Recent clinical studies show that the contour of the photoplethysmogram (PPG) wave contains valuable information for characterizing cardiovascular activity. However, analyzing the PPG wave contour is difficult; therefore, researchers have applied first or higher order derivatives to emphasize and conveniently quantify subtle changes in the filtered PPG contour. Our hypothesis is that analyzing the whole PPG recording rather than each PPG wave contour or on a beat-by-beat basis can detect heat-stressed subjects and that, consequently, we will be able to investigate the impact of global warming on human health. Here, we explore the most suitable derivative order for heat stress assessment based on the energy and entropy of the whole PPG recording. The results of our study indicate that the use of the entropy of the seventh derivative of the filtered PPG signal shows promising results in detecting heat stress using 20-second recordings, with an overall accuracy of 71.6%. Moreover, the combination of the entropy of the seventh derivative of the filtered PPG signal with the root mean square of successive differences, or RMSSD , improved the detection of heat stress to 88.9% accuracy. According to the Intergovernmental Panel on Climatic Change (IPCC), accelerated global warming is predicted due to increasing anthropogenic greenhouse gas emissions. There is a 3% increase in the death rates per 1 °C increase in temperature for all causes of mortality for hot and arid regions where the temperature of the warmest months exceeds 30 °C .et al. [The literature identifies two main heat stress indices: body core temperature (BCT) and heart rate variability (HRV). In terms of BCT, in 2002, the National Athletic Trainers’ Association recommended measuring rectal temperature as a standard criterion for recognizing exertional heat stroke . Five yeet al.  found thRegarding HRV, recent studies ,6,7 haveUsing these two heat stress indices is not practical, as measuring the BCT has an invasive nature, and calculating HRV requires electrocardiogram signals recorded over a long time. Therefore, there is a need to develop an easier and non-invasive method to aid in the management of human resources engaged in critical activities and to potentially prevent heat-related deaths. Accurate and simple-to-measure diagnostic biosignals are desirable, such as fingertip photoplethysmogram (PPG) signals. Advances in photoplethysmography may potentially lead to a new complementary tool for the management of vascular heat-related diseases.Although the clinical significance of the PPG wave contour has been investigated over many decades ,10,11,12The heat stress PPG data for this study were collected as part of a National Critical Care and Trauma Response Centre (NCCTRC) project to assess the physiological and perceptual responses of emergency responders to simulated chemical, biological and radiological (CBR) incidents in tropical environmental conditions to compare the efficacy of various cooling methods. The background of the NCCTRC’s thermal research can be found in . Forty hThe participants were normotensive (mean systolic blood pressure (BP) of 129.3 mmHg, range 110–165 mmHg) and had no known cardiovascular, neurological or respiratory disease. The range of systolic BP exceeds the usual normotensive range. However, the vast majority of guidelines recommend that hypertension be identified when systolic blood pressure exceeds 140 mmHg . TherefoPrior to the experiment, the participants provided information about their physical condition. Physical information, such as height and weight, was also measured for a reference and is summarized in An ingestible telemetric temperature sensor  was used to measure BCT by transmitting a signal proportional to the temperature of the gastrointestinal tract to a handheld receiver. Once located in the gastrointestinal tract, the thermosensitive pill is a valid index of core temperature when referenced to rectal or esophageal temperature ,33.Participants ingested a core temperature pill with fluid prior to breakfast, which preceded the commencement of data collection by approximately 4 h. This time frame was adopted to allow the pill to empty from the stomach and enter the small intestine while minimizing the risk of the pill being emptied from the body prior to the completion of the study . To contetc.). In this study, our goal is to systematically examine the derivatives of the PPG signal to determine the most informative derivative that can be used for heat stress assessment. In addition, we investigate whether filtering the PPG signals potentially improves the heat stress analysis.The PPG and its various derivatives define an ordered hierarchy of meaningful physiologic concepts of blood movement. There are special names for the derivatives of position  at the four time points (control and three interventions), where the signal status can be either before or after bandpass filtering. The extracted features are the normalized energy and the normalized Shannon entropy , as follp-values need to be appropriately corrected by applying Bonferroni post-correction [We calculated each time domain feature (energy or entropy) for each PPG signal recording. As we had 40 subjects, each feature set contains 80 values. Each feature set consists of 40 values calculated from subjects measured before the simulated heat stress induction and 40 values calculated from subjects measured after the simulated heat stress induction. For each derivative order, we compared the values within each feature set by applying the two-sided Wilcoxon-Mann-Whitney test, and rrection . Becauserrection .As the main objective of this research is to find the optimal derivative order for assessing simulated heat stress induction, we tested four classifiers: Mahalanobis distance, linear discriminant analysis (LDA), quadratic discriminant analysis (QDA) and the linear support vector machine (SVM). The classification rates were calculated using leave-one-out cross-validation. Two statistical measures were used for the output of the LDA analysis: sensitivity (SE), which was calculated using the formula Increased HR is due to both the direct and indirect effects of heat stress. Direct effects include heated blood perfusing the sinoatrial node of the heart, triggering a more rapid depolarization and, thus, more contractions per minute, and indirect effects include increased cutaneous blood flow, a reduction in total peripheral resistance without adequate compensation by other vascular beds and less blood returning to the heart. Therefore, extra heart beats are required to maintain blood flow . The HR The core temperature data confirm that the participants experienced substantial body heat storage. We would have seen a different HR response if we heated the participants without the use of exercise (sitting still in a sauna); however, that model has limited application to the assessment of heat stress in real-world settings. Fitness plays a role in HR recovery; those who are fitter have a finer regulation of blood flow and require fewer cardiac cycles per minute to restore the body to normal following exertion. Fitness was a constant in this study due to the same participants being assessed during each recovery period.It is known that when subjects are continuously subjected to dry heat, the heat induces a stress response as indicated by a significantly increased HR . This inMoreover, the significant reduction of RMSSD in subjects measured after exercises, shown in p-value) when they are applied to the unfiltered PPG signals to calculate the energy and entropy features. It was not clear which derivative order is optimal for distinguishing between at rest and after exercise, as the lowest p-value was obtained by a range of different derivative orders from 8 to 20.Here, the entropy and energy of different derivatives of 20-second PPG signals are calculated for 40 subjects measured before and after simulated heat stress induction. Based on the derivative optimization shown in p-values shown in To determine which derivative order is more informative for calculating the energy and entropy to detect heat stress, we employed four classifiers: Mahalanobis distance, LDA, QDA and SVM with leave-one-out cross-validation. The OA of the four classifiers in detecting after exercise measurements is presented in p-values for differentiating between at rest and after the three exercises were As a general observation in To ensure that the entropy of the seventh derivative of filtered PPG signals can be used to replace other measures of heat stress, it is important to compare its performance with another heat stress index, such as BCT or RMSSD. The problem is that we could not measure BCT immediately after exercise, as a few subjects had to wait, which cooled them down a bit . TherefoTo maximize the heat stress detection performance, we combined the optimal feature (entropy of the seventh derivative of filtered PPG) and the traditional HRV heat stress index (RMSSD). The progression of detecting heat stress continued, and the accuracy improved after Exercise 3, with an SE of 95% and a PP of 90.48%, as shown in It is important to emphasize that this study replicated “real-world” heat stress by simulating emergency responder work settings. Although the participant number may seem modest, the logistics of establishing a simulation of this size were very challenging and likely explain why there are few reports of the use of technology to assist in the detection and management of heat stress in field settings. We believe that the modest participant number (To our knowledge, there is no available PPG database containing data measured in tropical conditions or after heat stress that would allow a more thorough assessment and comparison of the tested algorithms. In future studies, it may be advisable to have multiple PPG systems to collect the signal immediately after exercise. In the present study, data were collected immediately after exercise; however, some participants may have cooled down whilst queuing for measurement. Furthermore, we used the standard definition of entropy in our analysis to measure the randomness of the PPG morphology, regardless if the PPG signal is noisy with high gain or fluctuations. An open question for future studies is to explore the effect of using alternative entropic measures for this purpose.The findings of this preliminary study indicate that heat stress can be assessed using unfiltered PPG signals without filtering and without beat-by-beat analysis. Moreover, filtering the PPG signal before differentiation slightly improves the heat stress assessment. The combination of RMSSD and the entropy of the seventh derivative of the filtered PPG signal can be used to detect heat stress measurements without the need for measuring BCT. The results of this study indicate that analysis of the whole PPG recording without detecting beats or wave amplitudes can be a potential modality for heat stress analysis and the identification of individuals at risk.


LineSets that enables biologists to fulfill these tasks more effectively.Biologists make use of pathway visualization tools for a range of tasks, including investigating inter-pathway connectivity and retrieving details about biological entities and interactions. Some of these tasks require an understanding of the hierarchical nature of elements within the pathway or the ability to make comparisons between multiple pathways. We introduce a technique inspired by Extended LineSets, to facilitate new explorations of biological pathways. Our technique incorporates intuitive graphical representations of different levels of information and includes a well-designed set of user interactions for selecting, filtering, and organizing biological pathway data gathered from multiple databases.We introduce a novel technique, Based on interviews with domain experts and an analysis of two use cases, we show that our technique provides functionality not currently enabled by current techniques, and moreover that it helps biologists to better understand both inter-pathway connectivity and the hierarchical structure of biological elements within the pathways. Pathways Commons [Much effort has been expended to organize the body of knowledge that is available regarding the structure and function of biological pathways. The Reactome Database  and the  Commons , allow b Commons , who strPathways are typically represented as directed graphs, where nodes in the graph represent biological "participants," such as proteins or protein complexes, and where the edges represent a biological functionality, such as a biochemical reaction. Often different shapes for arrows and nodes are used to differentiate between the different types of molecules or reactions. Though this type of visual encoding is the most familiar, node-link diagrams are known to have a number of issues. A main issue is scalability; as the number of nodes or edges increases, it quickly becomes more difficult to make sense of the data . In the The importance of dynamic visualization has been discussed by Hu et al.  and KlukCytoscape [The structure of biological pathways can be formalized as a hypergraph, a graph that contains hyperedges which can connect to any number of nodes. Traditional node-link diagrams cannot provide sufficient information to represent all of the complexities in a biological pathway data: biochemical reactions can involve multi-participant relationships; pathways can contain multiple subpathways; nodes within a pathway can represent a nested structure containing many biological entities; and links between nodes can convey different biological meanings. Some techniques for the visualization of pathways propose representations which limit the complexity of the data structure in favor of a simpler design. This is the case of the SIF format, which is often used to encode data for generating visualizations with tools such as ytoscape . This foExtended LineSets, that more accurately represents the intricacy of interconnected pathways and subpathway connections, and moreover, that helps to reduce visual clutter that can interfere with visual analysis tasks. Additionally, we describe a prototype implementation that makes use of this technique so that biologists can more effectively search, filter, visualize, and compare pathways data.This paper presents a novel technique, Extended LineSets.We interviewed seven domain experts in order to understand the type of tasks that could be usefully accelerated or augmented by visualization techniques. The experts are professors and researchers in different domains of cellular biology, molecular genetics, and informatics. While each of the experts have different research interests, we identified three high-level tasks that were considered to be important to all of them. Through defining these tasks, we were able to identify the primary requirements necessary for an effective visualization technique. These tasks are not meant to be comprehensive, but rather to provide insight into the motivation for the development and design of Task 1: Examine the upstream and downstream connections between two entities within a pathway. Understanding how entities within pathways are connected is of critical importance to all of the researchers we interviewed, and is essential to most research related to pathway data. When discussing directed paths between entities, one entity is said to be upstream or downstream of another. Understanding upstream and downstream relationships is particularly important to domains such as cancer drug research, where a drug may affect a small subset of genes or gene products, which in turn will affect various downstream processes. In most cases, a directed relationship is meant to represent a biochemical reaction, where one entity is consumed as a reactant and another is produced as a product. Thus, an upstream entity may be connected to a downstream entity through a chain of several directed links. In the most basic sense, the "entities" mentioned above are genes, gene products (such as proteins or complexes), or other small molecules within a cell. A researcher may be interested in understanding the path of reactions (or other relationships) that connects two entities.Task 2: Understand the hierarchical structure of protein complexes. Protein complexes are represented as nested hierarchies of proteins. These hierarchies can be very intricate and potentially involve dozens of proteins and sub-complexes. Since the proteins and the complexes which compose these compound structures might be involved in other relevant complexes and biological processes, understanding their structure and organization is generally important for pathway analysis tasks.Task 3: Curate, edit, query and merge pathway data files, or construct user-defined pathways. Several of the researchers mentioned the importance of various tasks related to the curation, maintenance, and understanding of pathway data. They expressed the need to create "personalized pathways" that only include a user-determined subset of entities and relationships. They are especially interested in mixing-and-matching information that is contained within multiple pathways, but then being able to filter this information so that only information relevant for a particular task is displayed.node duplication, which considerably reduces the overlapping of edges and enables a visually appealing arrangement of the nodes. However, as pointed out by Bourqui et al. [It can be challenging to represent multiple pathways simultaneously in traditional node-link diagrams, due to issues of scalability and also due to difficulties in defining a layout that accurately and efficiently displays the topology of these pathways. In order to reduce the visual clutter introduced by dense networks with many edge crossings, many visualization tools rely on i et al. , analysiTask 3, researchers may need to merge pathway components from multiple sources, while still retaining an indication of the original source. For this reason, we also review visualization techniques that are used for presenting category information.This section presents an overview of features implemented in popular pathway visualization tools. One feature missing from all of these tools is a specific visualization technique for presenting category data within node-link diagrams. Indeed, as indicated by Entourage [Reactome Pathway Browser [VisAnt [MetaViz [VitaPad [ChiBe [Broadly speaking, current tools make use of one of three primary visual representations: node-link diagrams, node-link diagrams with compound nodes, and adjacency matrices. ntourage , Reactom Browser , VisAnt  [VisAnt , MetaViz[MetaViz  and Vita[VitaPad , each usd [ChiBe  extends Reactome Pathway Browser is a tool for visualizing pathway diagrams included in the Reactome database. It offers basic navigation and limited interactivity, making use of side panels to enable the inspection of complexes structure and analysis data. Entourage is a tool for pathway visualization that enables the exploration of the "cross-talk" between pathways via an "artificial partitioning" of the pathway structure, helping to reduce the complexity of the visualization. Multiple pathways and sub-pathways are represented in isolated views, which preserves the pathway structure but introduces node redundancy. Other tools represent multiple pathways in a single view, such as MetaViz, which enables topological analyses across multiple metabolic pathways simultaneously. Our work is inspired by MetaViz, but our technique tries to organize the representation of all pathways according to a topological ordering and enables the interactive inspection of hierarchical structures of certain nodes. BioFabric [VisAnt enables the user to search for the shortest path between two nodes [VisAnt permits a dynamic exploration of a biological network composed by multiple pathways. However, if more than one pathway includes the same node, multiple instances of the node will be present in the representation.The ioFabric  also enawo nodes  and to iVitaPad allows the user to incorporate microarray data into pathways, Entourage enables the integration of experimental data, and VisAnt permits the user to create and view annotations of nodes within the network.Although the majority of these pathway visualization tools use conventional graph representations, many of them decorate the node-link diagrams with experimental data or other additional information. For example, Combining multiple pathways in a single visualization introduces the need for displaying the relationships between biological entities and the pathway or pathways they are participants of. That is, biochemical reactions, proteins, or other biological compounds can be included in more than one pathway. This raises the further challenge of effectively displaying this membership information on top of the traditional node-link diagram without introducing visual clutter.Euler Diagrams to display set-based annotations on biological networks. However, this may lead to visually confusing representations if the data contains complex set intersections. For this reason a range of alternative visualization techniques has been developed to display elements belonging to multiple sets [Bubble Sets [Line Sets [KelpFusion [Bubble Sets and KelpFusion prevent these techniques from being applicable to pathway visualization. For example, Bubble Sets displays set relations using isocontours, which can make it difficult to distinguish elements belonging to multiple sets.Dinkla et al.  propose ple sets . Bubble ble Sets , Line Seine Sets , and KellpFusion  all addrLineSets, developed by Alper et al. [Bubble Sets technique when multiple sets overlap. KelpFusion uses continuous boundaries made by lines and hulls. The visual appearance is generally comparable to LineSets, but this strongly depends on the spatial arrangements of elements. Hulls are used to group elements that are both spatially close to each other and that belong to the same set. This technique can become confusing when it is applied to biological networks, which have an arbitrary or dynamic spatial layout. LineSets, though it has been applied to geographic datasets, is a more extensible technique that is able to effectively represent pathway data.r et al. , is a teUpSet [OnSet [None of the above techniques scale well when the number of sets and number of intersections between sets increases. UpSet  and OnSet [OnSet  are two Extended LineSets, a modified version of the LineSets technique which introduces the inclusion of hierarchical structure and which is presented as a more abstract node-link diagram, without the need for elements to have a spatial reference. Our technique aims to enhance analysis tasks that make use pathway visualization tools. It adds an extra information layer to the node-link diagram that represents the intricate relationships within and between pathways, and at the same time it provides an approach that reduces clutter while avoiding node duplication. In the following section we include a detailed description of the Extended LineSets technique. After presenting a prototype implementation, we introduce two use cases that demonstrate how our technique enables effective, real-world pathway exploration.In this paper we introduce Extended LineSets technique. The first representation combines node-link diagrams with LineSets, allowing the user to concurrently identify set memberships in a network and to visualize directional relationships of between elements. The second representation displays the hierarchical layout of elements within the network, allowing the user to interactively inspect their nested structure. These two primary representations are integrated to support the effective interactive investigation of biological pathways. Our prototype utilizes our technique and implements a variety of user-driven interaction to enable pathway analysis tasks.In this section we describe the two primary representations used in the Our first visual component presents a directed graph where nodes and edges belong to one or more sets. We identify every set with a color, and every element is depicted as either a square or a circle. Edges linking elements are represented using colored lines with sharp-pointed tip. Figure LineSets implementation. First, every set is not represented by a single smooth curve, but is identified only by the color of the links and nodes. Second, the layout of the elements flows from the top to the bottom, matching, when possible, the direction of the relationships. A completely consistent representation cannot be achieved when the graph contains cycles; in this case some directed edges will point upwards, but the general layout will still mostly adhere to the flow from the top to the bottom of the viewport. Additionally, the layout of the elements is designed to minimize nodes overlapping and edges crossing. The resulting visualization combines the efficient identification of elements belonging to particular sets from the LineSets technique with a traditional node-link graph representation. Figure LineSets , the pruned tree is updated to reflect the hierarchy of elements from the root to that currently selected element. The display is "pruned" because it does not show the whole structure, but instead only displays the direct path from the root to the parent that contains the selected element. That is, the pruned tree enables the inspection of all the components directly included in the parent.G, which brings up a pruned tree that indicates the hierarchy of parent complexes that contain G, fist A, then B, and then the direct parent C. The right side of the pruned tree shows protein G and its siblings D, E and F. Figure B and its corresponding pruned tree.Figure ORC:origin, and the related complex and five proteins. For clarity, the hovered component in the symbolic view is underlined and emphasized in the pruned tree. This visual component is integrated in the node-link component previously described. For this reason, the color palette used to differentiate nested complexes is limited to one color hue and two different color intensities, as this minimizes the potential conflict with other colors used to visualize biological elements. Our technique aims to find an optimal balance between reducing the complexity of the displayed structure while at the same providing as much information as necessary for a particular task.The symbolic overview provides a compact overview of the structure of the complex, where the squares represent complexes and the white dots are proteins contained within those complexes. Figure Extended LineSets technique. It enables the user to narrow down a potentially large set of proteins and complexes contained within multiple pathways to an arbitrary small group of relevant components. That is, a biologist or bioinformatician can interactively expand or reduce the visible pathways and components that is relevant for his or her research. The prototype uses a force-directed layout [https://github.com/CreativeCodingLab/pathways. By default, for demonstration purposes, the prototype loads in six pathways related to the Cell Cycle and 80 sub-pathways. The pathways are referred to as the field of interest. The field of interest is comprised of the pathways that the biologist or bioinformatician considers relevant for his or her research. The field of interest initially needs to contain all of the pathways that might be required for the analysis task.We created a prototype application that implements our d layout  to help We designed a set of user interactions for exploring, extending and inspecting the pathways at different level of detail. The interaction tasks, discussed below, include the following:• Pathway highlighting and labelling;• Pathway hiding and unhiding;• Keyword filtering;• Upstream and downstream expansion;• Layout rearranging, zooming, and panning;• Complex structure inspection;• Finding intermediate steps between two nodes in a pathway.Task 1 and Task 3. By default, labels for all complexes and proteins are hidden. This reduces the potential clutter that might be introduced by having too many overlapping labels shown in the graphical representation. Instead, when the user hovers over a component with the mouse pointer, only the labels of the components which are included in the same pathway as the selected component are displayed. The pathways which include this component also stand out because components and reactions belonging to any other pathway are temporarily desaturated , the user can hide or reveal any pathway included in the field of interest by clicking on its name from the list on the left of the screen  whose name matches at least one of the searched keywords.upstream step and one downstream step, starting from the selected component. A downstream step of one protein or complex is composed of all the reaction relationships that start from the given component. Similarly, an upstream step involves all the reaction relationships that end at that component. To facilitate this operation, we place a triangle-shaped maker pointing upwards on the node if it has an hidden upstream step, and a triangle-shaped maker pointing downwards on the node if it has a hidden downstream step. This interaction technique enables the following workflow to support both Task 1 and Task 3 : first the user searches for a set of proteins and complexes, then he or she progressively explores the network of interconnections by revealing the upstream and downstream nodes.By clicking on a protein or complexes the tool will expand the visualization with one Considering that the number of complexes and proteins involved might lead the visualization to exceed the boundary of the screen, the user can interactively zoom in, zoom out, and pan across the viewport. Since the representation might occasionally lead to unavoidable edge intersections, the user can also drag and drop any component on a preferred location to improve viewability.Task 2), our prototype enables the hierarchical inspection of complex structures, as described earlier in this paper. A complex can be enlarged by double-clicking on it. This enlarged complex then reveals its inner structure; all the other components are pushed away to avoid unwanted overlapping. The pruned tree is updated on the right side of the screen when the user moves the mouse over different parts of the hierarchy structure. A single click on an enlarged complex will hide the hierarchical structure, causing it to shrink the complex to the default size. More than one complex can be enlarged at the same time, enabling the user to easily jump from the inspection of one complex structure to another, and to find the same sub-complex in multiple complexes. Indeed, when the user moves the mouse on a component inside a complex, the same component will be highlighted in all the enlarged complexes that contain it.In order to support the user's understanding of the hierarchical structure of protein complexes  [M/G1 Transition pathway and activates the Assembly of the pre-replicative complex (As-preRC) and Activation of the pre-replicative complex (Ac-preRC) subpathways. The user then searches for ORC1-6 and preRC in the text field. The visualization is updated with the six ORC proteins and the preRC complex that match the keywords entered by the user. The color coding shows that the ORC proteins are involved in the As-preRC pathway, whereas preRC in involved in both As-preRC and Ac-preRC.In this use case we explore some of the interactions involved in the assembly of the  (preRC) . We pre-ORC proteins to the pre-replication complex. Furthermore, the user expands the network to view the immediate downstream elements of preRC, revealing reactions involved in the Ac-preRC pathway, such as Mcm10:preRC, which is expanded again to show the upstream MCM10 protein and the downstream Mcm10:active preRC protein.The user then interacts with the prototype application to reveal the intermediate steps from the ORC:origin, revealing the MCM8 protein, and expands the downstream complex (CDC6:ORC:origin), revealing the upstream protein CDC6. Then the user chooses to inspect the hierarchical structure of preRC, ORC:origin, along with other two complexes. By hovering over a sub-complex of preRC, the user can see that these same sub-complexes appear in all the visible complex structures . This enables the user user to better understand the steps involved in the preRC assembly. This use case is depicted via the three screenshots shown in Figures The user then expands the upstream of Regulation of DNA replication (RDR), M-G1 Transition (MG1) and Miotic G1-G1/S phases (G1S). The user types into the textbox to search for the preRC complex and for proteins that match the ORC3 and Ubiquitin keywords. By selecting two nodes and dragging between them the user is able to explore the missing steps between, first, ORC3 and preRC and, second, between Ubiquitin and preRC. The user can then easily detect which proteins and complexes are involved in all three pathways, such as the DNA replication factor (Cdt1) and the Mini chromosome maintenance complex (MCM2-7) complex [In this use case, the user selects three pathways from the larger set of pre-loaded pathways:  complex .preRC and the other complexes, showing, for example, the location within the hierarchical structure of MCM2-7 and its six related polypeptides in all the expanded complexes. This use case is explained via the four screenshots shown in Figures Using the integrated visualization of the hierarchy of elements within protein complexes, the user can further inspect the structure of We received detailed feedback from two domain experts who confirmed that the design choices implemented in our prototype were effective for pathway analysis tasks. Both of the experts are professors in a biology department at a large public research university. Each of them told us that the ability to see elements shared across multiple pathways (via the different colors) is a very important feature of our prototype. Both experts found the integration of the hierarchical views within the network to be a novel and potentially useful feature. They also appreciated the two coordinated views (the overview within the expanded node and the pruned tree) for inspecting the hierarchical structure of biological complexes.BioPAX files or different databases. He also said that the ability to quickly reveal all of the steps between two nodes could be useful for his research into interconnected pathways.Additionally, we received positive feedback regarding the search-by-keyword functionality, and especially the step-by-step exploration of the network interconnections. One of the experts agreed that in some cases a more natural way of exploring the network is to first search for a set of known entities and then to freely explore the interconnections to their neighbors. He was especially excited by the fact that these neighbors could be located in different One expert argued that some direct labeling of the nodes would be necessary, at least as an option, as an alternative to the dynamic labelling described earlier. Furthermore, he asked for the possibility to interactively dim all the other pathways to focus on a single pathway. The other domain expert commented that it would be helpful to be able to display the name or type of the reaction between two nodes, perhaps when a user mouses over the link. Moreover, he suggested that it would be useful to include an "undo" operation for when a particular exploration of the network turned out not to be relevant; rather than starting over, he wanted to rewind his exploration to a previous state. For instance, after a node is expanded and its interconnections are revealed, the user might want to revert this operation and hide all of its neighbors. We plan to incorporate these suggestions in a future implementation.Extended LineSets was developed through an iterative process whereby design choices were informally evaluated through discussions with expert users. While we are for the most part happy with the visual encodings and interaction methods that are currently used, we will continue to experiment with alternative encodings and interactions in future iterations. We plan to conduct a thorough empirical evaluation in order to test the effectiveness of our design choices. Although our technique eliminates node redundancy, multiple edges often originate from the same biological reaction. New interactions and visual paradigms should be designed to better represent this information.BioPax 3 format, and a future improvement will be to integrate our tool with the Pathway Commons Web API [Our prototype will require further work in order to become a full-fledged application for enabling pathway visualization tasks. Currently our tool assumes that all relevant pathways are pre-loaded; but the selection of pathways could instead be dynamically loaded from a public database of pathways. Our prototype already reads the Extended LineSets is a novel technique that enables the interactive visualization of a network of multiple pathways. It introduces a novel graph representation and an effective interactive interface. The visualization aims to provide a better understanding of the biological components and reactions shared among different pathways. Colors are assigned to pathways for a clear visual encoding. The user can dynamically focus on a limited set of pathways to reduce the amount of information and the number of different colors displayed at the same time. User-driven interactions enable a procedural exploration of the network, with the aim of limiting the visual clutter of the graphical representation and minimizing excessive edge crossings. Furthermore, the dynamic labeling of components allows the user to focus on the pathway topology to further reduce the complexity of the visualization. Finally, the inspection of the hierarchical structure of complexes permits the user to change the level of detail of the representation, enabling the simultaneous display of different abstraction layers. Based on real-world use cases and expert feedback, our initial prototype of Extended LineSets has already proven to be an effective representation for a range of tasks common to domain experts in biology and bioinformatics.The authors declare that they have no competing interests.FP and AGF conceived of the interactive visualization technique. FP implemented the prototype application and applied it to the two case studies. FP and AGF drafted, read, and approved the final manuscript.


TransRate is a tool for reference-free quality assessment of de novo transcriptome assemblies. Using only the sequenced reads and the assembly as input, we show that multiple common artifacts of de novo transcriptome assembly can be readily detected. These include chimeras, structural errors, incomplete assembly, and base errors. TransRate evaluates these errors to produce a diagnostic quality score for each contig, and these contig scores are integrated to evaluate whole assemblies. Thus, TransRate can be used for de novo assembly filtering and optimization as well as comparison of assemblies generated using different methods from the same input reads. Applying the method to a data set of 155 published de novo transcriptome assemblies, we deconstruct the contribution that assembly method, read length, read quantity, and read quality make to the accuracy of de novo transcriptome assemblies and reveal that variance in the quality of the input data explains 43% of the variance in the quality of published de novo transcriptome assemblies. Because TransRate is reference-free, it is suitable for assessment of assemblies of all types of RNA, including assemblies of long noncoding RNA, rRNA, mRNA, and mixed RNA samples. High-throughput sequencing of RNA has revolutionized our ability to assess the genetic and quantitative basis of many complex biological traits. For organisms that have sequenced and annotated genomes, short reads can be directly mapped to these resources and quantitative estimates of gene expression (as well as splice-variants and mutations) can be determined using a variety of different methods. In the absence of an appropriate reference genome, de novo transcriptome assembly must be performed. These assemblies provide the primary data for gene discovery and evolutionary analyses, and facilitate quantitative assessment of differential gene expression. Given the importance of these applications to comparative biological research, several algorithms have been developed to produce de novo transcriptome assemblies from raw sequence data. Popular among these algorithms are Trinity , Oases , Trans-AIn addition to the considerable algorithmic flexibility, the data being assembled can be generated from multiple different RNA types. These can range from specifically amplified subpopulations of particular types of RNA, to total RNA encompassing all RNA types within the cell. Given the wide range of input data and assembly methods, there is a need to be able to evaluate the quality of any de novo transcriptome in the absence of a known reference and identify the set of parameters or assembly methods that best reconstruct the transcriptome from which the raw read data was generated. Moreover, there is a need to be able to identify within a given assembly the set of contigs that are well-assembled from those that are not, so that incorrect data do not influence downstream biological interpretation.Algorithms to assess the outputs of DNA-directed  assembly have been developed. These range in complexity from descriptive metrics  to expliTo date, the majority of de novo transcriptome assessment methods have exploited comparative approaches in which the assembled transcriptome is compared to a known reference data set . These cHere, we present TransRate, a novel method for evaluation of the accuracy and completeness of de novo transcriptome assemblies. TransRate assesses these features through two novel reference-free statistics: the TransRate contig score and the TransRate assembly score. The TransRate contig score provides a quantitative measure of the accuracy of assembly for each individual contig, and the TransRate assembly score provides a quantitative measure of the accuracy and completeness of the assembly.The aim of de novo transcriptome assembly is to accurately reconstruct the complete set of transcripts that are represented in the read data in the absence of a reference genome. There are several contributing factors that negatively affect the accuracy of this reconstruction process. These factors include error in the sequencing process, incomplete coverage of transcripts (due to insufficient sequencing depth), and real biological variability . Moreover, assembly errors can originate from algorithmic simplifications (such as representing the information contained in the reads as shorter words) and allowances  that are used to mitigate the computational complexity of the assembly problem. Together, these factors cause several common assembly artifacts, including hybrid assembly of gene families, transcript fusion (chimerism), spurious insertions in contigs, and structural abnormalities such as incompleteness, fragmentation, and local misassembly of contigs .TransRate is focused on a clear problem definition, i.e., to assess the accuracy and completeness of a de novo assembled transcriptome using only the input reads. TransRate proceeds by mapping the reads to the assembled contigs, proportionally assigning multimapping reads in a probabilistic manner to their contig of origin, analyzing the alignments, calculating contig-level metrics , integraTo calculate the TransRate contig score, a correctly assembled contig is assumed to have the following four properties: (1) The identity of the nucleotides in the contig will accurately represent the nucleotides of the true transcript; (2) the number of nucleotides in the contig  will accurately represent the number in the true transcript; (3) the order of the nucleotides in the contig will accurately represent the order in the true transcript; and (4) the contig will represent a single transcript. We propose that each of these four statements can be approximated through analysis of the reads that map to the assembled contigs and are encapsulated by the four metrics presented in s(Ccov)   observeds(Cord) and s(Cseg) (s(Ccov) and s(Cnuc)   produced s(Cseg) A, wherea s(Cnuc)  produced s(Cnuc) A. To det s(Cnuc) B, and thSupplemental Fig. S1). The Bayesian segmentation of coverage depth, s(Cseg), was also evaluated by inspection of coverage depth profiles  and simulation of artificial transcript chimeras. The latter was done by in silico fusion of randomly selected transcripts from the yeast transcriptome and assessment of s(Cseg) scores as a function of the difference in abundance between the fused transcripts . Here, the segmentation method was unable to distinguish chimeras between transcripts whose abundance differed by less than twofold . The individual score components are provided in the TransRate program output so that end users can gain insight into the common sources of error in their assembly.Manual inspection of reference-based results for the 30 lowest-scoring contigs according to each score component was consistent with the individual score components capturing their target properties . For comparison, we also applied RSEM-eval to the same data set . Nonparametric correlation measures were used here to enable unbiased comparison of TransRate and RSEM-eval scores because their score distributions differ in type, location, scale and shape.As the contig-level metrics provided discrete evaluation of assembled contigs, we sought to determine whether the geometric mean of these metrics . However, the additional penalty due to abundance weighting causes the overall scores to be much lower . Caution should be exercised by the user when using the abundance-weighted contig scores because they are not comparable between contigs. That is, a highly abundant transcript with an assembly error will have a lower score than a transcript with the same error that is expressed to a lower level.TransRate also provides an abundance-weighted contig score  against the appropriate Ensembl reference data set for each species. The TransRate scores for these contigs were compared, and the proportion of transcripts that had the highest TransRate score for each assembly was recorded E. No oneSupplemental Fig. S5). This automated cut-off method is consistent with the problem definition and overall aim of TransRate (to assess the accuracy and completeness of a de novo assembled transcriptome using only the input reads) as it automatically selects the subset of contigs that maximizes both accuracy and completeness. It should be noted that filtering contigs in this way may remove some accurately assembled low abundance transcripts that have incomplete coverage.Supplemental Fig. S6). Of the genes whose entire length was encompassed in a single transcript, ∼12% were discarded by TransRate . Although TransRate has identified these transcripts as poorly assembled, and caution should be exercised against using abundance level estimates for these contigs, they may contain regions that have utility in certain analyses .To provide an example of the results obtained from the application of the automated TransRate contig filtering, the 10 assemblies analyzed in Supplemental Table S2). All assembled transcriptomes were downloaded from the NCBI Transcriptome Shotgun Archive (http://www.ncbi.nlm.nih.gov/genbank/tsa) and were chosen for analysis if they met the following criteria: (1) The assembly program was listed; (2) the reads were Illumina paired-end reads; and (3) the published assembly contained at least 5000 contigs. TransRate assembly scores for this set of published assemblies ranged from 0.001 to 0.52  (r2 = 0.1) (r2 = 0.16)  G and the2 = 0.1) H. In Ill2 = 0.1) I, there  = 0.16) J. In sumHere, we present TransRate a novel method for reference-free assessment and filtering of de novo assembled transcriptomes. Our method is focused on a clear definition of an optimal de novo assembled transcriptome, that it should be a complete and accurate representation of the transcripts encompassed in the raw read data. TransRate avoids conflating assessment of de novo assembly quality with other criteria (such as coverage of expected reference transcript subsets) that are not equivalent to correct or complete assembly of the input reads. Moreover, the method is not biased by expression level of the transcripts, and each transcript is weighted equally in the overall transcriptome assessment . As the majority of published de novo assembled transcriptomes use Illumina paired-end sequencing, our analysis of the efficacy of TransRate is focused on this data type. However, the method is suitable for the analysis of other types of sequencing and thus is not restricted to use in the analysis of Illumina data.s(Cseg) scores are likely to represent chimeric transcripts. Here, although the transcript itself may be incorrectly assembled, the component segments of the transcript may themselves be correctly assembled and of utility if separated. To help users identify and diagnose likely assembly errors affecting low scoring contigs, TransRate provides each of the separate contig scores . This information can be used to help resolve assembly errors on a contig-by-contig basis. Further investigation by systematically exploring a large range of read mapping parameters across a large range of read mapping algorithms and assembly tools may yield new ways to improve the performance of TransRate. This may improve the s(Cord) and s(Ccov) measures that are affected by read coherency  would be better than 50% of published de novo assembled transcriptomes that have been deposited in the NCBI TSA. Through detailed analysis of these 155 published assemblies, we reveal that the quality of the input read data is the major factor determining the quality of any de novo transcriptome assembly, explaining more of the variance in quality between assemblies than the assembly method that is used.TransRate is a reference-free qualitative assessment tool for the analysis of de novo transcriptome assemblies. TransRate requires one or more transcriptome assemblies and the reads used to generate those assemblies. TransRate aligns the reads to the assembly, processes those read alignments, and calculates contig scores using the full set of processed read alignments. TransRate classifies contigs into those that are well assembled and those that are poorly assembled, by learning a score cut-off from the data that maximizes the overall assembly score.Reads are aligned to a given assembly using SNAP v1.0.0 . Alignmen = 5917) for the Saccharomyces cerevisiae genome were downloaded from http://www.yeastgenome.org/. The transcripts were quantified and mRNA abundances recorded using Salmon, and the same set of reads used in the de novo assembly evaluation is described in “Analysis of assemblies generated from real reads.” To simulate transcript chimeras, 1000 transcripts were selected at random without replacement from the complete set of transcripts. Pairs of transcripts (n = 500) were fused in silico by concatenation of two of the randomly selected full-length transcript sequences head-to-tail. These 500 transcript chimeras were placed back into the reference transcriptome file (replacing both of their constituent transcripts) such that the transcriptome submitted to TransRate contained the 500 chimeric transcripts and the 4917 transcripts that were not chimeric (n = 5417). The transcriptome was subject to assessment with TransRate using the same set of RNA-seq reads. This process was repeated 20 times to obtain the results for the analysis of 10,000 chimeras. The s(Cseg) score for each transcript chimera was compared to the difference in the relative abundance of the constituent transcripts in the chimera.The complete set of transcripts . This score measures the extent to which the nucleotides in the mapped reads are the same as those in the assembled contig. If the mapped reads do not support the nucleotides of the contig, then this is likely because either the nonsupportive reads should map to a different contig or to a contig that is not represented in the assembly , or the assembled sequence is incorrect. In the case of the former, a missing contig  will negatively affect the score of the contig to which its reads incorrectly map. Although the contig to which they map may be correctly assembled, the negative score for this contig can be justified because the incorrectly mapped reads will render the abundance estimate of the assembled contig invalid. In the case of the latter, disagreement between the reads and the contig must be due to misassembly. To ensure that stochastic read errors that result in disagreement between a read and a contig do not affect the overall score for that contig, support for an alternative nucleotide sequence needs to be provided by multiple reads (see below).The identity of the nucleotides in the contig will accurately represent the nucleotides of the true transcript s(Ccov). This score measures the proportion of nucleotides in the contig that have zero coverage, and thus have no supporting read data. If there are nucleotides in the contig that are not covered by any reads (regardless of the agreement between the reads and the sequence of the contig), then this should negatively affect the contig score.The number of nucleotides in the contig will accurately represent the number in the true transcript, s(Cord). This score measures the extent to which the order of the bases in contig are correct by analyzing the pairing information in the mapped reads. Here, if the orientation of the mapped reads does not conform to an expected mapping estimated from an analysis of a subsample of mapped read pairs, then these incorrectly mapping reads will negatively affect the contig score. Similarly, if the contig could have been extended, i.e., there are read pairs that map such that one read is present near a terminus of the contig and its pair is not mapped and would be expected to map beyond the scope of the contig, then such cases indicate that the contig does not use all of the available reads, and thus is incompletely assembled. This metric is informative for the identification of partially assembled transcripts.The order of the nucleotides in the contig will accurately represent the order in the true transcript, s(Cseg). This score measures the probability that the coverage depth of the transcript is univariate, i.e., that it represents an assembly of a single transcript and not a hybrid/chimeric assembly of multiple transcripts expressed at different expression levels. Here, the per-nucleotide coverage depth of the contig must be best modeled by a single Dirichlet distribution (described below). If the contig is better modeled by the product of two or more Dirichlet distributions, then this indicates that two or more contigs with different transcript abundances have been erroneously assembled together.The contig will represent a single transcript, The TransRate contig score is the product of the scores for each of these properties using the aligned reads as evidence. These four properties are evaluated next.TransRate outputs scores for every contig. Here, an assembly consists of a set of contigs s(Cnuc).The alignment edit distance is used to quantify the extent to which the contig sequence is correct. The alignment edit distance is the number of changes that must be made to the sequence of a read in order for it to perfectly match the contig sequence. Here, the edit distance of an aligned read This score is evaluated as the fraction of nucleotides in the contig that receive at least one mapped read irrespective of the agreement between the read and the contig.s(Cord) is then evaluated as the proportion of all mapped read pairs that are correct.The pairing information of the mapped reads is used to evaluate this score. To determine the parameters of the read library preparation, a randomly selected subsample of 1% of all mapped read pairs are analyzed. From these alignments, the orientation of the paired-end reads is determined, and the mean and standard deviation of the fragment size is inferred. All read pair alignments are then classified according to whether they are plausible given the estimated parameters of the library preparation and assuming that the assembled contig is correct. A read pair is considered correct if the following criteria are met: (1) Both reads in the pair align to the same contig; (2) the relative orientation of the reads in the pair is consistent with the inferred library preparation parameters; and (3) the relative position of the reads is consistent with the mean and standard deviation of the inferred fragment size. 2 of the read depth rounded to the nearest integer. As the probability will be a value between 0 and 1, this probability is used directly as s(Cseg).The per-nucleotide read coverage data is used to evaluate this score. To evaluate the probability that the contig originates from a single transcript , a Bayesian segmentation analysis of the per-nucleotide coverage depth is performed. For a correctly assembled contig, it is assumed that the distribution of per-nucleotide coverage values in that contig is best described by a single Dirichlet distribution, i.e., all nucleotides in the same transcript should have the same expression level, and thus should be best modeled as a stochastic sample from a single distribution. In contrast, a contig that is a chimera derived from concatenation of two or more transcripts will have per-nucleotide coverage values that are best described by two or more different Dirichlet distributions. The probability that the distribution of per-nucleotide read coverage values comes from a single Dirichlet distribution is evaluated using a Bayesian segmentation algorithm previously developed for analysis of changes in nucleotide composition . To faciT) is evaluated as the geometric mean of the mean contig score and the proportion of read pairs that map to the assembly such thatThe aim of the TransRate assembly score is to provide insight into the accuracy and completeness of any given assembly. Thus, the assembly score weights equally a summary statistic of the TransRate contig scores and the proportion of the input reads that are contained within this assembly. We note here that alternative methods for summarizing contig scores that weight contig scores by their expression level would produce different results. However, such schemes would not be consistent with the problem definition and aim of TransRate: to assess the accuracy and completeness of a de novo assembled transcriptome using only the input reads. This score assumes that an ideal assembly will contain a set of contigs that represent unique and complete transcripts to which all of the reads used to assemble those transcripts can be mapped. The TransRate assembly score (s(C) is as defined in equation TPM is the transcripts per million transcripts value assigned to that contig by Salmon. Under this framework; highly abundant transcripts that have assembly errors are penalized more heavily than low abundance transcripts with the same errors. The abundance-weighted assembly score (Tw) is thus evaluated asAn abundance-weighted contig and assembly score are also provided by TransRate. The contig score is evaluated asOryza sativa) and mouse (Mus musculus) using the Oases, Trinity, and SOAPdenovo-Trans assemblers (Homo sapiens) and yeast (Saccharomyces cerevisiae) that had been assembled with Oases and Trinity  (The full set of coding and noncoding transcripts for each species was downloaded from Ensembl Genomes version 25 (ftp://ftp.ensemblgenomes.org/pub/release-25/). Assembled contigs were then identified by BLAST searching the reference data set for the corresponding species using bidirectional blastn local alignment with an e-value cut-off of 10 2.2.29) . Only reFor each species, a total of 10 million mRNA molecules were simulated from the full set of annotated mRNAs from the Ensembl reference with exponentially distributed expression values using the flux simulator v1.2.1 . mRNA moF-score whereF-score and the TransRate contig score was calculated using R version 3.1.1 . De novo assembled transcriptomes were used in this analysis only if paired-end reads were provided, the assembler and species were named in the metadata, and the assembly contained at least 5000 contigs . For each of these test data sets, the assembly and reads were downloaded. TransRate was run on all assemblies, and FastQC version 2.3 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) was used to evaluate the quality of the read data sets.A survey of the range of assembly scores for published de novo transcriptome assemblies was conducted by analyzing a subset of transcriptome assemblies from the Transcriptome Shotgun Archive (http://github.com/Blahah/transrate and is released under the open source MIT license. Binary downloads and full documentation are available at http://hibberdlab.com/transrate. The software is operated via a command line interface and can be used on OSX and Linux. TransRate can also be used programmatically as a Ruby gem.TransRate is written in Ruby and C++ and makes use of the BioRuby  and BAMt


The presence of a foreign body in the periapical tissues can cause endodontic failure by triggering an inflammatory response and a subsequent foreign body reaction. This inflammatory response, which can occur to varying degrees, appears radiographically as a radiolucency that can remain asymptomatic for many years. A foreign object can reach the apical region by accident or iatrogenic procedures during dental procedures. The aim of the present case report is to describe the endodontic surgical treatment of an iatrogenic displacement of a foreign body  into the periapical tissues and to describe its clinical and radiographic follow-up over a period of 52 months. Root canal therapy is a reliable and predictable procedure with high short- and long-term success rates; nevertheless, endodontic failure can occasionally result from this treatment. Endodontic failure is usually associated with the persistence of bacteria within the root canal system ; howeverThe apical periodontium can encapsulate different irritants with collagen fibers and even has been shown to heal in the presence of a foreign object . NonetheForeign bodies can be forced into the periapical region by three different ways: (i) compacting food in teeth with a necrotic pulp and destroying the pulp chamber , (ii) a Our case is of a 25-year-old male patient who came into the clinic for a dental check-up of tooth number 21 , which presented a gradual colour change of the clinical crown with regard to the neighbor teeth over the past three years. Although this tooth was completely asymptomatic, the patient reported that he had suffered dental trauma 15 years ago when he fell while practicing sports, resulting in a complicated crown fracture. The day after the accident, his parents took him to see a dentist who simply “placed a screw and sealed the tooth with tooth-colored material.” The medical history revealed no history of systemic problems or allergies.Upon clinical inspection, a resin was observed at the palatal area and incisal edge, and the buccal and palatal mucosa appeared normal. There was no response to vertical or horizontal percussion or to palpation of the mucosa. Probing depth and tooth mobility were both within normal limits, and the adjacent teeth responded normally to both thermal  and electrical  tests. The radiographic inspection revealed the presence of an intraradicular post at the crown level, which was surrounded until the middle third of the root by sealing material . A wide Once all of the aforementioned information was collected, a pulpal diagnosis of previously initiated endodontic therapy with signs of infection was established, with periapical diagnosis of chronic apical periodontitis combined with iatrogenic displacement of a foreign body into the periapical tissues. Thereby, we decided to perform a surgical endodontic treatment of the tooth, including the retrograde removal of the foreign object, retrograde instrumentation, and retrograde obturation with a thermoplasticized gutta-percha injection. We concluded that trying to remove the foreign object using an orthograde approach could weaken the fragile immature root structure and could risk sending the object into a deeper anatomic area, such as the floor of the nasal cavity. The intentional replantation was considered only a last option due to the high probability of root fracture at the time of extraction. The initial prognosis for this patient was favourable.After performing a regional anaesthetic block with 1 : 100,000 articaine and waiting 10 min for deep action, a total thickness triangular flap was raised and the root apex was located with the use of long-shank round carbide bur number 4 mounted on a high speed hand piece with no direct air to the working area, under abundant saline irrigation delivered with a hypodermic syringe. After locating and exposing the periapical lesion, all tissue within the bony crypt was removed with a curette to locate the foreign body  , which wResin was detected inside the root canal using a retrograde approach with an endodontic explorer; thus, there was no need for a retrograde preparation of the entire length of the main canal. The ultrasonic retrograde preparation of the 3 mm apical portion was performed using ultrasonic tips . Low intensity and very light pressure were used to prevent the creation of microfractures in the thin walls of the immature apex, and abundant saline irrigation was provided drop by drop at a distance. After drying the retrograde cavity with absorbent paper points, the cavity was filled by retrograde obturation with thermoplasticized injected gutta-percha , which was compacted and its placement was confirmed radiographically once it had cooled . The flaOne month later, the patient returned completely free of any symptoms and signs of infection, and probing depth and tooth mobility were both within normal limits. It was decided to change the crown restoration to prevent the coronal leakage of saliva; however, the patient returned for a follow-up after 12 months, at which time proper radiographic healing of the periapical lesion was observed, and no symptoms or signs of infection were found  and clinThe expulsion of a foreign body to the periapical region is an unfortunate accident that can occur during dental procedures and could cause endodontic failure by triggering inflammation and a foreign body reaction , 5.In this case, conservative treatment using an orthograde approach implies high risks, due to the fact that an incomplete root formation is present, because the object can move into deeper anatomical areas, cause excessive wear of the main root canal, or even fracture the fragile structure of the immature root, despite the use of a dental operating microscope. Fortunately, these types of accidents are rare, and relatively few have been reported in the literature , 9, 10. For many years, the appropriate selection of retrograde filling material has been the subject of debate. The use of gutta-percha for this purpose was considered a good choice because it is biocompatible with human tissues, can be adapted to the irregular walls of the retrograde cavity, is malleable, can absorb some moisture of the periapical tissues, and does not corrode , 12. GolA well-established alternative, mineral trioxide aggregate (MTA), provides good clinical and histological results, which makes it the retrograde filling material of choice, largely due to its physical/chemical/biological properties . HoweverThis case shows that the iatrogenic displacement of a foreign body into the periapical tissues through an immature root canal can be treated successfully through surgical endodontic treatment, as shown by the clinical and radiographic follow-up after 52 months.


The aim of the proposed study is to perform a comparative analysis of functional training effects for the paretic upper limb with and without transcranial direct current stimulation over the primary motor cortex in children with spastic hemiparetic cerebral palsy.P ≤ 0.05).The sample will comprise 34 individuals with spastic hemiparetic cerebral palsy, 6 to 16 years old, classified at level I, II, or III of the Manual Ability Classification System. Participants will be randomly allocated to two groups: (1) functional training of the paretic upper limb combined with anodic transcranial stimulation; (2) functional training of the paretic upper limb combined with sham transcranial stimulation. Evaluation will involve three-dimensional movement analysis and electromyography using the SMART-D 140® system (BTS Engineering) and the FREEEMG® system (BTS Engineering), the Quality of Upper Extremity Skills Test, to assess functional mobility, the Portable Device and Ashworth Scale, to measure movement resistance and spasticity, and the Pediatric Evaluation of Disability Inventory, to evaluate performance. Functional reach training of the paretic upper limb will include a range of manual activities using educational toys associated with an induced constraint of the non-paretic limb during the training. Training will be performed in five weekly 20-minute sessions for two weeks. Transcranial stimulation over the primary motor cortex will be performed during the training sessions at an intensity of 1 mA. Findings will be analyzed statistically considering a 5 % significance level (This paper presents a detailed description of a prospective, randomized, controlled, double-blind, clinical trial designed to demonstrate the effects of combining transcranial direct current stimulation over the primary motor cortex and functional training of the paretic limb in children with cerebral palsy classified at level I, II, or III of the Manual Ability Classification System. The results will be published and evidence found may contribute to the use of transcranial stimulation for this population.RBR-6V4Y3K. Registered on 11 February 2015.ReBEC  Cerebral palsy is a term used for motor development disorders stemming from a primary brain lesion that are permanent and mutable, causing secondary musculoskeletal problems and limitations regarding activities of daily living . Motor iDepending on the topographic distribution, motor impairment is categorized as tetraplegia when all limbs are affected, which accounts for 9–43 % of cases, diplegia when upper limb involvement is milder than lower limb involvement, which accounts for 10–33 % of cases, and hemiplegia when only one side of the body is affected, which accounts for 25–40 % of cases. The upper limbs are affected in 50–70 % of individuals with cerebral palsy , 7. TogeChildren with hemiparesis have limitations regarding the use of the affected upper limb and, consequently, two-hand coordination; this exerts a negative impact on activities of daily living and participation at school, in the community, and in family life . SpasticPrimary upper and lower limb impairment due to spasticity, muscle weakness, and deficient motor control can give rise to secondary musculoskeletal complications, such as contractures and deformities, resulting in restricted movements. Functional limitations can result in motor deficiency, as well as impairments with regard to sensation, perception, cognition, behavior, and communication , 11. AccTranscranial direct current stimulation is a promising non-invasive therapeutic resource for the treatment of children with cerebral palsy in which the motor cortex is stimulated using a low-intensity (1–2 mA), monophasic, direct, electrical current through surface electrodes. The advantages of transcranial direct current stimulation over other transcranial stimulation methods are the longer-lasting modulating effect on cortex function, ease of administration, and lower cost. Moreover, this type of intervention allows better placebo stimulation, thereby conferring greater specificity to scientific findings , 17.The effects of stimulation are achieved by the movement of electrons. The two electrodes are an anode with a positive charge and a cathode with a negative charge. An electrical current flows from the positive to the negative pole and has different effects on biological tissues. Although most of the current is dissipated among the tissues over the cortex during transcranial direct current stimulation, a sufficient amount reaches the cortex structures and modifies the membrane potential of local cells , 19.Transcranial direct current stimulation has short-term effects on cortex excitability when administered for short periods and longer-lasting effects related to plastic mechanisms when administered over a longer period . A numbeIn the rehabilitation process, the aim of neuromodulation techniques is to increase local synaptic efficacy, thereby altering the maladaptive plasticity pattern that emerges following a cortex lesion. The possibility of combining physical therapy modalities is one of the advantages of transcranial direct current stimulation. Stimulation is a way to modulate cortex activity by opening a path to enhance and prolong functional gains achieved through physical therapy. Stimulation allows a change in a dysfunctional excitability pattern through the activation of specific neural networks so that physical therapy can mold a functional cortex activity pattern . StudiesAnalyzing the effects of transcranial magnetic stimulation, a recent study  reports The primary objective of the proposed study is to analyze manual ability before and after functional training of the paretic upper limb with anodic and sham transcranial direct current stimulation over the primary motor cortex  in children with spastic, hemiparetic cerebral palsy, classified at levels I, II, or III of the Manual Ability Classification System .Functional reach training of the paretic upper limb with anodic transcranial direct current stimulation over the primary motor cortex will achieve greater effects in comparison to functional reach training with sham transcranial direct current stimulation in children with spastic, hemiparetic cerebral palsy classified at level I, II, or III of the Manual Ability Classification System .A prospective, paired, randomized, controlled, double-blind clinical trial is proposed Fig. . The proIndividuals with hemiparetic cerebral palsy will be recruited from the physical therapy clinics of the Nove de Julho University, São Paulo, Brazil, and will be selected based on the following eligibility criteria.Diagnosis of spastic, hemiparetic cerebral palsyFunctional classification at level I, II, or III of the Manual Ability Classification System Aged 6 to 16 yearsAdequate understanding and cooperation during the proposed activitiesStatement of informed consent signed by a legal guardianHaving undergone surgical procedures or neurolytic block in the 12 months prior to the onset of the training sessionsOrthopedic deformity with indication for surgeryEpilepsyMetal implant in the skull or use of hearing aidsThe sample size was calculated using the STATA 11 program, using a study conducted by Choudhary et al.  as the bChildren who meet the eligibility criteria will be randomly allocated to one of the two study groups using block randomization.This group will receive the intervention, that is, functional reach training of the paretic upper limb with range manual activities combined with active transcranial direct current stimulation over the primary motor cortex of the ipsilesional hemisphere.This will be the ‘control’ group and will receive functional reach training of the paretic upper limb with range manual activities combined with sham transcranial direct current stimulation over the primary motor cortex of the ipsilesional hemisphere.The children will be randomly allocated to the two groups. To minimize the risk of an imbalance in the size of the groups, a randomization list will be generated using five blocks of six participants, with three participants in each block randomly allocated to each group, and one block of four participants, with two participants in each block randomly allocated to each group. The allocation sequence will be stipulated in sequentially numbered, sealed, opaque envelopes. Following the baseline evaluation, each participant will be allocated to one of the groups by opening an envelope. This process will be performed by a member of the research team who is not involved in the recruitment process or other aspects of the study.Pre-treatment evaluationEvaluation immediately following a single intervention sessionPost-treatment evaluationEvaluation 1 month after the end of treatmentEvaluation 3 months after the end of treatmentThe evaluation process will be conducted by two physiotherapists with experience in the evaluation procedures and blinded to the allocation of the participants to the different groups. Evaluations will be conducted in the following manner:Evaluations will be conducted on three non-consecutive days with a maximum period of 1.5 hours per day.Upper-arm movement kinematics will be evaluated using the SMART-D 140® system , which involves eight cameras sensitive to infrared light with a sampling frequency of 100 Hz and a synchronized video system. Passive markers will be positioned on anatomic reference points following the SMARTup protocol Fig. . The marThe positioning of the upper limb will be reconstructed using markers. Twelve markers will be positioned bilaterally in the upper limbs: acromion, lateral epicondyle of the humerus, styloid process of the ulna and radius, head of the second metacarpus and nail of the index finger. The head position will be reconstructed using four markers: two symmetrically in the frontal bone near the timeline and two symmetrically at the back of the head. The trunk position will be estimated using markers positioned on the C7 spinous process and in the jugular notch. An additional marker will be positioned on the target of the movement trajectory.SMART Analyser software program . To evaluate changes after the intervention, the following variables will be identified in each session and the mean of the results will be calculated [Based on data published by Menegoni , the movlculated , 33, 35.This is calculated as the total time required for completing each task. The duration of the three phases  will also be computed.This is the mean velocity of the marker positioned on the index finger.This is the length of the path described by the fingernail in the adjusting phase; it is a measure of the adjustments made to reach the final position and represents the degree of precision.This is the ratio of the length of the fingernail path to the linear distance between the initial and the final pointing position; representative of movement smoothness during the going phase.This is the mean value of the acceleration derivative (jerk) according to the equation:x(t), y(t) and z(t) are the x, y and z coordinates of the fingernail and T is the movement duration. It has been shown that the average jerk index decreases with the increase in the smoothness of movement. This index is often used as a measure of the quality of selective motor control [in which  control , 37.This is calculated as the difference between the maximum and minimum values of the angle between the frontal plane (shoulder) and sagittal plane (elbow and shoulder) during the going phase , 33, 3515 Ω//0.2 pF and 60/10 Hz 92 dB, respectively. The motor point of the muscles will be identified and the skin will be cleaned with 70 % ethanol to minimize bioimpedance, following the recommendations of SENIAM 8 [Electromyography is the most widely used assessment tool for the study of muscle activation during exercise and the intensity of contractions . The eleSENIAM 8 . ElectroThis test will be used for the evaluation of manual function. It was developed to evaluate the movement patterns of normal development, which are the basis for the determination of functional upper limb performance. Evaluation of upper limb skills is divided into four areas: dissociated movements (19 items), grasp (6 items), weight bearing (5 items), and protective extension (3 items) . ParticiThe Modified Ashworth Scale  will be Elbow extension and flexion will be analyzed. The scale will be administered by physiotherapists blinded to the allocation of the participants to the different groups. The Biomedical Technology Laboratory of the Polytechnic University of Milan has developed a portable device to measure the angle: momentum ratio to allow a high degree of precision in the evaluation of hypertonia. This device consists of two inclinometers and a strain gauge momentum sensor , which r2 moistened in saline solution. The participants will be randomly allocated to active or sham transcranial direct current stimulation over the primary motor cortex following the procedures described by Fregni et al. [Transcranial direct current stimulation will be administered using a DC-STIMULATOR  with two sponge  electrodes measuring 25 × 25 cmi et al. . The anoi et al.  will be Cross-sectional analysis will be conducted to determine the immediate effects of transcranial direct current stimulation. For this, the participants will be submitted to a single 20 min session of transcranial direct current stimulation over the primary cortex while at rest one month prior to the onset of the main intervention. The participants will first be instructed regarding the procedure and will remain at rest for 20 min with the electrodes in place (as described in the previous item). The evaluations will involve QUEST, three-dimensional movement analysis, surface electromyography of the biceps and triceps brachii muscles and the measurement of spasticity of the paretic upper limb. A first evaluation will be made after 20 min of rest and a second evaluation will be made after 20 min of transcranial direct current stimulation (active or sham). The raters will be blinded to the allocation of the participants to the different groups.The therapy will be based on manual reach with the induced constraint of the non-paretic limb during the transcranial direct current stimulation session following the protocol described by Hoare et al. . In eachAfter the transcranial direct current stimulation intervention protocol and until the final evaluation (three-month follow-up), the patients will receive physical therapy in two 1 hour sessions per week, directed at the functional activities of gait as well as passive stretching of the trunk, elbow, and wrist of the paretic upper limb to maintain the ranges of motion. No training or unilateral or bilateral stimulation of the upper limbs will be performed, to avoid any conflicts in the expected results of the study.t test for continuous variables. Analysis of covariance (ANCOVA) will be employed to evaluate the effects of transcranial direct current stimulation on the outcome variables of upper limb function. For all analyses, the fixed independent variables will be group  and evaluation . The dependent variables at baseline will be considered covariables: QUEST, outcomes obtained from three-dimensional movement analysis, electromyography, and spasticity. The ANCOVA will be used to compare effects after the interventions , adjusting individual performance prior to transcranial direct current stimulation. This model was considered to be an advantageous approach for statistical analysis considering the nature of the study . Cohen’s d (effect size) will be calculated based on the difference in values between baseline and the post-intervention evaluation, with the comparison of the groups. Statistical significance will be considered for P < 0.05. Data will be organized and tabulated using the Statistical Package for the Social Sciences .Statistical analysis will be performed using intention-to-treat analysis. If data losses occur during the study, ‘last observation carried forward’ analysis will be employed to adjust the missing data in the follow-up evaluations. The data will be expressed as mean and standard deviation values. The two intervention groups will be analyzed for differences in anthropometric characteristics as well as clinical and functional variables using the chi-square test for categorical variables and the This paper presents a detailed description of a prospective, randomized, controlled, double-blind, clinical trial designed to demonstrate the effects of combining transcranial direct current stimulation over the primary motor cortex and functional training of the paretic limb in children with cerebral palsy classified at levels I, II, or III of the Manual Ability Classification System. The results will be published and evidence found may contribute to the use of transcranial stimulation for this population.This study protocol used defining standard protocol items for clinical trials. SPIRIT 2013 Statement .Patients are being recruited at the time of submission.ANCOVA, analysis of covariance; CONSORT, Consolidated Standards of Reporting Trials; QUEST, Quality of Upper Extremity Skills Test; ReBEC, Brazilian Registry of Clinical Trials; SPSS, Statistical Package for the Social Sciences