal relationship. Further analyses using a time lag should be carried out to explore this result. While the a effect, which estimates the intervention's impact on the psychosocial mediators, indicates that the intervention significantly impacted three of the thirteen psychosocial variables . While largely consistent, it should be noted that these results of the intervention's impact on the psychosocial mediators differ somewhat from those of the main outcome paper. This is most likely due to the fact that the analyses for this study were carried out on only a subset  of the study cohort and thus have less statistical power. For a detailed description of the intervention's impact on the psychosocial outcomes for the entire cohort, see the main outcome paper [The me paper . c′, was in the expected direction. All c′ effects indicate, there is still a reduction in sexual debut in the intervention arm relative to the control condition after adjusting for each psychosocial mediator. However, only two of the psychosocial mediators significantly mediated the intervention effect, Knowledge of STI signs and symptoms, and refusal self-efficacy. This demonstrates the importance of refusal self-efficacy skills training for this age group to delay sexual initiation. Multiple behavioral science theories  propose that self-efficacy is an important construct leading to behavior change. Thus, including activities that enhance students' self-efficacy to refuse to engage in unhealthy behaviors  is a key strategy in many effective sexual education programs. Many middle school youth find it hard to use effective refusal skills , so they benefit from activities like role plays that let them practice these skills in a safe environment and to get feedback from the instructor and peers.The program's impact on the behavior adjusting for each mediator,  Neither of the two significant mediators explained a high proportion of the intervention effect. This, combined with the lack of significance of mediating effects for the majority of psychosocial variables examined, indicates that the intervention's impact on behavior is likely influenced by mediators not measured in this study.Glassman et al. (submission pending)found similar results. The only other study found to investigate mediators of a school-based HIV/STI prevention program was conducted for middle school youth in Tanzania; they also identified only one significant mediator (peer norms regarding having sex), accounting for 16% of the total intervention effect stigler . As thisThis mediation analysis is only the first step to understanding all aspects of the complex process of behavior change resulting from a theory-based prevention intervention such as IYG . These sResults of this study should not be viewed in isolation, rather taken as a piece of a larger puzzle to aid in the overall understanding of how to tailor and augment current HIV/STI pregnancy prevention programs that have already proven to be successful. Through targeting and refining these interventions for specific populations, the end goal is to achieve the largest, cost-effective risk reduction that is feasible within the confines of a school-based curriculum. This study is not without limitations. Outcome measures were self-report, and it is unknown the ability to generalize beyond the study population. Additionally, some of the mediators had low internal consistency. More work needs to be done in the area of measurement around these potential mediators. Authors in  also notThis study did not conduct mediation analyses across subgroup populations . These population characteristics were controlled for in the overall analyses; however, separate models carried out within each of these groups could provide useful and cost-effective information when tailoring an intervention to a specific population. Although costly, a much larger-scale study needs to be conducted to both improve the ability to generalize as well as understand the mediating pathways and how they differ across subpopulations. More accurate methods for testing mediation and building confidence intervals (CIs) can be obtained using the actual distribution of the product or bootstrap methods , 25; howResults of this study suggest specific areas for deepening the focus of these types of HIV/STI/pregnancy prevention interventions for this type of middle school population. In addition, the relatively small proportion of intervention effect mediated by each of the psychosocial measures is suggestive that additional mediators not measured in this study may also be driving the intervention's effect on behavior change. Augmenting existing theories may help bolster intervention effects. Additional mediation analyses are needed that examine different aspects of the underlying mechanisms of interventions for different populations. Different stories are beginning to emerge about mediators for similar theory-based interventions when implemented in different populations . As the body of literature in this field begins to grow, interventionists will be better equipped to begin to understand these complex processes and how to tailor intervention curriculum through targeted instruction for different populations to gain maximum risk reduction.


The two underlying subspaces are orthogonal by construction and their direct sum is the space of full motions. Using Functional Mode Analysis, we were able to identify a collective coordinate within the tertiary-only subspace that is correlated to the most dominant motion within the quaternary-only motions, hence providing direct insight into the allosteric coupling mechanism between tertiary and quaternary conformation changes. This coupling-motion is substantially different from tertiary structure changes between the crystallographic structures of the T- and R-state. We found that hemoglobin's allosteric mechanism of communication between subunits is equally based on hydrogen bonds and steric interactions. In addition, we were able to affect the T-to-R transition rates by choosing different histidine protonation states, thereby providing a possible atomistic explanation for the Bohr effect.Hemoglobin is the prototypic allosteric protein. Still, its molecular allosteric mechanism is not fully understood. To elucidate the mechanism of cooperativity on an atomistic level, we developed a novel computational technique to analyse the coupling of tertiary and quaternary motions. From Molecular Dynamics simulations showing spontaneous quaternary transitions, we separated the transition trajectories into two orthogonal sets of motions: one consisting of intra-chain motions only (referred to as  Hemoglobin transports oxygen from our lungs to other tissues. Its effectiveness in binding and unbinding of oxygen is based on a type of regulation called allostery. Thereby, already bound oxygen molecules in either of the four binding sites of hemoglobin increase the likelihood of oxygen binding in the other sites; the sites act cooperatively. It is known that the four protein chains of hemoglobin – each containing one oxygen binding site – need to rearrange globally or the cooperativity is lost. But how do the individual chains communicate with each other in this rearrangement? This is still not resolved on the molecular level. By applying the computational technique of molecular dynamics simulations we were able to simulate the motions of the individual atoms of hemoglobin during such a global rearrangement. We present a novel method that allows to directly analyse the coupling of motions within and between the four protein chains and thereby reveal the allosteric mechanism. That allows us to classify which amino acids are important for the cooperativity and in what way: by either pushing other amino acids away or attracting them. Also, we have observed in simulations how pH differences could affect the cooperativity. Hemoglobin (Hb) consists of two Today, many high-resolution structures are available, including oxy states, deoxy states, carbon monoxide bound structures and structures of a large number of point mutations In addition to experiments, Molecular Dynamics (MD) simulations have provided insight for shorter timescales from Recently, Hub and co-workers observed for the first time spontaneous reproducible transitions from the T- to the R-state during MD simulations In the present study we investigated how the local intra-chain motions couple to the global inter-chain motions on a molecular level. To this end, we enhanced the statistical basis to 21 transition trajectories, and developed a method which allows to characterize the coupling between global and local motions. For this purpose, we first separated global from local motions and then identified the coupling mechanism between them. We analysed the resulting coupling collective coordinate on the level of molecular contacts, shedding light on the molecular allosteric mechanism of hemoglobin. In addition, by using a different set of histidine protonations to mimick simulations at different pH, we were able to show a reduction in the number of transition trajectories. This constitutes a possible explanation for the Bohr effect in Hb From the Hb simulations carried out by Hub and co-workers Starting from our T-to-R transition trajectories, to analyse the interplay of local and global motions, we separated local from global motions as the first step. Here, the MD transition trajectories were decomposed into two trajectories: quaternary-only (Q) and tertiary-only (T). The first consists of inter-chain motions with the Hb chains translating and rotating as rigid bodies and the second contains intra-chain motions, omitting the global movements. The combination of the Q and T trajectories yields the full MD trajectories. For a visual explanation of the basic idea of the decomposition see We simplified the Q trajectory by only considering the first eigenvector of a Principal Component Analysis (PCA) as the most dominant motion (referred to as cQ). For obtaining a collective coordinate within T that maximally correlates to cQ, we applied Functional Mode Analysis ((FMA),  see . This meWithin the FMA framework it can be desirable to reweigh the individual latent vectors with their contribution to the overall variance see : While tTo elucidate the mechanism of coupling between T and Q, we focused on the hyperplane spanned by T and Q. In this plane we chose a specific path starting from the T-state and moving first parallel to cQ and then parallel to cTew. The path is marked in white in In order for the information of a local conformation to flow from one protein chain to another, it has to cross the corresponding interface. It is therefore of interest to investigate interactions at the subunit interfaces. We focused on inter-chain van der Waals overlaps and general distance based contacts to investigate the interactions underlying the allosteric coupling mechanism.To estimate the influence of interatomic repulsion due to steric interactions as a driving force for the allosteric coupling, we calculated the overlap of atomic van der Waals (vdW) spheres. We did this for structures in the plane spanned by cQ and cTew, and took into account only inter-chain overlaps.  The higher the overlap in a specific structure, the more energetically unfavourable it is. As can be seen in For a broader view including also attractive interactions, we monitoredinter-chain atom pairs showing a distance smaller than 3 nm. This analysis was carried out along cQ-cTew which allows us to classify the contacts according to when the contact is or is not formed along this specific path. For specific interaction types following contact patterns are expected. The two residues in contact are either:pulling at each other, breaking the contact when in the off-diagonal intermediate artificial states , scalar products between the coordinates are summarized in The correlation of The model that we used to describe the coupling of quaternary and tertiary motions is of linear nature. On the one hand, since there is no necessity for a linear coupling, non-linear models could be more suitable. On the other hand, the fact that we found a linear model with an substantial correlation in the cross-validation makes us confident that already a major part of the coupling can be described linearly. We investigated only instantaneous cQ to T coupling, although in reality there might be a lag-time due to the time required for the signal to pass. Our results, however, show that the coupling can already be identified from an analysis of instantaneous correlations. Future work may include non-linear models as well as delayed responses for the coupling, especially for systems in which the order of events is known.An analysis of the identified coupling mode focused on the protein chain interfaces revealed key interaction residues. We suggest that the allosteric coupling between local and global motions in Hb consists of an interplay of repulsive and attractive interactions at the subunit interfaces on a similar scale.The interaction picture we derived from our vdW overlap analysis suggests that vdW overlaps are a global driving force for the allosteric coupling. The fact that MD trajectories projected onto the plane spanned by cQ and cTew coincide with the region around the diagonal in Nevertheless, efforts to break down the global interactions to individual repulsive contact pairs did not yield a conclusive picture. This suggests that the vdW overlap at Hb's inter-chain interfaces does not act on a residue level, but on a broader, collective scale.The contact analysis along cQ-cTew allowed us to classify contacts according to their behaviour along this path. By picking this path in the plane spanned by cQ and cTew, we ensure that the observed contacts are important for the coupling. If any contact pair did not play a role in the allosteric coupling, it would not have been part of the coupling of global and local motions. For a number of contacts we observed also the symmetry-related residue-pairs , which further indicates the significance of these contact pairs. The similar number of pulling and pushing interface interactions suggests that both types contribute to the allosteric coupling on a similar scale. The fact that steric repulsions and hydrogen bonds could not be unambiguously traced down to a residue level individually, but could in the generalized contact analysis, points at an interplay of repulsive and attractive interactions.In our analysis the two residues Further, In this study, we assigned different roles in the coupling mechanism  to individual amino acids. Mutagenic studies of these amino acids provide a direct means to validate the predicted role of individual amino acids in the allosteric mechanism. The observed contacts which are caused by van der Waals overlap may be reduced by mutating to residues with smaller side chain sizes. In the case of charged side chains introducing an additional charge of the same sign may increase the repulsion. Contacts including hydrogen bonds can be suppressed by using unfavourable mutations.We suggest mutations affecting the “hinge” and “switch” contacts The repulsive interaction of the two Further, we suggest a mutation of the In our simulations, we were able to lower the T-to-R transition probability by mimicking a pH shift, thereby providing a molecular picture of the Bohr effect. Future mutational studies may verify our predicted interactions and consolidate the molecular interaction mechanism of hemoglobin's allosteric coupling.The measured histidine protonations in the T-state by Kovalevsky et al. Even though our simulations are not long enough to have reached equilibrium, our observations can be taken as a proof of concept of the electrostatic interactions stabilizing the T-state and thereby underlying the pH-driven Bohr effect. During the T-to-R transition residues at the ions see , are locTo estimate if our choice of force-field influences the transition process, we carried out further simulations using the CHARMM27 force-field The starting structure for all simulations was the Hb T-state X-ray structure  coordinates, we superimposed the coordinates of each chain individually onto the respective chain in the T-state X-ray structure. This yielded an artificial trajectory with all chains having the same center-of-mass and orientation as in the T-state, not moving relative to each other and displaying only subunit-internal fluctuations. All four subunits were superimposed individually, reducing the degrees of freedom (DOF) of each by six  and resulting in 3N-24 DOF for the T trajectory (with N = 4556 being the number of non-hydrogen atoms).The complementary quaternary-only (Q) coordinates were obtained by doing the opposite and superimposing the individual chains of the rigid T-state structure onto the respective chains of the MD structures, thereby leaving out the internal motions and only keeping the rigid body motions of the chains relative to each other. Thus each chain was represented by a rigid body (six DOF) and lost all information on internal coordinate changes. Since the six global DOF for the whole protein were removed, the Q motions sample 18 DOF. For the coupling analysis, we simplified the Q motions further using PCA. The eigenvalue spectrum shows a steep decrease among the 18 non-zero eigenvalues with the first eigenvector describing 22% of the total variance of the Q motions. This principal mode (referred to as cQ) was used for FMA.Functional Mode Analysis (FMA) identifies collective motions in a coordinate space (in our case T) maximally correlated to a specific functional property (in our case cQ). To avoid overfitting the high dimensionality of the T space needs to be reduced. The original implementation of FMA For both analyses, we reassembled the coupled tertiary and quaternary motions in the following way: The MD trajectories were projected onto the plane spanned by cQ and cTew. The space between the minimal and maximal projections was divided equidistantly into 20 parts, yielding Note that one would expect a vdW overlap of zero for any full-dimensional MD structure. The fact that we did observe non-zero overlap values arises from the reduced dimensionality of the coupling plane: The vdW overlaps have been calculated for structures in the cQ-cTew hyperplane, which is a two-dimensional representation of the coupling process. Any such projection cannot fully cover the full-dimensional motion (and overlap) of atoms and therefore only shows the global trends. The fact that the maximal correlated coordinate from T (cTew) is reducing the vdW overlap when moving along cQ at the same time, points at (the prevention of) vdW overlaps as a global driving force for the coupling mechanism.For each of these 400 structure the inter-chain vdW overlap was computed using a modified version of the dist program from the CONCOORD software


The human visual system is highly sensitive to biological motion and manages to organize even a highly reduced point-light stimulus into a vivid percept of human action. The current study investigated to what extent the origin of this saliency of point-light displays is related to its intrinsic Gestalt qualities. In particular, we studied whether biological motion perception is facilitated when the elements can be grouped according to good continuation and similarity as Gestalt principles of perceptual organization. We found that both grouping principles enhanced biological motion perception but their effects differed when stimuli were inverted. These results provide evidence that Gestalt principles of good continuity and similarity also apply to more complex and dynamic meaningful stimuli. Human vision has highly efficient mechanisms to recognize actions of others rapidly and without apparent effort, even when the stimulus is defined solely by a few moving light points Recent studies that have attempted to identify key features of the point-light stimulus largely focused on the importance of local motion signals for the perception of biological motion. For instance, Casile and Giese Beside the rather strong evidence that motion is important, the relative contribution of shape information has also been emphasized. Several studies found evidence for the accurate processing of biological motion stimuli in which local motion was removed by jittering the individual dots' locations from frame to frame along the limbs While the controversy about key features and their relative contribution to biological motion perception still remains unresolved, the apparent contradiction might at least partly be reconciled: Inconsistent findings often originate in the nature of the tasks that are used and the availability of cues that are required for optimal performance in a particular task For both, representations based on dynamic and representations based on static information, the visual system manages to group the elements into a meaningful whole: How are the disparate dots making up a point-light stimulus connected? This recovery of the connectivity structure of a point-light action is a form of grouping. Grouping and perceptual organization were central on the research agenda of Gestalt psychology. Indeed, Gestalt psychologists described a number of laws in visual perception, defining what is necessary to group a number of elements into one object. A stimulus object that can be organized according to these rules represents a ‘good Gestalt’ and hence the object will be salient and easily recognized. The question arises to what extent well documented Gestalt laws of perceptual grouping facilitate the recovery of the connectivity structure during the perception of biological motion. To investigate this issue, we implemented two well-known Gestalt principles, that of good continuation and similarity, by presenting a human walker by means of oriented Gabor patches instead of non-oriented dots.Wertheimer Collinearity is probably the strongest grouping cue in the context of biological motion perception, since the local orientations of the elements are consistent with the underlying shape to which the elements belong. The alignment of the elements might thus reinforce the shape signals and enhance the perception of a human figure.In addition, similar elements also tend to group together, regardless of the origin of their resemblance, the motion pattern, luminance, or orientation in space. In our study, we introduced similarity into the point-light stimulus by orienting the Gabor patches making up the walker in the same direction (similarity by isolinearity). This similarity could support the segmentation of elements from the display and hence facilitate their interpretation. Isolinearity is most likely a less strong grouping principle in this context than collinearity. Although all elements have the same orientation, their orientation does not reveal the underlying structure at a local level, and therefore we might expect lower rates of recognition. By employing these grouping cues, we can investigate how much they might facilitate the recovery of the connectivity structure of a point-light figure.An additional aim of the present study is to further investigate the configural nature of biological motion processing and the possible role of grouping cues in configural processing. Tadin, Lappin, Blake, & Grossman Fifteen participants  from the undergraduate psychology program at the University of Leuven conducted the experiment for course credit. They all had normal or corrected-to-normal vision. The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Psychology, University of Leuven. All participants gave their written informed consent.collinear condition, the orientation of each Gabor patch was calculated relative to the underlying body-parts (as in a stick-figure presentation of the human figure), aligned with that body-part and was thus updated each frame. The orientation change was the largest for the Gabor patches representing wrists and feet, and smaller for some other patches, such as shoulders and hips. In the isolinear condition, all Gabor patches that constitute the walker had the same spatial orientation. In the random condition, the orientation of each Gabor patch was randomly chosen in the first frame and was updated each frame such that the change in orientation of individual Gabors across the whole presentation was the same as in corresponding Gabors in collinear walkers. The orientation of the head patch in the collinear condition was fixed to the vertical orientation. A scrambled counterpart was created for each of the three walker conditions, in which the initial frame of each separate Gabor element was randomly chosen within a walking cycle, but the motion remained the same as in the corresponding elements of the intact figure, as did the Gabor orientations  and Psychophysics toolbox extensions ntations . For all2. The basic design employed a two-alternative forced choice paradigm with a temporal succession of two presentations, one containing an intact point-light walker (Participants were seated approximately 57 cm from the screen (Dell 19″ CRT monitor) on which stimuli were displayed at a resolution of 640×480 at 60 Hz, resulting in the stimulus subtending about 5° of visual angle in height and 2° in width. Background was light-gray with a luminance of 60 cd/mt walker  and the t walker  with theUnlike the standard detection in noise task To examine whether a particular arrangement (orientation) of the Gabor patches affects the perception of biological motion, we first employed dynamic presentations of the experimental stimuli, with collinear, isolinear, and random element arrangements within a walker, as well as their inverted counterparts. Participants had to indicate which of the two presentations in each trial contained an intact representation of a human figure by means of a key-press. The collinear orientation of the Gabor patches might facilitate grouping and enhance the perception of the underlying body connectivity structure. The isolinear stimulus does not give clues to the underlying shape, at least not at the local level, but does contain other cues that can support the integration of the individual elements. The similarity of the Gabor patches with regard to their orientation might be employed by the visual system to integrate them into an organized global shape and hence facilitate perception.To further investigate the contribution of grouping in biological motion perception, we employed static presentations  of postures from the walking cycle Usually, biological motion has the status of a special stimulus for the visual system. However, paradigms used to investigate its perception most often involved a point-light representation of a human figure in one particular orientation . Therefore, the question of generalization and ‘ecological validity’ issues were often raised To further explore the contribution of the two implemented organizational principles, we asked the participants to repeat the same experiment with 45° view, while the figures were defined by small bright dots, as in the classical representation of the point-light walker. Their size was approximately 0.1° visual angle. The point-light stimulus does not contain  cues as the collinear and isolinear condition and could be thus used as a base-line. This was necessary to examine whether the manipulations in previous conditions lead to improvement or decrease in performance relative to this condition.Participants had to indicate in which of the two presentations of each trial they perceived an intact point-light representation of a human figure, while we manipulated the orientation of individual Gabor-elements that constituted the figure. For data analysis, 75% correct performance thresholds were estimated by fitting a Gaussian distribution to the data  better than for the classic point-light walker. The Gestalt principle of similarity states that elements with similar features tend to be grouped together In the present study, all dynamic displays contained the same motion irrespective of the local orientation of the individual elements. Motion is a useful cue in the perception of point-light displays, especially the opponent motion of the pairs of limbs, which is often put forward as a key feature for the recognition of biological motion as such e.g., . One intTo disentangle the influence of orientation cues from motion, we also employed static displays. In his 1973 report Johansson suggested that static point-light displays (‘snapshots’) do not contain enough information to correctly interpret an action, and research that followed largely assumed that motion is indeed crucial. However, Thirkettle et al. A characteristic inversion effect was revealed in worse performance in both dynamic and static displays in our study. Once the walker is presented upside-down, the human figure is not as easily recognized as such anymore The primary goal of Gestalt psychologists was to understand and define principles responsible for the perceptual organization of our visual impressions of everyday objects . Stimuli used in most studies of perceptual grouping were usually artificial patterns, shapes or contours and in that sense they represented meaningless objects. While their use in the study of grouping is valuable, the principles should also apply to more complex, meaningful stimuli. We here show that the perception of biological motion, represented by point-light figures, is also enhanced when perceptual grouping of the elements is enhanced.Movie S1The collinear walker. The orientation of Gabor elements are corresponds to the body-lines.(WMV)Click here for additional data file.Movie S2The isolinear walker. All Gabor elements have the same spatial orientation.(WMV)Click here for additional data file.Movie S3The random walker. The orientation randomly chosen in the first frame for each element.(WMV)Click here for additional data file.


MEDLINE citations are manually indexed at the U.S. National Library of Medicine (NLM) using as reference the Medical Subject Headings (MeSH) controlled vocabulary. For this task, the human indexers read the full text of the article. Due to the growth of MEDLINE, the NLM Indexing Initiative explores indexing methodologies that can support the task of the indexers. Medical Text Indexer (MTI) is a tool developed by the NLM Indexing Initiative to provide MeSH indexing recommendations to indexers. Currently, the input to MTI is MEDLINE citations, title and abstract only. Previous work has shown that using full text as input to MTI increases recall, but decreases precision sharply. We propose using summaries generated automatically from the full text for the input to MTI to use in the task of suggesting MeSH headings to indexers. Summaries distill the most salient information from the full text, which might increase the coverage of automatic indexing approaches based on MEDLINE. We hypothesize that if the results were good enough, manual indexers could possibly use automatic summaries instead of the full texts, along with the recommendations of MTI, to speed up the process while maintaining high quality of indexing results.We have generated summaries of different lengths using two different summarizers, and evaluated the MTI indexing on the summaries using different algorithms: MTI, individual MTI components, and machine learning. The results are compared to those of full text articles and MEDLINE citations. Our results show that automatically generated summaries achieve similar recall but higher precision compared to full text articles. Compared to MEDLINE citations, summaries achieve higher recall but lower precision.Our results show that automatic summaries produce better indexing than full text articles. Summaries produce similar recall to full text but much better precision, which seems to indicate that automatic summaries can efficiently capture the most important contents within the original articles. The combination of MEDLINE citations and automatically generated summaries could improve the recommendations suggested by MTI. On the other hand, indexing performance might be dependent on the MeSH heading being indexed. Summarization techniques could thus be considered as a feature selection algorithm that might have to be tuned individually for each MeSH heading. MEDLINE®; citations are manually indexed using the Medical Subject Headings (MeSH)®; controlled vocabulary. This indexing is performed by a relatively small group of highly qualified indexing contractors and staff at the US National Library of Medicine (NLM). MeSH indexing consists of reviewing the full text of each article, rather than an abstract or summary, and assigning descriptors that represent the central concepts that are discussed. Indexers assign descriptors from the MeSH vocabulary of 26,581 main headings (2012), which are often referred to as MeSH Headings (MHs). Main heading descriptors may be further qualified by selections from a collection of 83 topical Subheadings (SHs). In addition there are 203,658 Supplementary Concepts  which are available for inclusion in MEDLINE records.Since 1990, there has been a steady and sizeable increase in the number of articles indexed for MEDLINE, because of both an increase in the number of indexed journals and, to a lesser extent, an increase in the number of in-scope articles in journals that are already being indexed. The NLM expects to index over one million articles annually within a few years . The weight of an edge e representing an is_a relation between two vertices, vi and vj (where vi is a parent of vj), is calculated as the ratio of the depth of vi to the depth of vj from the root of their hierarchy. The weight of an edge representing any other relation  between pairs of leaf vertices is always 1.•The second step, topic recognition, consists of clustering the UMLS concepts in the document graph using a degree-based clustering method similar to PageRank  at intervals of 0.1, have been tested using a set of 150 biomedical articles different from those used in the experimentation. The combination of weights that, according to ROUGE metrics , produceROUGE is a commonly used evaluation method for summarization which uses the proportion of n-grams between a peer and one or more reference summaries to compute a value within . Higher values of ROUGE are preferred, since they indicate a greater content overlap between the peer and the model. The 1.2 version of ROUGE is used and the ROUGE-2 and ROUGE-SU4 metrics are used for evaluation. ROUGE-2 counts the number of bigrams that are shared by the peer and reference summaries and computes a recall-related measure. Similarly, ROUGE-SU4 measures the overlap of skip-bigrams. As model summaries, we use the articles’ abstracts. Even though using more than one single reference summary would report more accurate results, previous experiments have shown that, when the size of the evaluation collection is large enough, using a single reference summaries produces reliable results .F1). See Additional file The evaluation of the indexing process is carried out by comparing the MeSH headings recommendations by the different indexing methods  on the different types of documents  and the actual indexing of the articles by the MEDLINE indexers for the 1413 articles in the evaluation collection, and using text categorization measures: precision (P), recall (R), and F-measure , while achieving a recall only slightly better than that of the MEDLINE citations. The high recall of the full text is expected since it contains more details than the summaries or MEDLINE citations.Table Gr-sum) produces better F1 than the concept frequency-based summarizer (CF-sum). Graph-based summaries are more precise. However, recall is higher for the frequency-based summaries. The reason seems to be that, on average, frequency-based summaries are longer than graph-based ones, since the frequency-based summarizer tends to select longest sentences. Among the summaries, the ones at the 15% compression rate present the lowest recall but the highest precision, so achieving a higher F1 for micro average. On the other hand, F1 is slightly higher for macro average.Regarding the use of automatic summaries, it is observed that the graph-based method , we have limited the reported experiments to the top 30 more frequent MeSH headings. See Additional file We have compared the results of these three representations with MTI, MMI, PRC and two machine learning algorithms. We have included learning algorithms like SVM with linear kernel and AdaBoostM1, both from the WEKA package . PrecisiTable This performance might be due to the capabilities of the full text to provide disambiguation features that other methods, like MMI, are not using, similar to the increased performance of PRC on full text. In contrast to other works, summaries do not offer better performance compared to full text. On the other hand, further tuning of the set of parameters for the summarization process might improve summary performance . From thGlobally, results for SVM and AdaBoost are better than MMI and PRC. This has been already seen in previous work with learning algorithms and very frequent MeSH headings. On the other hand, it has been shown  that lesCohort Studies appears under the E  and N  branches. We have used this MeSH structure to group the results by tree branches, according to the MeSH headings in those branches. The idea is that, for instance, the indexing of terms in branch C (Disease) will be different to the indexing of terms in branch G (Phenomena and Processes). See Additional file MeSH terms are organized in a tree structure. The top nodes of this tree define broad topics within the medical domain. Each branch is identified by a letter, and Table Comparing both summary types and MeSH branches, we observe, as above, that graph-based summaries achieve higher precision but lower recall compared to the frequency-based summaries. We find that the larger differences between the two types of summaries occur in the B, M, N and Z branches.Organisms) and M (Named Groups) branches, terms like Humans, Mice, and Animals are most frequent terms in the results of each method. This result is similar to the one observed in full text articles. These terms belong to a special category denominated Check Tags (CTs) [In the case of the B (gs (CTs) . Recall gs (CTs) . SummariHealth care) branch, terms like Cohort Studies are predicted by forced rules. These rules are encoded into MTI to comply with the indexing policy at the NLM and are supposed to improve the quality of indexing based on indexer feedback. Terms like cohort indexes the citation with the MeSH heading Cohort Studies, which seem to be more frequent in frequency-based summaries.In the case of the N  branch, the difference is larger, but becomes more similar as the size of the summary increases. The Z branch presents the highest recall but the lowest precision in the full text. On the other hand, the summaries do not exhibit this behavior. Examples of high recall but low precision in full text are: United States, , PMID “20473639”, and Germany, Rapid DNA ligation kit was from Roche , PMID “19609521”. In these cases, the country was mentioned as a reference in the full text. Neither the MEDLINE citation or the summaries contain mentions to them.In the case of the Z (Named Groups) shows a slight advantage in favor of MEDLINE citations. The M branch contains a limited number of MeSH headings and some of them overlap with the Check Tags for which we have trained learning algorithms.If we compare the summaries to MEDLINE citations, the trend is higher recall but lower precision. Only the M branch (Anatomy) and D . We find that terms in these branches are identified using the Related Citations which predicts the MeSH heading if there is enough evidence in similar documents. In this case, the summaries seem to be more similar to previously indexed citations.Comparing the recall of the summaries and the full text we find that, as expected, in most of the cases the full text has a higher recall. However, we have identifiedtwo MeSH branches for which the summaries achieve higher recall compared to full text. The branches are A  when applied on (1) summaries of different lengths generated with two different summarization methods (2) full text articles and (3) MEDLINE citations.Our results show that automatic summaries produce better indexing than full text articles. Summaries produce similar recall to full text but much better precision, which seems to indicate that automatic summaries can efficiently capture the most important contents within the original articles. Compared to MEDLINE abstracts, they allow for higher recall but lower precision. With respect to the different types of summaries, the best results are obtained by a graph-based method with a compression rate of 15%.There are several reasons for the lower precision of summaries and full text compared to MEDLINE citations. In many cases, it is the use of specific techniques which were tuned for MEDLINE citations. This tuning provides a higher recall in summaries and full text due to the higher probability of triggering the rules. We have evaluated indexing without the forced rules and machine learning algorithms. Without these rules, both the precision and recall dropped. A revision of the forced rules for the summaries and full text might improve the indexing performance.Furthermore, it must be noted that summarization algorithms are tuned based on ROUGE. Tuning of the summarization algorithms based on MeSH indexing could also provide better performance.Even with full text, the indexing recall is still low in some cases. We have looked into frequent example terms, and one of the reasons for low recall is that in some cases the terms are not explicitly mentioned in the citations or appear with a different term, e.g., synonym not covered by MeSH or the UMLS. The PRC and machine learning algorithms try to address this problem.In previous work, machine learning has been evaluated on some of the MeSH headings and MEDLINE with mixed results ,50. We hThe authors declare that they have no competing interests.AJ participated in the development of the MTI system and carried out the indexing evaluation experiments. LP developed the methods for automatic summarization and generated the summaries. JM is the lead developer of the MTI system. AD and AA participated in the design of the experiments and reviewed the manuscript. All authors read, commented and approved the final version of the manuscript.Evaluation benchmark. The first column is the PubMed identifier (PMID) of the article. The second column is a MeSH heading used to index the article.Click here for fileEvaluation of MeSH term ranking per document. The first sheet shows a summary of the results. The following sheets show the results according to the method used to index the full text, the summaries and MEDLINE. The data has been obtained using the trec_eval evaluation program.Click here for fileResults for the 30 more frequent MeSH headings. The first sheet shows a summary of the results. The following sheets show the results according to the method used to index the full text, the summaries and MEDLINE. Machine learning experiments include ML - SVM for SVM with linear kernel and ML - AdaBoostM1 for AdaBoost experiments.Click here for fileAverage results per MeSH 2012 top level branch code. The first row of results corresponds to the full text and MEDLINE results. The following one corresponds to the graph-based summaries results. The final row of results corresponds to the frequency-based summaries results. For each row of results the following values are shown: top level branch code (Branch), number of unique MeSH headings (MH Count), number of positives (Pos), number of true positives (TP), number of false positives (FP), micro precision, micro recall, micro F1, macro precision, macro recall and macro F1.Click here for file