The increasing use of ontologies highlights the need for a library for working with ontologies that is efficient, accessible from various programming languages, and compatible with common computational platforms.owlcpp, a library for storing and searching RDF triples, parsing RDF/XML documents, converting triples into OWL axioms, and reasoning. The library is written in ISO-compliant C++ to facilitate efficiency, portability, and accessibility from other programming languages. Internally, owlcpp uses the Raptor RDF Syntax library for parsing RDF/XML and the FaCT++ library for reasoning. The current version of owlcpp is supported under Linux, OSX, and Windows platforms and provides an API for Python.We developed owlcpp is significantly more efficient in terms of memory usage and searching RDF triple stores. owlcpp performs strict parsing and detects errors ignored by other libraries, thus reducing the possibility of incorrect semantic interpretation of ontologies. owlcpp is available at http://owl-cpp.sf.net/ under the Boost Software License, Version 1.0.The results of our evaluation show that, compared to other commonly used libraries, The online version of this article (doi:10.1186/s13326-015-0035-z) contains supplementary material, which is available to authorized users. Ontologies are being increasingly recognized as important information resources that capture descriptive information in a standardized, structured, and computable form. One of the most widely used approaches for representing ontologies is the family of languages referred to as the Web Ontology Language (OWL) . The OWLWorking with OWL ontologies involves several common procedures, including parsing ontology documents, storing them as RDF triples and axioms, querying and serializing their in-memory representation, passing the axioms to a reasoner, and performing logical queries. Given the increasing size of ontologies, it is extremely important to have software for working with OWL ontologies that can perform these procedures efficiently.During the last decade, many open-source libraries useful for working with OWL ontologies written in RDF+XML format have become available. These, however, fail to fully meet the needs of software developers building software for working with OWL ontologies. First, existing libraries do not scale well enough to support ontologies of over a few million triples , 7. In aowlcpp, is designed to support a common workflow where OWL ontologies written in RDF/XML are loaded from the file system and submitted to a reasoner for processing  supports fast loading and searching of large ontologies, (ii) has a small memory footprint, (iii) provides cross-platform compatibility, and (iv) can be accessed from multiple programming languages. The resulting library, ing Fig. . owlcpp on Table .Fig. 1Woowlcpp were based on providing the four key features listed above. Specifically, owlcpp is developed in ISO-compliant C++03 [owlcpp was designed to be concise without sacrificing usability and performance. In the interest of clarity and thread-safety, class methods and function arguments were declared const wherever possible.The design choices in implementing nt C++03 , which ent C++03 , and comowlcpp with different platforms was verified by compiling the library and executing the unit tests on the following platforms and compilers: Linux, Ubuntu 14.04 64-bit ; Windows 7 64-bit ; Mac OS X 10.6.8 (i686-apple-darwin10-gcc 4.2.1); Windows XP 32-bit .The compatibility of owlcpp comprises three modules, rdf, for storing and searching RDF terms and triples; io, for loading ontology documents; and logic, for converting triples into axioms and passing them to a reasoner. The io module depends on the Redland Raptor [logic module depends on FaCT++ [io and logic modules have different external dependencies and can be built and used separately from each other. owlcpp also uses many of the Boost libraries, e.g., iterator, multi-index, and file system [Currently, d Raptor , libXML2d Raptor , and icod Raptor  librarien FaCT++ , 25. Thee system .owlcpp, as well as those of other key libraries, are shown in Table The basic features of rdf module implements classes and methods as needed to support the RDF standard. To accommodate the demands of working with large ontologies, the design priorities for the module were compact in-memory representation of RDF terms and triples, and their efficient search and retrieval. The Triple_store class is the main container provided by the module. It supports storing, retrieving, and searching for prefix IRIs, RDF terms, ontology document descriptions, and RDF triples  store Node_id’s for the ontology and version IRIs and for the file system path for the ontology document.The Triple class represents RDF triples by defining a combination of subject, predicate, and object terms. Since each term may appear in many triples, the Triple class stores a light-weight ID rather than a value for each of the terms. In addition, Triple stores the ID of the source document.Since term IDs do not distinguish between different types of terms, the Triple class cannot, by itself, enforce the type restrictions on its terms. For example, it is possible to create a triple where the subject term ID refers to a literal or where the predicate term ID refers to a blank node. While such triples will not be created during normal ontology parsing, it should still be noted that the Triple class implements a generalized RDF triple .owlcpp stores triples in several indices. Within an index, triples are separated into bins according to one term and sorted within each bin according to the other terms. For example, an index in which triples are binned by subject and sorted by predicate, object, and document can help to efficiently identify the triples matching a subject and a predicate. On the other hand, if matching an object and a predicate is required, an index configured to bin by object and sort by predicate, subject, and document is expected to perform better.Searching stored triples is a frequent, performance-sensitive operation. The types of searches are application dependent and may involve matching any combination of subject, predicate, object, and document, while leaving other elements unspecified. To efficiently perform the required types of searches, owlcpp uses two indices: (i) bin by subject and sort by predicate, object, and document, and (ii) bin by object and sort by predicate, subject, and document. These defaults were selected because they were empirically found to perform best for axiom generation, as described below.Since the indices have a significant memory footprint, the user can define the number and type of indices during compilation. By default, Searching stored RDF triples is done using the find_triple method provided by Triple_store. The method is designed to automatically select the optimal search procedure based on the type of query and the available triple indices. The results of the search are returned as an iterator range , which iio module provides several methods for loading RDF/XML ontology documents to a triple store. The documents can be loaded from the C++ Standard Library input streams using method load or directly from the filesystem using load_file. Loading an ontology document involves reading data from a stream, parsing XML structures, interpreting them as RDF statements, converting the statements into RDF terms and triples, and inserting them into a triple store. Currently, the Redland Raptor library is used as an RDF parser [The F parser , which, F parser  for parsF parser  for chario module is designed for early detection of and recovery from errors. The errors may originate at different levels of the document loading process, such as opening a file or parsing XML, RDF, or OWL. An error occurring for any reason during document loading aborts the process by throwing an exception containing detailed information about its causes. In addition, instead of producing a triple store that contains an uncertain number of triples from the document that caused the error, the exception leaves the triple store in a valid state with none of the document’s content in the triple store. This behavior allows the user to utilize the existing triple store content or to attempt loading the document again.The io module provides a mechanism to automatically load the imports from the file system. To be able to locate the documents, the module implements a Catalog class that stores document descriptions and maps the document, ontology and version IRIs to file system paths. The module also provides a method for scanning file system directories for ontology documents and adding their metadata to the catalog. Supplying a Catalog object to the load or load_file methods causes the module to also load the imported documents to the triple store. Automatically loading documents from the Internet is not currently supported by the module because this feature introduces a significant uncertainty to the success and performance of ontology loading and adds complex operating system-specific dependencies to the library.OWL documents often import the contents of other documents, identifying them by their ontology IRIs and version IRIs. The logic module is responsible for translating RDF triples into OWL axioms and facilitating interaction with reasoners. Translation of triples to axioms is implemented by following the W3C Recommendation [The endation . If the Frequent search operations make configuration of triple indices an important factor affecting axiom generation performance. The optimal configuration was identified empirically by comparing axiom generation times using eight hand-picked index configurations and three different ontologies: the Ontology of Biological Pathways (OBP) , the Ontlogic module works with FaCT++, which is, to our knowledge, the only open-source C/C++ reasoner library for OWL DL [Currently, the r OWL DL . Logicalowlcpp does not provide explicit support for concurrency, similar to C++ Standard Library containers, it is designed to maximize the number of thread-safe operations without penalizing performance. Operations that do not change the state of owlcpp containers  are guaranteed to be thread-safe. On the other hand, if multiple threads concurrently access an owlcpp container object and at least one of the threads modifies its state , the behavior is undefined. Therefore the user is expected to ensure that a modifying thread obtains exclusive access to owlcpp containers.Although owlcpp is based on Boost.Build and is compatible with both Unix-like and Windows platforms [owlcpp Python bindings, and produces API documentation using Doxygen [The build system for latforms , 33. It  Doxygen .owlcpp modules. The tests for the io and logic modules make use of many small sample ontology documents that are part of the project source tree. Some of the documents are designed to test error detection at the XML, RDF, or OWL level. Some of the sample documents used by the logic module tests were adapted from the OWL 2 Test Cases [Unit tests comprise approximately 20 % of the library source code and cover most of its functionality. A separate test suite is implemented for each of the st Cases . The tesowlcpp includes bindings for Python developed using the Boost.Python library. The functionality of the bindings is verified by a separate unit test suite. The bindings and their dependencies are packaged by the build system into a distributable Python module. APIs for other programming languages can also be exposed from owlcpp relatively easily and with minimal overhead. In future versions of owlcpp, these will be provided with the help of the SWIG library [The current version of  library .owlcpp, we compared the document loading time, triple query time, and memory foot print of owlcpp with those of Redland, Jena, and OWL API. Evaluation was conducted using five ontologies of varying size and composition: the Vertebrate Taxonomy Ontology (VTO) [To evaluate gy (VTO) , Medicalgy (VTO) , Drug Ongy (VTO) , Biomodegy (VTO) , and Opegy (VTO) . In addiowlcpp was compiled with gcc v4.8 using the default triple index configuration. Redland v1.0.13 with a hashed in-memory store, Jena v2.12.1 with an in-memory RDF store, and OWL API v4.0.1 were used for comparison, also with their default settings. The source code for performance tests can be found in Additional file owlcpp v0.3.5.To test the ability of the library to process large ontologies on off-the-shelf hardware, the tests were conducted on an underpowered, by current standards, computer. Performance was tested on a laptop with an Intel Core2 Duo T7700 2.40GHz CPU, 3GB of RAM, running Linux Ubuntu 14.04 64-bit. The performance of Java libraries was tested using Oracle Java JDK v1.7.0_51 with a 2 GB maximum memory pool (-Xmx2048m). Unless noted otherwise, owlcpp ranges from 3.1 to 6.9 MB/s, while the range for the other libraries is from 2.6 to 7.2 MB/s. The owlcpp loading rate is faster than that of Jena for the five ontologies Jena could load and faster than that of Redland for two of the three ontologies Redland could load. Redland had a faster loading rate than owlcpp for MeSH. In addition, the owlcpp loading rate is faster than that of OWL API for four of the six test ontologies. OWL API has a faster loading rate for both OpenGALEN full and part.To evaluate ontology loading performance, each of the six ontologies was loaded into each of the four libraries. Some libraries were unable to load the larger ontologies. Jena was unable to load the complete OpenGALEN ontology, while Redland failed to load the complete OpenGALEN, Biomodels, and DRON ontologies. An attempt to load the complete OpenGALEN into Redland on a system with 32GB of RAM was also unsuccessful. The loading rates (size of the ontology file system footprint divided by the recorded loading time) is shown in Fig. owlcpp had the smallest memory footprint for all ontologies ranging from 1.8 to 3.2 bytes of RAM required for each byte on the file system. The same ratio ranged from 7.0 to 11.5 for Redland, from 6.8 to 10.7 for Jena, and from 2.6 to 7.0 for OWL API.The amount of memory required by each library during ontology loading was estimated by probing the resident set size of the process virtual memory. The peak RAM utilization normalized by the size of the ontology on the file system is shown in Fig. owlcpp library showed significantly higher search rates ranging from 3.0 to 3.2 million queries per second (MQ/s). Jena showed significantly lower rates from 0.53 to 0.83 MQ/s. The rates for Redland ranged from 0.2 to 0.69 MQ/s. Note that OWL API was not included in this evaluation because it stores axioms rather than triples.Searching by subject and predicate is the most common triples search during axiom generation. Therefore, the triple search performance of the libraries was tested by repeating queries where the subject was selected at random, and the predicate was rdfs:subClassOf. For each query, all matching triples were identified and counted. The number of queries for each test was selected so as to keep the test time at about one minute. The number of queries performed by each library divided by the elapsed time is shown on Fig. owlcpp, we wanted to assess the accuracy of parsing and axiom generation. This was done using the OWL 2 Test Cases [owlcpp unit tests. Further testing was done during the development of the Ontology of Biological Pathways (OBP) [In addition to evaluating the performance of st Cases , some ofys (OBP)  by execuys (OBP) . The resowlcpp, helping to avoid incorrect semantic interpretation of ontologies and facilitating their development. Examples of errors detected by owlcpp but ignored by OWL API and Jena are undeclared property and annotation predicates and misspelled standard OWL terms.Strict error checking has proven to be an important feature of owlcpp is a C++ library providing support for storing and searching RDF terms and triples, for loading RDF/XML documents along with their imports into a triple store, for generating OWL axioms based on stored triples, and passing axioms to the FaCT++ reasoner. To the best of our knowledge, owlcpp is the first C++ library for working with OWL ontologies.owlcpp to have a concise and expressive C++ API and an efficient Python API. For example, loading an ontology file into an owlcpp triple store can be accomplished with just two lines of code, whereas the same operation through the Redland Raptor library API requires over a dozen lines [owlcpp, a single method, Triple_store::find_triple, can be used to search for triples matching a specific subject, predicate, object, document, or any combination thereof. The search is performed without sacrificing performance by selecting the most suitable triple index at compile time. The result of the search, an iterator range, can be used transparently to determine whether the triple store contains a triple matching the specified condition, to retrieve the first matching triple, or to iterate over all matching triples. On the other hand, the triple stores of both Redland and Jena define over ten different methods for searching triples.Our primary goal was to design a library for software developers that would scale well for working with large ontologies. To facilitate use by software developers, we designed en lines . The APIowlcpp is ensuring its scalability for use with large ontologies. Thus, we designed owlcpp to have a compact, in-memory storage of RDF terms and triples, efficient indexing of stored triples, and no virtual machine requirement. The latter facilitates owlcpp’s deployment in HPC environments. To evaluate the scalability of owlcpp, we compared its memory footprint, ontology document loading time, and triple query time with those of Jena, Redland, and OWLAPI. We found that owlcpp has a smaller memory footprint than the other three libraries for all ontologies tested : Cross-platform Programming language: C++, PythonOther requirements: Boost, libxml2, iconv (under Windows), Raptor, FaCT++License: Boost Software License–Version 1.0Any restrictions to use by non-academics: none


The results indicate that the epidendroid taxa include most of the clades that are successively sister to the grade of clades representing previously recognized tribes. Calypsoeae comprising four well-supported clades with 12 genera (except for the previous temporarily placed Wullschlaegelia) is supported as a monophyletic and sister clade to Epidendreae (excluding Coeliinae). The new orchid is nested in Calypsoeae and is a sister to Dactylostalix and/or Calypso. This new holomycotrophic orchid presents a subumbel inflorescence that grows underground, and flower with a long pedicel reputing the ground to open and two fragments at the base of the hook, which are obviously morphologically different from those of Calypsoeae. To accommodate this species in the current generic circumscription, a new genus Yunorchis was created.We identified a new holomycotrophic orchid that is related to the myco-heterotrophic Calypsoeae. Because chloroplast genes are primarily lacking or are highly divergent, key morphological characters are either reduced or lost from many myco-heterotrophs, and the phylogenetic relationships of weakly supported paraphyletic Calypsoeae within Epidendroideae have been poorly understood in previous molecular systematic studies. Using chloroplast  Nonphotosynthetic mycorrhizal plants have long attracted the attention of botanists and mycologists and have been the target of unabated controversy and speculation . WorldwiDanxiaorchis singchiana. The species of the Calypsoeae tribe are typically terrestrial or myco-heterotrophs  with nodes on a pseudobulb, a shrimp ridge velamen, and an occasionally lateral inflorescence with several to many flowered four pollinia in two pairs, except for Wullschlaegelia, which is characterized by two pollinia and was temporarily placed in Calypsoeae by Pridgeon et al. [Corallorhiza (including approximately 11 species), Govenia (20), Oreorchis (16), and Tipularia (7), which occupy a majority of the species of this tribe, and other genera are monotypic or oligotypic, especially the saprophytic genera. Calypsoeae are distributed from Europe, northern Asia and the Americas and absent from Africa, Australia, and the islands of the East Indies and Pacific Ocean [The tribe Calypsoeae  comprises approximately 12 genera and 70 species . Recentln et al.  based oneorchis 1, and TipWullschlaegelia and Govenia in Gastrodiinae and Cymbidieae, respectively [Yoania australis, a species that is endemic to New Zealand, was renamed Danhatchia australis and placed in Cranichideae (Orchidoideae) based on the anatomical features [Calypso, Changnienia and Tipularia, and the second group comprising Aplectrum, Corallorhiza, Cremastra, Danxiaorchis, Govenia, and Oreorchis. However, the relationships between the two groups in the context of the remainder of Epidendroideae remain unclear [Because of the reduction or loss of key morphological characters, the taxonomic affinities of many myco-heterotrophic Calypsoeae have remained elusive for many decades . The posectively . In addifeatures . The molfeatures . The resfeatures ,12,13,14Gastrodia, with a PP of 52 based on 26 genera samplings  in Epidendroideae. Although Xiang et al. [rbcL, matK and psaB), these authors sampled only one supported groups  of Calypsoeae, and the results indicated the two genera is sister to Dendrobieae/Malaxideae in a Bayesian inference with a PP of 0.80 and unsupported in Maximum parsimony analyses. Additionally, Pridgeon et al. [Dactylostalix and Ephippianthus form a well-supported clade within Calypsoeae based on the morphological characters. The family-wide analyses of Zhai et al. [Calypso clade based on the combined molecular markers  and morphological character matrix; however, the tribal-wide analyses formed a sister clade to the Calypso and Govenia clades. ITS was popular to permit systematic comparisons of low levels of taxa and chloroplast genes were preliminary lacking or divergent in many myco-heterotrophic Calypsoeae, both of which caused biases in inferred phylogenies [The presence of unusual winter leaves in some of the members of the two supported groups  is indicative of a close relationship . The resg et al.  sampled n et al.  indicatelogenies . These wrbcL, psaB and matK and the nuclear ITS and low-copy protein-coding gene Xdh to determine the circumscription and systematic positions of new myco-heterotrophic orchids and the Calypsoeae tribe in Epidendroideae and to infer molecular phylogenetic relationships within Calypsoeae.In this study, we used DNA sequences for the chloroplast genes psaB from the new myco-heterotrophic orchid (Yunorchis pingbianensis) failed. It is possible that this region is lacking. A total of 1047 bp of rbcL and 1458 bp of matK sequences (GenBank accession KM526763 and KM526774) were assessed, and none obvious deletion/addition loci were observed among the sequences.The general features of the DNA regions that were used are presented in rbcL and matK, 87 of psaB, and 70 of Xdh were obtained. The most variable dataset was Xdh with 47.25% potentially informative sites (excluding uninformative characters). Parsimony analyses resulted in a parsimonious tree of 2150 steps (TL) with a consistency index (CI) = 0.4647 and a retention index (RI) = 0.5843. The second variable dataset was matK , which had the lowest CI = 0.3560 and RI = 0.5182. The third most variable dataset was rbcL with 16.05% potentially informative sites. The least variable dataset was psaB with 11.75% potentially informative sites, which had the highest CI = 0.5835 and RI = 0.6291. In the combined three chloroplast markers  matrix, which comprised 4646 aligned nucleotides with 931 potentially informative sites (20.04%), TL = 5140, CI = 0.4811 and RI = 0.5309. In the combined chloroplast and nuclear Xdh markers (963 bp) matrix, which comprised 5609 aligned nucleotides with 1386 potentially informative sites (24.71%), TL = 7333, CI = 0.4739 and RI = 0.5438.In the subfamily-wide matrix, 104 DNA sequences of rbcL, 33 of matK, and 32 of ITS were obtained. The most variable dataset was ITS, with 30.46% potentially informative sites. Parsimony analyses resulted in a parsimonious tree of 641 steps with the lowest CI = 0.7285 and RI = 0.7573. The second most variable dataset was matK with 12.27% potentially informative sites, which had the highest CI = 0.8390 and RI = 0.9378. The least variable dataset was rbcL with 8.72% potentially informative sites. In the combined two chloroplast markers  matrix, which comprised 3000 aligned nucleotides with 322 potentially informative sites (10.73%), TL = 913, CI = 0.8160 and RI = 0.8918. In the combined two chloroplast and nuclear ITS markers (673 bp) matrix, which comprised 3673 aligned nucleotides with 527 potentially informative sites (14.35%), TL = 1593, CI = 0.7659 and RI = 0.8362  are indicated at each node. The branches that collapse in the strict consensus tree are indicated by “-”. In the nuclear Xdh analysis , is supported as monophyletic with weak support  and sister to Epidendreae (excluding Coelia).The results from the single and combined analyses were presented by the Bayesian trees. The values of the Bayesian Posterior probabilities (PP) and Bootstrap percentage  was performed. The trees of this analysis are shown in No strongly supported, incongruent patterns of relationship were detected in the individual chloroplast markers analyses, so a combined analysis of all of the chloroplast data  of the two supported groups indicate a close relationship. Therefore, the chloroplast and nuclear DNA were combined into a single dataset for the phylogenetic analyses , sister to the second group Calypsoeae (excluding Wullschlaegelia) with a stable topology, the third group Coelia (PP = 0.95) with an unstable topology, and the fourth group Agrostophyllinae with strong support (PP = 1.00 and BS >75). The new holomycotrophic orchid, as a sister to Calypso, is a member of the tribe Calypsoeae.In the Lower or “basal” Epidendroideae, highly supported clades include a monophyletic clade (PP = 1.00 and BS = 100) consisting of Neottieae sister to all of the remaining Epidendroideae, followed by Sobralieae (PP = 1.00 and BS > 50) and the other four tribes with a relatively weak bootstrap and an unstable topology. Higher Epidendroideae taxa include most of the clades that are successively (PP > 0.90 and BS > 50) sister to the grade of clades representing previously recognized tribes. Arethuseae are monophyletic as sister to all of the other Higher Epidendroideae with strong support (PP = 1.00 and BS > 90), followed by Cymbidieae (PP = 0.90 and BS = 65), and then Malaxideae plus Dendrobieae with a relatively weak bootstrap and an unstable topology. The next most supported clade (PP = 0.99) is divided into three tribes: Collabieae, Podochileae and Vandeae (excluding Agrostophyllinae) and strongly support (PP > 0.90 and BS >75) were observed between them. The last clade is divided into four groups, comprising the first group Epidendreae (excluding Oreorchis) receiving a paraphyly  and stable topologies in bootstrap analyses (BS = 100) between this group and others were observed. The new orchid Yunorchis pingbianensis is a sister to Dactylostalix ringens with a PP of 0.96, BSML of 64 and BSMP of 85.Based on the ITS sequence data, the relationships in the strict consensus tree for most of the genera are resolved and well supported (PP > 0.95 and BS > 75) with only one genus  and is followed by the others. Yunorchis pingbianensis, as a sister to Dactylostalix ringens, has been recognized as a natural genus within this tribe. Clade II includes only the genus Govenia, which is strongly supported  and is followed by a complex group (clade III), which includes Aplectrum, Cremastra, Danxiaorchis, Oreorchis, and Yoania. The topologies of this group were different, with a PP of 1.00 and a relatively weak bootstrap and an unstable topology of ML and MP phylograms (no show), and were subdivided into four subclades , with only 59190 bp, is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellate [Corallorhiza than in other leafless angiosperms that have been examined. The rbcL and matK have not been found in Rhizanthella [Corallorhiza [Yoania [Danxiaorchis and the new orchid. This gradually lost pattern seems that the leafless habit of orchids has developed several times and at least three times within Calyposeae.The chloroplast genome loci agellate . Freudenagellate  found muanthella  and a lollorhiza  and YoanrbcL, matK, and psaB sequences into a single dataset for the analysis of Epidendroideae, but the sampling and data support for Calypsoeae was weak. In this study, the data for the combined chloroplast genes  indicate that the tribe Calypsoeae is divided into two supported groups . The results of these chloroplast analyses are consistent with those that were obtained in previous studies [matK and ycf1 [rbcL, matK, psaB, and Xdh into a single dataset for the phylogenetic analysis of Epidendroideae. The sister relationship of Calypsoeae and Epidendreae was also recovered in the result of Nuebig et al. [ycf1 using parsimony analysis.The use of multiple genes is helpful in the accuracy of phylogenetic reconstruction ,20. Xian studies ,13,14. G studies  indicateand ycf1  and alsoand ycf1  and thosand ycf1 , but theand ycf1 . The Caland ycf1 . These rg et al.  based onCoelia (Coeliinae) to other genera have been problematic for more than a century. Based on multiple DNA  analyses, van den Berg et al. [Coelia is a sister to Calypsoeae. However, Pridgeon et al. [Coelia as a subtribe within Epidendreae based on their unpublished analyses. In our analyses  was far from the core Vandeae, being a sister to Epidendreae plus Calypsoeae. In our chloroplast analysis , Agrostophyllinae was a sister to one of the groups of Calypsoeae  and Calypsoeae in Epidendreae and the system position of Agrostophyllinae warrant further study.The relationships of g et al.  describen et al.  placed Canalyses , Coelia g et al.  indicateg et al.  recoverelypsoeae . In the lypsoeae  and comb results , the AgrWullschlaegelia has long been in trouble. Szlachetko [rbcL and matK, Pridgeon et al. [Xerorchis within the “lower” Epidendroideae. Szlachetko [Wullschlaegelia is a holomycotrophic plant [Wullschlaegelia has walls in a striate-rugulate pattern with free-standing baculae between them or coarsely verrucate-striate or gemmate [Wullschlaegelia is two, and the members of Calypsoeae are four. These data suggest that this genus and Calypsoeae are not closely related. Therefore, we conducted analyses with Wullschlaegelia, which is a sister to Xerorchis, based on our results , and Yoaniinae, which were circumscribed by Zhai et al. [The present phylogenetic study was primarily conducted based on the molecular dataset that was described by Zhai et al. . In thisi et al. , as fourCorallorhiza, Danxiaorchis and Yoania) holomycotrophic genera of Calyposeae. The Corallorhiza comprising 11 species that are confined to the northern hemisphere of the New World except for one circumboreal species (C. trifida), is one of the largest leafless genera in Orchidaceae [Danxiaorchis is founded in Guangdong, China, and the species possesses a labellum with a large Y-shaped callus and fleshy seeds [Yoania is sister to Danxiaorchis in the phylogeny of combined chloroplast rbcL and matK  of Calypsoeae. The genera Calypso, Dactylostalix, and Yunorchis have a trilobed labellum. The lip lobe of Calypso occupies the first half of the sac border, the lobe base has tufted setae, and the labellum tip has two calcariform objects. This green leaf plant has an erect raceme and is primarily distributed in temperate regions. While the lip lobe of Yunorchis is small and is located in the middle of the labellum sac, the sac border before the lobe closes upward and forms the unciform tip. The entire plant, including the inflorescence, grows underneath the ground, and the flower stretches toward the earth’s surface when blooming; therefore, only a few flowers are observed scattering on the ground, and this plant is completely saprophytic and is distributed in tropical regions. Both genera have obvious plant morphology and habitat differences and are different from Changnienia and Tipularia, which have a distinct calcar in the labellum, green leaves, a scape growing along the ground, and inflorescences with one or many tiny flowers.The new orchid, lostalix  and/or C and that Wullschlaegelia is not a member of Calypsoeae. Based on the combined sequences of the chloroplast genes rbcL and matK and the nuclear ITS gene, the subfamily-wide molecular analysis revealed that Calypsoeae comprises four well-supported clades with 12 genera. The newly identified orchid has several distinct features, and molecular analyses indicate that this plant represents an independent lineage under the tribe Calypsoeae. This lineage should be treated as a new genus in the Calypso Clade with the following classification:Based on the combined sequences of the chloroplast genes Subfamily: EpidendroideaeTribe: CalypsoeaeYunorchis pingbianensis Z. J. Liu, G. Q. Zhang et M. H. Li, gen. et sp. nov. , except for the new orchid and Agrostophyllum, which include two markers (rbcL and matK). The Dactylostalix is sampled for the first time in a broad molecular phylogenetic study. The voucher information and GenBank accession numbers are listed in To reconstruct a robust phylogeny for determining the circumscription and systematic positions of Calypsoeae, we sampled as many taxa as possible from all of the tribes recognized within Epidendroideae by Chase et al.  and PridrbcL and matK) and quickly (ITS) evolving genes and DNA regions was used to infer the phylogenetic relationships. The analysis is primarily based on the molecular dataset that was published by Zhai et al. [rbcL, four of matK, and five of the ITS were newly obtained) excluding the highly homoplasious morphological characters for molecular phylogenetic analyses. This tribal-wide matrix included 34 in-group species and two outgroups . The voucher information and GenBank accession numbers are listed in A second series of analyses focused on the tribe Calypsoeae. A composite of slowly  and the MP and ML methods. We performed the BI analysis using MrBayes 3.1.2 . For theThe electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants; therefore, the new names that are contained in the electronic publication of a PLoS ONE article are effectively published under that Code from the electronic edition alone. There is no longer any need to provide printed copies.http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.In addition, the new names that are contained in this work have been submitted to IPNI, from where they will be made and are available to the Global Names Index. The IPNI LSID 77145331–1 can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix S1 FigML middle, and BSMP right). “*” indicates that the node is 100% supported. “-” indicates that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.The numbers near the nodes are Bayesian posterior probabilities and bootstrap percentages Click here for additional data file.S2 FigML middle, and BSMP right). “*” indicates that the node is 100% supported. “-” indicates that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.The numbers near the nodes are Bayesian posterior probabilities and bootstrap percentages Click here for additional data file.S3 FigML middle, and BSMP right). “*” indicates that the node is 100% supported. “-” indicates that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.The numbers near the nodes are Bayesian posterior probabilities and bootstrap percentages Click here for additional data file.S4 FigML middle, and BSMP right). “*” indicates that the node is 100% supported. “-” indicates that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.The numbers near the nodes are Bayesian posterior probabilities and bootstrap percentages Click here for additional data file.S1 TableA dash (-) indicates missing data, an asterisk (*) denotes the sequences that were obtained in this study (the voucher and location are listed in (DOC)Click here for additional data file.S2 TableA dash (-) indicates missing data, an asterisk (*) denotes sequences that were obtained in this study, and the remaining sequences are from GenBank.(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


The recognition of protein folds is an important step for the prediction of protein structure and function. After the recognition of 27-class protein folds in 2001 by Ding and Dubchak, prediction algorithms, prediction parameters, and new datasets for the prediction of protein folds have been improved. However, the influences of interactions from predicted secondary structure segments and motif information on protein folding have not been considered. Therefore, the recognition of 27-class protein folds with the interaction of segments and motif information is very important. Based on the 27-class folds dataset built by Liu et al., amino acid composition, the interactions of secondary structure segments, motif frequency, and predicted secondary structure information were extracted. Using the Random Forest algorithm and the ensemble classification strategy, 27-class protein folds and corresponding structural classification were identified by independent test. The overall accuracy of the testing set and structural classification measured up to 78.38% and 92.55%, respectively. When the training set and testing set were combined, the overall accuracy by 5-fold cross validation was 81.16%. In order to compare with the results of previous researchers, the method above was tested on Ding and Dubchak's dataset which has been widely used by many previous researchers, and an improved overall accuracy 70.24% was obtained. With the accomplishment of the Human Genome Project, the “post genome era” has presented a large number of protein sequences that have challenged to develop a high-throughput computational method to structurally annotate the sequences coming from genomic data. One of these critical structures, the protein fold, reflects a key topological structure in proteins as it contains three major aspects of protein structure: the units of secondary structure, the relative arrangement of structures, and the overall relationship of protein peptide chains .A protein can only perform its physiological functions if it folds into its proper structure. Abnormal protein folding causes molecular aggregation, precipitation, or abnormal transport, resulting in different diseases. For example, the prion protein (PRNP) accumulates in the brain and causes neurodegenerative diseases, such as scrapie, Creutzfeldt-Jakob disease, Parkinson's disease, Huntington's disease, and mad cow disease. And the PRNP is a pathogenic protein caused by the abnormal folding of proteins. Thus, the correct identification of protein folds can be valuable for the studies on pathogenic mechanisms and drug design –8. Thus,Proteins with similar sequences tend to fold into similar spatial structures. When proteins have close evolutionary relationships, the similarity-based methods can achieve very reliable predicted results , 10. HowAfterwards, based on the dataset built by Ding and Dubchak and the feature parameters, some researchers suggested algorithm improvements for the identification of folds. For example, Chinnasamy et al.  introducBased on the dataset built by Ding and Dubchak, other previous researchers suggested the selection of feature parameters for the identification of protein folds. For example, Shamim et al.  used theBased on the dataset built by Ding and Dubchak , many piOther previous researchers have constructed new 27-class fold datasets and performed corresponding researches. For example, based on the Astral SCOP 1.71, with sequence identity below 40%, Shamim et al.  construcIn this paper, based on the dataset built by Liu et al.  in our ghttp://202.207.29.245:8080/Ha/HomePage/fzxHomePage.jsp.The dataset used in this paper was built by Liu et al.  in our gThe dataset built in our group was according to Ding and Dubchak's description about the construction of protein folds dataset in literature . The numThe distributions of the 20 amino acid residues in protein sequences for different protein folds are obviously different, and previous researches have shown that amino acid composition is associated with protein folding information , 17, 22.http://meme.nbcr.net/meme/cgi-bin/meme.cgi). Motif information (M) includes functional motifs and statistical motif.A motif is the conserved local region in a protein during evolution , which o1) Functional Motif. The PROSITE dataset was used to gather protein sequence patterns with notable biological functions. PS_SCAN packets provided by the PROSITE dataset and compiled by a Perl program were used as a motif-scan tool to search the sequences of 27-class folds training set; 45 functional motifs were obtained and selected. For an arbitrary sequence in dataset, the frequencies of different motifs in the sequence were recorded. If a motif occurred once, the corresponding frequency value was recorded as “1”; if the motif occurred twice, the value would be 2, and so on; otherwise if the motif did not occur, the corresponding frequency value was recorded as “0.” Thus, the frequencies of different functional motifs in a protein sequence were converted into a 45-dimensional vector.(2) Statistical Motif. For statistical motifs, MEME was applied as the motif-scan tool. MEME has been widely used to search protein motifs and DNA sequences [(equences . In thishttp://bioinf.cs.ucl.ac.uk/psipred/) was used as the tool to obtain the predicted secondary structure of each protein sequence in the 27-class protein fold dataset. As a protein fold is a description based on the secondary structure, the interaction between secondary structures plays an important part on the folding of protein. In this paper, we extracted the average interactions between predicted secondary structure segments as a feature parameter for the recognition of protein folds.A previous study showed that predicted secondary structure information is a main feature parameter for the identification of multiclass protein folds , 21, 22.1) The Calculation of Autocross Covariance (ACC). The autocross covariance (ACC) [β-hairpins [i is one kind of secondary structure segment , L is the number of the secondary structure segments in a protein sequence, and Si,j is a property value of secondary structure segment i at position j. i along the whole sequence.(ce (ACC)  has beence (ACC) , G-protece (ACC) , 35, proce (ACC) , and β-hhairpins . Howeveri contains m residues, and Si,j represents the summation of hydrophobic values for the m residues. The dimension of AC variables is 3 ∗max (lg).For example, given the hydrophobicity values  for 20 ai1 and i2 are two different types of secondary structure segments  and Si1,j is a property value of secondary structure segment i1 at position j. i1(i2) along the whole sequence. The dimension of CC variables is 3∗2∗lg.CC variables describe the average interactions between different types of secondary structure segments, which can be calculated according to the following equation:2) The Selection of the Maximum Value of lg. The statistical analysis of the number of the secondary structure segments in the 27-class folds dataset is shown in (3) The Values of Physicochemical Properties. In this paper, four physicochemical properties used by many researchers were selected as ACC feature values to calculate the interactions of segments: hydrophobicity (H1), hydrophilicity (H2), polarity (PL), and solvent accessible surface area (SASA). The values of physicochemical properties for the 20 amino acid residues were taken from the literature of Guo et al. [ was used to get the predicted secondary structure of each protein sequence.As the protein fold is a description based on the secondary structure, the formation of secondary structure in sequence influences the folding of protein. From the researches of published literatures , 22, 25,m , and (2) the number of decision trees represented by k . The Random Forest algorithm has been successfully used for the prediction of antifreeze proteins [β-hairpins [http://www.r-project.org/) was used to perform the Random Forest algorithm by calling the randomForest program package.Random Forest is an algorithm for classification developed by Breiman . The basproteins , DNA-binproteins , the metproteins , β-hairphairpins , and so For the 27-class fold dataset, amino acid composition, motif frequency, predicted secondary structure information, and the interaction of secondary structure segments were extracted as feature parameters, with the combined feature vector as input parameters for the Random Forest algorithm. The overall accuracy of the testing set in the dataset measured up to 78.38% by independent test.For further comparison, identification results from the gradual addition of relevant feature parameters are listed . Furtherhttp://202.207.29.245:8080/Ha/HomePage/fzxHomePage.jsp).When only one feature parameter, amino acid composition, was used, the overall accuracy was 43.66% . After aTo test the efficiency of our method, with the same feature parameters, classification strategy, and algorithm used above, the 27-class folds dataset built by Ding and Dubchak  was alsoAccording to Shen and Chou's  descriptIn early 2001, Ding and Dubchak built the 27-class folds dataset and started research on the identification of 27-class protein folds with multiple feature groups. Researchers have since been devoted to the improvement of feature parameters, algorithms, classification strategies, and the datasets for the identification of protein folds and have achieved good identification results. Based on previous researches, we combined sequence information, structural information, and functional information as input feature parameters of the Random Forest algorithm for protein folds identification and obtained better results. Therefore, the addition of segment interactions and motif information for recognizing 27-class protein folds is a valid and novel approach.Given the same dataset, when different feature parameters are used, the same sequence can be correctly or falsely classified. Here, our approach achieved the improved results with the following possible reasons. First, in considering the correlation at the level of secondary structure segments, we calculate the interaction information of secondary structure segments, which reflects the segments-order and long-range correlation information of the sequence and has a major influence on protein folding. Second, in considering the local conservation of kernel structure in protein folds, motif information was extracted, including functional motifs and statistical motifs. Finally, the Random Forest algorithm is a combination classifier of convenience and high efficiency whose final classification results are decided by votes from decision trees.In our future work, with the same parameters and classification strategy, testing methods  would be used. Different physiochemical properties also would be analyzed for the recognition of protein folds. Moreover, Precision-Recall curves or ROC curves for individual folds could be presented.


Hemodialysis (HD) patients have a high risk of infections. The uremic milieu has a negative impact on several immune responses. Online hemodiafiltration (HDF) may reduce the risk of infections by ameliorating the uremic milieu through enhanced clearance of middle molecules. Since there are few data on infectious outcomes in HDF, we compared the effects of HDF with low-flux HD on the incidence and type of infections.We used data of the 714 HD patients  participating in the CONvective TRAnsport STudy (CONTRAST), a randomized controlled trial evaluating the effect of HDF as compared to low-flux HD. The events were adjudicated by an independent event committee. The risk of infectious events was compared with Cox regression for repeated events and Cox proportional hazard models. The distributions of types of infection were compared between the groups.P = 0.42. No difference was found in the occurrence of the first infectious event . Of all infections, respiratory infections  were most common, followed by skin/musculoskeletal infections .Thirty one percent of the patients suffered from one or more infections leading to hospitalization during the study (median follow-up 1.96 years). The risk for infections during the entire follow-up did not differ significantly between treatment arms , HDF as compared to HD did not result in a reduced risk of infections, larger studies are needed to confirm our findings.NCT00205556ClinicalTrials.gov  Infections are an important cause for morbidity and mortality in hemodialysis (HD) patients . The ureTheoretically, amelioration of the uremic milieu by improving clearance of middle molecules by convection might reduce this risk of infections. The HEMO study however, comparing low-flux with high-flux HD, did not show a decrease in the risk of infections for patients treated with high flux membranes . Online urea ≥ 1.2. Exclusion criteria were age < 18 years, treatment by HDF or high-flux HD in the 6 months preceding randomization, severe incompliance defined as non-adherence to the dialysis prescription, a life expectancy < 3 months due to causes other than kidney disease, and participation in another clinical intervention study evaluating cardiovascular outcome. All patients were randomized centrally by a computer-based randomization service  into a 1:1 ratio for treatment with online hemodiafiltration or continuation of low-flux hemodialysis, stratified per participating center (permuted blocks). Because of the nature of the intervention, it was not possible to blind the patients, the local study nurses, or the investigators to the treatment assignment. The laboratory samples were measured in routine clinical care; hence, personnel were unaware of treatment assignment. The event adjudication committee was blinded to the treatment assignment. Patients were enrolled from June 2004 until January 1st 2010 by nephrologists and research nurses in participating centers. Follow-up ended December 31st 2010. The study was conducted in accordance with the Declaration of Helsinki and centrally approved by the Medical Ethical committee VU University medical Center, Amsterdam, The Netherlands, for all the participating hospitals on the 31st of July 2003, an amendment was approved on 28th of June 2007. Local approval was obtained from the Medical Ethical Committees of all participating centers, which are listed in the acknowledgements. The authors confirm that all ongoing and related trials for this intervention are registered. Written informed consent was obtained from all patients prior to randomization. The study was registrated in the trial registries after the first patients were enrolled, immediately by the time the investigators knew trial registration was obligatory. The assessment of difference in risk of infections between dialysis modalities was one of the predefined secondary outcomes [The CONvective TRAnsport STudy (CONTRAST)  is a randomized controlled trial, conducted in twenty-nine dialysis centers in The Netherlands (n = 26), Canada (n = 2), and Norway (n = 1), that compared the effects of low-flux HD and online post-dilution HDF on all-cause mortality and cardiovascular morbidity and mortality ,10. Patiurea was below 1.2. Online HDF was performed in the post-dilution mode; target volume was 6 L/h. For HDF, synthetic high-flux dialyzers were used . HD patients were treated with synthetic low-flux dialyzers . All patients were treated with ultra pure dialysis fluids, defined as less than 0.1 colony forming units per mL and less than 0.03 endotoxin units per mL. Routine patient care was performed according to national and international Quality of Care Guidelines.Treatment times were fixed during follow-up in both treatment arms, unless single pool Kt/VAt baseline standardized forms were used to collect demographical, clinical and laboratory data. Type of vascular access, duration of dialysis , and medical history (presence of diabetes mellitus (DM) and previous cardiovascular disease (CVD)), were also recorded. CVD was defined as a history of angina pectoris, myocardial infarction, prior coronary revascularization, stroke or transient ischemic attack and/or peripheral vascular disease. Dialysis vintage was determined as the sum of time patients were treated with HD or peritoneal dialysis (PD) before inclusion in CONTRAST.At each three monthly visit, data on clinical events (including infections), dialysis treatment, medication, and laboratory values were recorded. Infectious events were registered for all patients before drop out due to transplantation, switch to PD, move to another clinic or stop for other reasons. All infectious events were adjudicated by an independent Endpoint Adjudication Committee, whose members reviewed source documentation and were not aware of the treatment assignments. Infections were adjudicated as categorized definite or probable when patients were admitted to the hospital with a clinical picture of an infection, and with laboratory results suggesting an infection  or when infection was proven by culture. Infections were also adjudicated when these occurred during an admission for another cause. Only definite and probable infections were used for this analysis, possible infections  were considered as no infections. A report of two or more infections within a timeframe of 14 days was counted as one infection. If the second infection was fatal, that infection was used in the analysis. Infections were grouped as graft or fistula infection, catheter sepsis, sepsis, respiratory, urinary and ‘other infections’. Those that were categorized as ‘other infection’ have been subdivided into gastro-intestinal, skin/musculoskeletal, cardiac and miscellaneous infections retrospectively. Written diagnoses from the adjudication committee were used for this categorization.In HDF patients, infusion volumes (liters per treatment) were reported as the mean value of three consecutive treatment sessions. Convection volumes (liters per treatment) were calculated by the sum of the intradialytic weight loss and the substitution volume. Patients with a urinary production of less than 100 mL per day were considered anuric.Routine laboratory values were measured in the different participating hospitals using standard techniques.st fatal or non-fatal infection and the risk for 1st cause specific infection. Furthermore we studied the distribution of cause specific infections on the event level .The primary study outcome was the risk of hospitalizations due to infection during the follow-up period. The secondary outcomes were mortality due to an infectious cause, the risk for the 1All infectious events that occurred after randomisation up to censoring were used in our analyses. Censoring could be due to death, due to end of study or due to dropping out. Dropping out means that a participant either stopped because of a renal transplantation (n = 151), switch to PD (n = 11), transfer to another hospital which did not participate in CONTRAST (n = 24) or due to other reasons (n = 53).Data were reported as means with standard deviations, medians with interquartile (IQR) ranges, or proportions when appropriate.The difference in risk for hospitalization due to infections between patients treated with HDF and HD during the follow-up was evaluated with a Cox proportional hazards model for repeated events, which is a Prentice-Williams-Peterson (PWP) conditional model . In thisst infectious events  between HDF and HD was analyzed with Cox proportional hazard models. The distribution of infectious events from different causes was compared on the event level. The analyses were conducted in SPSS software  and in R .The difference in risk for 1P = 0.42. In addition, no statistically significant differences were found between the two treatment arms for the first occurring infection , sex (P = 0.49), DM , previous CVD (P = 0.14), RKF  and dialysis vintage , or country of residence . We found a trend towards a different effect of HDF in patients with high or low serum albumin levels (P for interaction = 0.05). The HR (HDF vs. HD) for infections was 1.31  in patients with a baseline serum albumin above 40.5 g/L (median), and was 0.86  in patients with a baseline serum albumin below the median.No difference in the effect of HDF was found in subgroups of age , P = 0.41, highest convection tertile HR 0.88 (0.64–1.20), P = 0.42 . This result remained unaltered after adjustment for age, sex, CVD, DM, hematocrit, serum albumin, treatment time, blood flow rate, vascular access and site.Compared to HD, the incidence of infections did not differ across tertiles of convection. Lowest convection volume tertile HR 1.10 (0.84–1.45), Of all infections, respiratory infections  were most common, followed by skin/musculoskeletal infections  Tables .P = 0.23) nor in Canada . Since the proportion of patients treated with grafts in the Netherlands was larger in the HDF group, we adjusted for access type at baseline. The HR’s for the Netherlands and for Canada remained however comparable  and 0.93  respectively.In this large randomized controlled trial we found that 31% of the participants suffered from one or more infections leading to hospitalization during the study (median follow-up 1.96 years). Our results suggest that treatment with HDF does not reduce the risk of mortality and hospitalization due to infections as compared to HD. The most common infections in both treatment arms were respiratory infections and skin/musculoskeletal infections.To our knowledge, this is the first study investigating the risk of infections in patients treated with HDF as compared to patients treated with low-flux HD. Our data indicate that alteration of the uremic milieu by convective clearance does not reduce the risk of infections. Our data are in line with the results of the HEMO study, which showed that high flux HD did not decrease the risk of infectious outcomes . Also inIn our study we found a trend towards a higher risk of sepsis in HDF patients. However, it may be a chance finding, since the number of patients in whom a sepsis or catheter sepsis occurred was small. Also, if we would take multiple testing into account, the association would be far from statistically significant. We do not have a theoretical explanation for the trend towards an increased number of sepsis in the HDF group. We neither have an explanation for a potentially different effect of HDF in patients with an albumin below or above the median, this might be a chance finding as well.The impact of various dialysis modalities on immune function is speculative. There is no evidence that alteration of dialysis efficiency or improving clearance of middle molecules from the blood would have a beneficial effect on immune processes on the tissue level. HDF might enhance the clearance of deleterious molecules such as complement factor D, granulocyte inhibitory protein II (GIP II) and immunoglobulin free light chains, which have been shown to have an in vitro depressant effect on degranulation, chemotaxis and phagocytosis of mainly polymorphonuclear leukocytes . AlternaThe groups of respiratory infections 25–28%) and skin/musculoskeletal infections (13–21%) were the most common infections in our study. These infections occurred more often in the same patients as well. In the HEMO study the contribution of respiratory disease as the cause for an infection related hospitalization was 22% as well –28% and , which wThe strengths of our study are the randomized design and the prospective collection of the data on infectious events, including the categories of infection. Furthermore, all infectious events were adjudicated and all infections during the on treatment follow-up were taken into account in the data-analyses. A limitation is the fact that this study was powered on mortality and not on infections, so the study is underpowered to detect smaller, clinically relevant, differences. So a beneficial or harmful effect of treatment with HDF cannot be ruled out completely based on our findings. As the 95% of the HR (1.09) was 0.88–1.34, the effect of online HDF may vary between a 12% reduction and a 34% increase in hospitalization for infections, both extremes being clinically relevant. Most important in this superiority trial, a small beneficial effect for HDF can still exist. Furthermore the lack of cause specific registration of fatal infections, the retrospective categorization of the category ‘other infections’ and the generalizability to non-European countries, with a different composition of vascular accesses might be limitations.More and larger studies are needed to confirm our findings. These studies should be powered on the incidence on infections, to rule out that the lack of effect we found, is not due to a lack of power. These studies should be designed as inferiority trials, to explore the trend towards more sepsis in the HDF group. The FINESSE trial, comparing high-flux HD with HDF, will investigate episodes of septicaemia as a secondary safety outcome . FurtherIn conclusion, as compared to HD, our data suggest that treatment with HDF does not reduce the risk of infectious episodes in patients with end stage renal disease.S1 CONSORT Checklist(DOC)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


Arterial stiffness is an independent risk factor for cardiovascular morbidity and can be assessed by applanation tonometry by measuring pulse wave velocity (PWV) and augmentation index (AIX) by pressure pulse wave analysis (PWA). As an inexpensive and operator independent alternative, photoelectric plethysmography (PPG) has been introduced with analysis of the digital volume pulse wave (DPA) and its second derivatives of wave reflections.The objective was to investigate the repeatability of arterial stiffness parameters measured by digital pulse wave analysis (DPA) and the associations to applanation tonometry parameters.b/a and e/a, showed good repeatability. Overall, the correlations to AIX were better than to PWV, with correlations coefficients >0.70 for EEI, AI and b/a. Considering the level of repeatability and the correlations to tonometry, the overall best DPA parameters were EEI, AI and b/a. The two pansystolic time parameters, ejection time compensated (ETc) by DPA and LVET by tonometry, showed a significant but weak correlation.112 pregnant and non-pregnant individuals of different ages and genders were examined with SphygmoCor arterial wall tonometry and Meridian DPA finger photoplethysmography. Coefficients of repeatability, Bland-Altman plots, intraclass correlation coefficients and correlations to heart rate (HR) and body height were calculated for DPA variables, and the DPA variables were compared to tonometry variables left ventricular ejection time (LVET), PWV and AIX. No DPA variable showed any systematic measurement error or excellent repeatability, but dicrotic index (DI), dicrotic dilatation index (DDI), cardiac ejection elasticity index (EEI), aging index (AI) and second derivatives of the crude pulse wave curve, b/a are suitable, for large artery stiffness EEI, and for small arteries DI and DDI. The only global parameter, AI, showed a high repeatability and the overall best correlations with AIX and PWV.For estimation of the LV function, ETc, EEI and  Increased arterial wall stiffness secondary to endothelial dysfunction and arteriosclerosis is a risk factor for cardiovascular morbidity ,2. ArterArterial stiffness parameters can be determined by applanation tonometry methods, where pressure sensors are placed on superficial arteries, e.g. the carotid, radial and femoral arteries. The carotid-to-femoral PWV is regarded the gold standard to assess arterial stiffness . AlthougA crude PPG recording allows analysis of the amplitude of the PW but not much of details of the contour itself. A mathematical ‘remodelling’ of the PW components with second derivatives enables analyses of the anacrotic and dicrotic notches of the crude PW curve. However, a full understanding of the second derivative wave details is still lacking, though several DPA parameters correlate to vascular pathology ,11,13–19The study was performed at the Medical Research Unit, Department of Internal Medicine, Skåne University Hospital in Malmö, Sweden. It was approved by the Regional Research Ethics Committee in Lund (Dnr. 532/2006) and all participants gave their oral and written informed consent.Volunteers recruited for the study were men and women age 64 years and above recruited from an ongoing screening study at the Medical Research Unit, and non-pregnant women up to 64 years and pregnant women recruited from the Department of Obstetrics and Gynecology . The phywww.atcormedical.com). The apparatus and the method have been described in a white paper by Skinner et al. [P<0.001) [Davies & Struther  have desr et al. . The SphP<0.001) ,22. The P<0.001) . The forP<0.001) , but dueP<0.001) . For thiThe PWV were recorded with the tonometer placed on the carotid and femoral arteries. By ECG synchronization and measurements of the distances between the recording points with a vernier calliper, the heart-to-carotid and heart-to-femoral PWVs could be calculated and expressed in m/s.The LVET , i.e. the pansystolic time expressed in milliseconds (ms), is the time elapsing from the start of the LV contraction . A clip with a light-emitting diode (LED) on the nail side and a light receiving photodiode on the opposite side was placed on the left index finger. The DPA works by emitting infrared light at a wavelength of 940 nm through the finger to the photodiode and on its way the light is absorbed relative to the oxyhemoglobin content (red cell density) in the tissue (and the expansion of the vascular and tissue volume); blood volume changes in the finger can thus be recorded by measuring the non-absorbed light and a PPG PW be formed. A crude PPG recording allows analysis of the amplitude of the PW but not of the contour itself. A mathematical ‘remodelling’ of the PW components by second derivation of the PW curve recorded by PPG was therefore proposed by Ozawa [We used the terminology for digital PPG proposed by Elgendi . Millassby Ozawa . Thus, aby Ozawa  in both The Meridian DPA automatically analyses HR and 16 different PW parameters. A recording takes 70 seconds to perform. For accurate analysis at least 80% of the PWs must be recognized and a prerequisite for good PW recognition is a good pulse height (PH). Poor PH can be caused by nail polish, acrylic nails or cold hands. When needed, nail polish was removed and the hand warmed by a heating pad.The physiological explanations of the different PPG and APG indices were obtained from the manufacturer’s manual , by personal email communication with Mr. Saint Myeong and Mr. Steve Kim at the Meridian Company, and from the literature. To facilitate the readability of the article, references to personal communication are not indicated in the text. Much research related to the APG has been performed in Japan and South Korea and much is published in the native languages, which restricts our possibility to go back to the original sources of publication. The reference intervals for normal values provided by the manufacturer were not used in our study.a through e of the APG refer to curve points as shown in the lower panels. The waveform elements are: S = starting point of arterial pulse wave. The aortic valve opens and blood is ejected from the LV; P = percussion (forward) wave. The LV blood ejection is transmitted by the elastic arterial wall. A higher peak means stronger LV ejection and higher compliance of large arteries [The capital letters S, P, T, C, and D of the PPG refer to curve points as shown in the upper panels of arteries ; T = tidarteries ; C = incarteries ; D = dicIn general, the pulse amplitude relates to the LV ejection performance, cardiac stroke volume and large artery distensibility –30, thoubps expressed in milliseconds (ms), where HRbps denotes heart beats per second (bps). ETc corresponds to the LVET, i.e. the time from onset of the systolic upstroke limb to the closure of the aortic valve (points S to C in ETc = (time from S to C)/√HREI = P/T. EI is a measure of LV ejection capacity and arterial stiffness and compliance (elasticity) of large arteries. A low EI indicates vasoconstriction, arteriosclerosis or LV ejection insufficiency; a high EI indicates arterial dilation or increased ejection power of the LV.EEI = (P/T) × (-b/a). EEI is an index for LV ejection capacity and compliance/elasticity of large arteries. A low EEI indicates vasoconstriction, arteriosclerosis or LV ejection insufficiency; a high EEI indicates vasodilation of large arteries, anemia, increased ejection power, hyperthyroidism or congested heart failure.DI = C/PH. DI represents the peripheral circulation (capillaries and veins) and indicates peripheral resistance; DI decreases at vasodilation.DDI = (PH-C)/PH = 1-C/PH = 1-DI. DDI is an index for elasticity in small arteries. A low DDI indicates vasoconstriction or arteriosclerosis, a high DDI indicates vasodilation.DEI = (D-D’)/C, where the distance D-D’ is the overshoot of the dicrotic wave over a straight line from C to S. DEI is an index for compliance/elasticity of small arteries/arteriole or venous blood flow. A low DEI indicates arteriolar constriction, arteriosclerosis or environmental effects such as cold, caffein, nicotine, or emotions with increased sympathetic tone; a high DEI indicates arteriolar dilation, low sympathetic tone, vasodilatory drugs, or venous insufficiency.a to d) and one diastolic peak (denoted e) are obtained on the APG . In 1998 Takazawa and coworkers [By the second derivative, four separate systolic wave peaks –(c/a)–(d/a)–(e/a) = (b-c-d-e)/a. AI represents the ‘vascular age’, i.e. arterial stiffness. The lower value, i.e. the more negative, the better; AI increases with age and arteriosclerosis [AI = . Since the d wave mainly reflects the intensity of the reflected PW from small peripheral arteries [a–d time might indicate vasodilation or low vascular tone.The intervals arteries , one canarteries , and a pagreement between repeated measurements is a characteristic of the method or instrument and is not dependent of characteristics of the population investigated, whereas the reliability depends on both the magnitude of measurement errors (bias) and the heterogeneity in the population. Since a DPA measurement is to just press a button, there was no need to take inter-observer variations into consideration. Reproducibility refers to measurements during changing conditions [We applied the recommendations by Bartlett & Frost  and Vaz nditions  and was Agreement quantifies how close measurements made on the same subject are under identical conditions over a short period of time [ of time . We desc of time ,37. The Reliability relates to the magnitude of the measurement error and was expressed as the intraclass correlation coefficient (ICC) of model 2 for individual ratings [http://department.obg.cuhk.edu.hk/index.asp?scr=1920). The ICC takes dimensionless values between zero and one [ ratings , calcula and one ,35, wher and one .p value of <0.05 was regarded significant.The mean value of multiple measurements was used in the comparisons with demographic characteristics and between the equipments. Associations between continuous variables were tested with Spearman’s rank correlation coefficient (rho); to reveal linear correlations, simple and second order of polynomial linear regression analyses with Pearson’s correlation coefficient (r) were performed. A two-sided As stated by Jang et al. , linear The adjustments of DPA variables were performed in a hierarchical order, where adjustments for HR were first performed and then each adjusted variable@75 was compared with body height in new linear regression analyses and the DPA variable@75 adjusted accordingly to a variable@75@170. The adjusted variables were then compared with the SphygmoCor tonometry variables PWV and AIX@75.The DPA variable ETc and the tonometer variable LVET were compared separately because these variables are the only ones directly corresponding to each other, measuring the pansystolic time; although the ETc is automatically adjusted to a HR of 60 bpm by Bazett’s equation, the LVET values were adjusted to a HR of 60 bpm (LVET@60) by the obtained linear regression equation see .The StatView version 5.0.1  and MedCalc  computer softwares were used for the statistical calculations.c/a and d/a , a moderate reliability for EI, c/a, d/a, a-b, and a-e (ICC 0.53–0.72), whereas a poor reliability was found for ETc, DEI, a-c, and a-d (ICC 0.26–0.46).A good reliability was found for DI, DDI, EEI, AI, p = 0.56; simple linear regression, p = 0.98) and these variables were therefore considered to be independent of each other.There was no significant correlation between body height and HR   ; the rele/a, but at linear regression analysis also DEI @75, and at linear regression also for DEI@75.In analogy with the strategy for adjusting the DPA variables for HR and/or body height, adjusted variable@75 values were compared with height in new regression analyses . Then, Sa-d), (a-d)@75, and (a-e)@75@170, and for PWV also for (a-e).a-b)@75 related to PWV, and for DEI@75@170, AI@75, e/a@75@170 and (a-b)@75 related to AIX@75, in higher Pearson correlation coefficients, but in no instance did the coefficient increase by more than 0.05. There were minor changes in the levels of significance, but for (a-e) adjusted to (a-e)@75 the significant correlation with AIX@75 disappeared. Overall, the linear correlations were stronger for AIX@75.Adjustments for HR/height of the DPA variables resulted, with exceptions for AI@75 and  but not with body height ; to compare the DPA variable ETc and the tonometer variable LVET, LVET was adjusted to a HR of 60 bpm, denoted LVET@60. A significant correlation was found between ETc and LVET@60   .A Bland-Altman plot  revealedb/a. The two pansystolic time parameters, ejection time compensated (ETc) by DPA and LVET by tonometry, showed a significant but weak correlation. For estimation of the LV function, ETc, EEI and b/a are suitable, for large artery stiffness EEI, and for small arteries DI and DDI. The only global parameter, AI, showed a high repeatability and the overall best correlations with AIX and PWV.Considering the levels of repeatability and the correlations to tonometry, the overall best DPA parameters were EEI, AI and a-c and a-d, and strong for EEI, AI, b/a and e/a, but comparing parameter ICCs between studies is not meaningful unless the studies have equal population heterogeneity [We deliberately chose to perform the study in a heterogeneous population sample, comprising both men and women and both pregnant and non-pregnant women of different ages. Such a population sample is expected to cover most components of the arterial stiffness concept, i.e. stiffness due to increased vascular tone, endothelial dysfunction, and structural elastic changes in the arterial wall. On the other hand, differences in age and gender have impacts on the accuracy of the pulse contour analyses , indicatogeneity . As illuogeneity , a measuc/a and d/a. After exclusion of the three persons from analyses, the CoRs for these parameters improved from 0.40–0.54 to 0.17–0.34.The agreement analyses were performed with Bland-Altman plots, which reflects the analysis method or instrument precision . We founAs emphasized by Bland & Altman , repeatab/a represent in varying grades the LV ejection capacity, the parameters EI, EEI and b/a the stiffness in large arteries, and the parameters DI, DDI, DEI, d/a and probably also a-d the stiffness in small arteries and arteriole. AI, aging index, is a general arterial stiffness parameter. To select representative parameters with the best repeatability of the different vascular segments along the cardiovascular system, without discussing their functionality and clinical relevance, we judge that for the LV ejection capacity and large arteries they are EEI and b/a, and for small arteries it is DI and DDI that had the best reliability figures, i.e. highest ICCs. Also the reliability of AI was high (ICC 0.84), but for the only parameter representing the whole systole, ETc, it was poor (ICC 0.46).The parameters ETc, EI, EEI and a-c and a-d, is that they all represent time axis measurement points occurring during late systole (c and d waves) or end of systole (point C). The c, d and e waves may not always be salient or exist as they are smoothed or merged, and false positive waves may occur due to high signal noise [c/a, d/a) contain these inflections points, but in contrast to the other variables these variables represent quotas, which may be less sensitive to inexact positioning along the time axis.The repeatability was generally stronger for DPA parameters representing the early systolic phase than for indices recorded in late systole or diastole. A common feature of three of the four variables with poor reliability in the present study, i.e. ETc, al noise , which qThe PPG signal is distorted and contaminated by noise from electrical circuits of the equipment and a correct identification of the APG waves is essential for calculation of wave peaks and wave crest time intervals, but there are few studies focusing on the automatic detection of these waves ,43. The a and b wave detection algorithms in a database of recordings and found large differences between the algorithms to correctly detect the waves. Detection of the c, d and e waves is even more elusive [There are no standard PPG databases available to evaluate different APG algorithms ; Elgendi elusive . The dra elusive .e/a also with body height. Although the associations were highly significant, they were for most variables weak and for no variable did Spearman’s rho or Pearson’s correlation coefficient exceed 0.66. Regardless, in the comparisons with the tonometric variables the DPA variables were accordingly adjusted for differences in HR and body height. However, the adjustments resulted in only minor differences in significance levels and Pearson correlation coefficients compared to unadjusted DPA values. The question whether the DPA variables need to be adjusted for HR should be evaluated in each situation, and especially in situations where interventions influence the HR.The tonometric variables are influenced by changes in HR, though there is confusion whether HR should be controlled for or not . In the a-d and a-e variables were significantly associated with both AIX@75 and PVW, but the associations with AIX@75 were in general stronger. The strongest correlations were found for EEI, AI, and b/a (r 0.75–0.78); notably, all these variables contain b/a, representing the rising systolic slope of the PPG. An inverse relation exists between myocardial contractility and arterial compliance [b/a ratio and the AIX reflect these entities. The strongest correlations with PWV were found for EEI, AI, and d/a (r 0.61–0.65). Weak to moderate correlations with PWV have been shown in other studies [b/a ratio but would not be detected as a change in PWV [All DPA variables except the mpliance  and both studies ,48 and we in PWV . Hashimoe in PWV  write thNo Pearson correlation coefficient was outstanding, but among the DPA variables it was AI that showed the strongest correlations with both AIX@75 and PWV .With exception for the ETc and LVET, the DPA and tonometer variables are not directly interchangeable since they represent different technical methodologies measuring different physiological circulatory modalities. While the PWV is essentially reflecting the distensibility of the aortic wall and depends passively on arterial pressure, the DPA parameters depend on both central and peripheral vascular properties . Moreoveb/a and d/a with arterial stiffness and cardiovascular risk are well established in the literature [It was outside the scope of the study to relate the DPA variables to clinical conditions, but the associations of AI, terature ,14–18.Among the DPA and tonometer parameters, only the systolic time parameters ETc by DPA and LVET by tonometry directly correspond to each other. Already in 1971, Chirife et al.  stated tAn additional uncertainty in the ET comparison is the corrections for differences in HR. In the Meridian DPA apparatus the ETc is automatically HR-adjusted by Bazett’s equation , originaWe performed multiple statistical analyses including numerous PW variables, exposing the study to a risk of a type I error, but we consider that risk insignificant because the study showed consistent and no conflicting results.b/a and e/a. No DPA variable showed systematic errors or skewness along the range of measurements. Almost all DPA variables were HR dependent, whereas almost none correlated with body height. In the comparisons with tonometry variables, adjustments of DPA variables for HR and height made no difference in the interpretation of the associations between parameters. The overall highest correlations were found with AIX, where EEI, AI and b/a were the only parameters showing a Pearson correlation coefficient >0.70; with PWV, a coefficient >0.60 was found only for EEI, AI and d/a. Considering the level of repeatability and the correlation with the established parameters for PW analysis, the best overall DPA parameters were EEI, AI and b/a. The only parameters defined equally by both equipments were ETc by DPA and LVET by tonometry. The correlation between these parameters was significant but fairly weak, which probably much depends on the poor repeatability of ETc, which in turn might be due to methodological difficulties to identify the end of systole. The DPA apparatus we used contained advanced mathematical algorithms to distinguish between LV performance, large artery stiffness and small artery stiffness; thus, for estimation of the LV function we regard ETc, EEI and b/a to be suitable, for large arteries EEI, and for small arteries DI and DDI. The only global parameter, AI, showed a high repeatability and the overall best correlations with the AIX and PWV. Although no DPA variable is a surrogate for applanation tonometry variables AIX and PWV, our validation of the DPA against the gold standard method suggests that DPA is a valuable tool for estimation of vascular status. Since the DPA method is fast, relatively inexpensive and operator independent, it could implicate advantages in clinical practice and screening.In summary, this study was performed in a heterogeneous population because we wanted to test the limits of repeatability of a commercially available DPA. The results should be interpreted from that perspective. No DPA variable showed excellent repeatability; a good repeatability was found for DI, DDI, EEI, AI,


Prophylactic use of amoxicillin and amoxicillin/clavulanic acid, although controversial, is common in routine clinical practice in third molar surgery.Our objective was to assess the efficacy of prophylactic amoxicillin with or without clavulanic acid in reducing the incidence of dry socket and/or infection after third molar extraction. We conducted a systematic review and meta-analysis consulting electronic databases and references in retrieved articles. We included double-blind placebo-controlled randomized clinical trials published up to June 2015 investigating the efficacy of amoxicillin with or without clavulanic acid on the incidence of the aforementioned conditions after third molar extraction. Relative risks (RRs) were estimated with a generic inverse-variance approach and a random effect model using Stata/IC 13 and Review Manager Version 5.2. Stratified analysis was performed by antibiotic type.p< 0.001; 95% CI 0.214 to 0.574). We found no evidence of heterogeneity . The number needed to treat was 18 (95% CI 13 to 29). Five studies reported adverse reactions . The RRs were 0.563 for amoxicillin  and 0.215 for amoxicillin/clavulanic acid .We included 10 papers in the qualitative review and in the quantitative synthesis . The overall RR was 0.350 (Prophylactic use of amoxicillin does not significantly reduce the risk of infection and/or dry socket after third molar extraction. With amoxicillin/clavulanic acid, the risk decreases significantly. Nevertheless, considering the number needed to treat, low prevalence of infection, potential adverse reactions to antibiotics and lack of serious complications in placebo groups, the routine prescription of amoxicillin with or without clavulanic acid is not justified.Key words:Meta-analysis, amoxicillin, infection, removal, dry socket, third molar. Third molar extraction is a common procedure in oral surgery. There still is controversy over the need to routinely use systemic antibiotics for the prevention of infectious and inflammatory complications associated with this type of surgery ,2.In a survey in 2014 , we founThe objectives of this study were; 1: to assess the efficacy of the use of amoxicillin with or without clavulanic acid to prevent infection and/or dry socket, compared to a control group given placebo, in third molar surgery patients; and 2: to carry out stratified analysis of the efficacy of amoxicillin and amoxicillin/clavulanic acid. We designed a meta-analysis testing the null hypothesis that the use of amoxicillin with or without clavulanic acid is not effective.This study is reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and the Institute of Medicines’ guidelines. The literature search was based on questions structured in the Patient, Intervention, Comparison, and Outcome (PICO) format.- Eligibility criteria: We selected studies including patients of any age and sex who underwent extraction of third molars with any degree of impaction. Regarding type of intervention, we included trials that analysed the efficacy of amoxicillin with or without clavulanic acid at any dose or regimen, and regarding comparisons, we exclusively included randomized double-blind placebo-controlled (RDBPC) clinical trials, not excluding those with split-mouth designs. With respect to outcome, we excluded studies that did not investigate the incidence of dry socket, infection, and both conditions concurrently, but did not apply restrictive criteria for the definition of infection or dry socket. Results of interest: The search was not restricted by language. The last search date was 1 June 2015.- Sources of information. The electronic databases consulted were: Medline/PubMed, Scopus, ScienceDirect, Web of Science, Evidence-Based Dentistry, ClinicalTrials.gov, the EU Clinical Trials Register, the Cochrane Central Register of Controlled Trials, the Spanish General University Board database of doctoral theses in Spain (TESEO) and Spanish National Research Council (CSIC) bibliographic databases.- Search strategy: The search terms selected are descriptors of each of the PICO components: extraction, removal; third molar; antibiotic, amoxicillin, clavula*; infection; and dry socket. The filters used were: humans, clinical trials, meta-analysis, randomised, and controlled trials. The electronic search in the Medline/PubMed database was carried out using MeSH strings and search algorithms connected with Boolean operators as key words for titles and abstracts. Specifically, we used the following search strategy:   OR (doubl* OR trebl* OR tripl*) AND (mask* OR blind*) OR (“Latin square” ) OR placebos OR placebo* OR random* OR research design OR comparative study OR evaluation studies OR follow-up studies OR prospective studies OR cross-over studies OR control* OR prospectiv*  AND (third molar) AND (antibiotic OR amoxicillin OR clavula*) AND (infection OR dry socket) AND . For the Spanish language databases, we used the following Spanish terms: (antibiótico OR amoxicilina OR clavulan*) AND  AND (exodoncia OR extracción). The references in each paper were reviewed, and we also searched for conference abstracts.- Selection of studies: Two researchers independently performed the searches in the databases with the aforementioned criteria. After applying the filters, we obtained the following: 26 papers from PubMed; 123 from SCOPUS; 668 from ScienceDirect; 69 from the Web of Science; 65 from Evidence-Based Dentistry, 42 from the Cochrane Library, 4 from TESEO, and 2 from IME-Biomedicina (a CSIC database). The databases not listed did not yield any relevant publications. Having removed duplicate publications and any for which it was clear from the title and abstract that they did not met the inclusion criteria, 75 papers were retrieved, and these were reviewed by two of the authors. - Data extraction process: Data were extracted on 13 variables from each of the studies -19. Each- List of data: The types of data collected are listed in Risk of bias in individual studies. Qualitative and quantitative data were collected on potential sources of bias in each of the studies  . To ass1- Generation of the random sequence: we assessed whether the method for generating the random sequence was appropriate.2- Concealment of allocation: we assessed how patients were allocated to each group  and how allocation was concealed.3- Blinding of participants and personnel: all the studies included claimed to be double blind, but they did not report the method for blinding of participants and personnel.4- Blinding of outcome assessment: as above, though all the studies included claimed to be double blind, they did not specify the method for blinding assessment of the outcome.5- Handling of data: we identified whether patients lost to follow-up were included in the analysis and whether the analysis was carried out on an intention-to-treat basis.6- Selective reporting: we checked whether data were in fact reported for all the variables and outcomes that authors had planned to report a priori.7- Other sources of bias: we sought to identify other potential sources of bias.For each study, all of these factors were analysed and the study was then assigned to one of three categories  based on the estimated risk of bias.- Summary measure of efficacy: the analysis of efficacy was based on the relative risk (RR) or cumulative incidence ratio in the treatment vs. control groups. In clinical trials that compared several experimental groups using the same antibiotic under different regimens with a single control group, the data considered were the total numbers of surgical interventions and complications in the experimental groups, without considering them as independent clinical trials. To assess the clinical significance of the treatment effect, we used the difference in the incidence or attributable risk of infection and calculated the number needed to treat (NNT) to prevent one case of infection.- Synthesis of results: All the analyses were carried out using StataCorp 2013 Stata Statistical Software: Release 12  and Review Manager (RevMan) Version 5.2 . We studied the heterogeneity of the different studies using the I2 statistic, an expression related to Cochran’s Q test. The overall relative risk, the result from combining data from the different studies, was calculated using an inverse-variance approach with a random effect model. Empirical correction was used for the studies with zero effect sizes in one of their arms. Any studies with a zero effect size in both arms were excluded. The clinical significance was analysed by calculating the NNT for each study and overall.- Risk of between-study bias. The publication bias was assessed graphically using a funnel plot and quantitatively with the methods of Egger and Macaskill. The number of unpublished studies was estimated with Rosenthal’s method.- Additional analysis: We also carried out meta-analysis stratified by the type of antibiotic and cumulative meta-analysis by publication date, as well as analysing adverse reactions.- Selection of studies: Out of the 75 studies retrieved, qualitative and quantitative analysis was performed on 10 RDBPC studies. - Characteristics of the studies: - Risk of bias in the studies: Fig.  illustra- Results of the individual studies: The forest plot Fig.  is a gra- Synthesis of the results:p<0.0001) and different from 1, indicating that treatment with amoxicillin with or without clavulanic acid prevents the development of infectious complications .Analysis of the overall efficacy of amoxicillin with or without clavulanic acid: The quantitative analysis included 1997 extractions: 1072 in experimental groups and 925 in control (placebo) groups, with 27 and 74 reported events of dry socket and/or infection respectively. The overall RR was found to be 0.350, with a 95% CI of 0.214 to 0.574, this being significant (p=0.470), supporting the assumption of homogeneity among the studies. Further, there is no sign of heterogeneity in the L’Abbé plot , and Egger´s more sensitive method also suggested a lack of publication bias . Macaskill’s more specific procedure yielded a slope that was close to 0 and non-significant (p=0.489), confirming the lack of publication bias, both when using the sample size (n) as the independent variable, as proposed by Peters, and when the regression uses the inverse of the sample size (1/n) as the independent variable (p=0.330). Lastly, with Rosenthal’s method, it was estimated that it would be necessary to add 79 non-significant studies to cause the results of this meta-analysis to become non-significant.The Begg method suggested a lack of publication bias . We found no evidence of heterogeneity . The NNT was 40, meaning that about one in every 40 patients would benefit from the treatment. The 95% confidence interval for the NNT ranged from 22 to 274.- Amoxicillin: We included 7 studies . The RR was 0.563 . Again, we found no evidence of heterogeneity . The NNT was 10, meaning that about one in every 10 patients would benefit from the treatment, and the 95% confidence interval for the NNT ranged from 7 to 16.- Amoxicillin/clavulanic acid: We included 3 studies . The RR was 0.215 . The adverse reactions were generally mild and short lived. The number needed to harm (NNH) was 26, meaning that 1 in 26 patients given the prophylactic antibiotics would have an adverse reaction.Analysis of adverse reactions: Five studies reported adverse reactions  with a tOur meta-analysis includes 10 RDBPC clinical trials that assess the efficacy of amoxicillin with and without clavulanic acid to prevent dry socket, infection and both conditions concurrently after third molar extraction. These studies yielded a total of 1997 third molar extractions. We only selected trials that used placebo in the control group. It is important to highlight that we have not taken into account the antibiotic regimen used. As noted in the qualitative analysis, the studies included are not free from individual bias, but we have not detected publication bias. Adverse reactions were more frequent in the experimental group but were generally mild.In the quantitative analysis, we used a multiplicative relative risk model and estimates were weighted by the inverse of the variance. We opted to use a random effect model, which assigns a fixed constant coefficient of variability to all studies, and this gives more importance to studies with smaller sample size; however, this is the most appropriate type of model when analysing fewer than 20 studies, provided there is no publication bias.p=0.082), while for amoxicillin and clavulanic acid, the result was statistically significant . We should emphasize that only three studies were included in the amoxicillin/clavulanic acid subgroup analysis, and that two of these were conducted by the same research team , finding this variable to be relevant. The hypothesis of the analysis by subgroups was established a priori and the variable type of antibiotic used for weighing was defined prior to randomisation in all of the studies. This stratified meta-analysis has found that the statistical significance of the RR differs between the subgroups: in the case of amoxicillin alone, the CI of the RR (95% CI 0.295 to 1.08) includes 1 and the result was not significant  or amoxicillin/clavulanic acid (2.79%). In contrast, there were notable differences analysing absolute risk reduction, with values of 2.50% (95% CI 0.37 to 4.64%) in the amoxicillin group and 10.12% (95% CI 6.37 to 13.88%) in the amoxicillin/clavulanic acid group. This discrepancy is understandable given the difference in rates of the conditions considered in patients treated with placebo in the two subgroups, with higher rates of infection in the case of studies using amoxicillin/clavulanic acid than those using amoxicillin  . This uet al. . In our case, the relative risk was somewhat lower, but only half of the studies had recorded adverse reactions . Lodi et al. (Other meta-analyses have been published on the efficacy of antibiotics for the prevention of inflammatory and infectious complications after third molar extraction. In 2007, Ren et al.  studied  (et al.  includedIn our case, the results should also make us think. To avoid one patient having an infectious complication using amoxicillin prophylactically, we would need to treat 40 patients. Moreover, as mentioned earlier, amoxicillin does not significantly reduce the risk of infection and/or dry socket. For this reason, we believe that its use is not justified.In the case of amoxicillin/clavulanic acid, we would have to treat 10 patients to avoid 1 case of infectious complication. It is important to analyse the clinical significance of these results. First, we should take into account the low rate of infectious complications and the lack of serious complications. On the other hand, the risks of antibiotic use are widely documented, in relation to increases in antibiotic resistance at the population level ,21, as wFor all these reasons, and given our results, we conclude that there no basis for recommending the prophylactic use of amoxicillin without clavulanic acid for preventing infection and/or dry socket after third molar extraction in healthy patients. Regarding amoxicillin/clavulanic acid, although the null hypothesis was rejected and prophylactic use was statistically significantly effective, taking into account the NNT, low rate of infectious complications, adverse reactions in experimental groups and lack of serious complications reported in controls, the prescription of this combination of antibiotics cannot be justified either.