Searching a biological sequence database with a query sequence looking for homologues has become a routine operation in computational biology. In spite of the high degree of sophistication of currently available search routines it is still virtually impossible to identify quickly and clearly a group of sequences that a given query sequence belongs to..We report on our developments in grouping all known protein sequences hierarchically into superfamily and family clusters. Our graph-based algorithms take into account the topology of the sequence space induced by the data itself to construct a biologically meaningful partitioning. We have applied our clustering procedures to a non-redundant set of about 1,000,000 sequences resulting in a hierarchical clustering which is being made available for querying and browsing at Comparisons with other widely used clustering methods on various data sets show the abilities and strengths of our clustering methods in producing a biologically meaningful grouping of protein sequences. With the overwhelming growth of biological sequence databases, handling of these amounts of data has increasingly become a problem. Protein sequences constitute one such data type. The number of unique entries in all protein sequence databases together exceeds now about a million. However, biological evolution lets proteins fall into so-called families, thus imposing a natural grouping. A protein family contains sequences that are evolutionarily related. Generally, this is reflected by sequence similarity. Therefore, one aims at organizing the set of all protein sequences into clusters based on their sequence similarity.Clustering a large set of sequences as opposed to dealing only with the individual sequences offers several advantages. A frequent problem is the identification of sequences that are similar to a new query sequence. This task can be executed much quicker when only one comparison to an entire cluster has to be performed rather than one comparison per database sequence. Another application lies in the possibility of analyzing evolutionary relationships among the sequences in a cluster and of the species they come from. Moreover, the presence or absence of sequences of a group of species can give useful information about their evolutionary relationship, if their complete set of protein sequences is known.single linkage clustering. Starting with the pair of data points of least distance, one incrementally merges single data points or already existing clusters. Such a hierarchical clustering can be viewed as a tree, called the single linkage tree. The leaves represent the individual data points, while the root of this tree corresponds to just one large cluster representing the whole data set. All other layers in between can be seen as cluster sets at different levels of similarity. However, it is not clear which layers give a meaningful partitioning of the data. They should be chosen so that they neither produce small trivial clusters nor form huge uninformative clusters.The aim of clustering protein sequences is to get a biologically meaningful partitioning. One of the simplest well-studied and computationally cheap methods to construct a clustering of data points is et al. [et al. [et al. [Several approaches already deal with the problem of partitioning a protein sequence database into protein families. Automatically generated cluster sets like ProtoMap , ProtoNeet al.  recentlyet al.  is a met [et al.  and Liu  [et al. .superfamily. Each superfamily typically covers several closely related protein families. They can be determined by revealing the connectivity of the sequences belonging to a superfamily. Since the single linkage tree is built using only the smallest distances connecting subtrees, information about the connectivity within these subtrees is lost in the hierarchy. For each superfamily, we construct a superfamily distance graph by including only those nodes labeled with sequences belonging to the respective superfamily. These graphs are then split at reasonable cut sites into highly connected subclusters. For historical reasons [SYSTEmatic Re-Searching.In our approach we first exploit the branching structure of the single linkage tree, which elucidates an unexpected structuring of the sequence space. Traversing the tree from a leaf towards the root we inspect the sizes of the merging subtrees. First one notices relatively small increases that correspond to very similar proteins. Later on, sequences merging in correspond to weakly related proteins. At one point, however, we observe an enormous increase in the size of the subtree, where a large part of the database merges in. All sequences below this point in the tree are assumed to belong to the same  reasons , we calldomains. They again can be grouped independently of a certain order in a protein sequence. For this level we rely on one of the currently established domain databases, i.e., the Pfam database [Up to that point, the hierarchy consists of superfamily and family clusters. However, protein sequences are built up of smaller entities, called database . To alloHere, we explain in more detail the SYSTERS algorithms developed to determine superfamily and family clusters. Each step is illustrated by an example. We report our results on clustering the non-redundant protein sequence space consisting of about 1,000,000 sequences. An overview of the availability and accessibility of the cluster set is given. Finally, we present a comparison of our clustering method with two other currently available and widely used clustering methods, namely single linkage clustering and TribeMCL.We have applied our algorithms as described in the Methods Section to a sequence set consisting of all known protein sequences from the Swiss-Prot Rel. 41 and TrEMBL Rel. 23 databases , and fro. There it is possible to explore the protein sequence space by navigating through the complete hierarchy consisting of superfamilies, family clusters, and domains. For the last layer in the hierarchy, the domain level, we rely on one of the currently established domain databases, namely the Pfam collection of protein domains. It is possible to enter the hierarchy at any layer, e.g., by searching for a keyword, choosing a species, or selecting a domain. For each family cluster a consensus sequence is generated. All consensus sequences together build a database searchable by BLAST. Thus, a clear assignment of a new protein or nucleotide sequence to a family and a superfamily is possible. Additional information like a multiple alignment or a phylogenetic tree is given for each of the family clusters. Whenever possible, links to external resources are provided to allow for further information, e.g., about structural properties or underlying genes.The SYSTERS cluster set  is availFor the validation of our clustering procedures we needed on one hand a "true" biologically verified cluster set and on the other hand results of other clustering procedures on this data set. Unfortunately, for large scale analyses such validated data is not available. Thus, we decided on performing our evaluations on two biologically meanigful data sets, namely well characterized sequences from Swiss-Prot and TrEMBL with (a) Pfam domain annotations and (b) ENZYME annotations.Clustering of such large data sets is not an every day routine. Normally the software to handle such data sets is not publicly available and only the results of their application are published. Although these results are mostly publicly available for browsing on the web the underlying primary data differs in all of these data sets. Additionally a systematic, unbiased and independent comparison would be intractable on a large scale by querying the web.One of the simplest well-studied and computationally cheap methods to construct a clustering is single linkage clustering. We implemented procedures to perform a single linkage clustering on the two data sets at various different cutoffs. This corresponds to performing single sequence searches with a certain E-value cut-off for all sequences in the data set with subsequent determination of the connected components of the results. Additionally we decided to compare our clustering procedure to one of the most widely used and publicly available methods for large scale protein sequence clustering, namely TribeMCL.We applied the single linkage clustering as well as the SYSTERS clustering to the Pfam data set and computed the Jaccard coeffcient, the sensitivity and the selectivity of the clustering results in comparison to the "true" cluster set as described in the Methods Section. All clusterings were performed on the non-redundant data set as described under Preprocessing in the Methods Section. After the clustering, redundant sequences were added again to the cluster sets to allow for a comparison with the "true" cluster sets.For the Pfam cluster set the best single linkage clustering with respect to the "true" cluster set can be achieved at an E-value cutoff of 1e-53 , without any knowledge of the biological information represented. Such self-structuring properties have also been observed in other large data sets such as phone-call or web-link graphs .et al. [An alternative approach for cluster determination is presented by Sharan et al. . Their CAlthough the vast majority of cases we looked at are in agreement with biological knowledge, there exist some inconsistencies due to peculiarities in the data. Distinct protein families may end up erroneously in the same superfamily because of a fusion protein covering sequence information from both families. The same effect can be seen at multidomain protein families linked together by a single highly conserved common domain. Although the subclustering in most cases splits these superfamilies again into distinct families, we would prefer to take care of these cases already in the process of superfamily determination. Nevertheless, comparisons with other methods showed that our clustering methods are able to produce a biologically meaningful clustering.Thus far, our hierarchy consists of two layers representing protein superfamilies and families. For the third layer located at the domain level, we currently rely on well-established domain databases, but intend to follow our methodology also in the direction of deriving so far unknown domains.tree of life. We plan to combine the evolutionary information given by each of the protein clusters to extend the knowledge about the relationship between different groups of species.Future plans also include a regular update of the SYSTERS cluster set. Since the most time consuming part are the all-against-all sequence searches, new sequence similarities will be incrementally added instead of recalculating all similarity values. The clustering procedures themselves rely on the topology of the whole sequence space and can be run on the whole data set whenever the underlying sequence set changes. Other future developments will be in the direction of the so called Here we present the methods that we use to compute our clustering of protein sequences, i.e., selecting superfamilies and dividing them into reasonable family clusters. Figure The total number of entries in all protein sequence databases together now exceeds about a million. This number includes fragmental as well as identical (sub-)sequences from different resources. To reduce the amount of data without losing information we exclude redundant information in the form of identical and nearly identical (sub-)sequences from the data set prior to the clustering.S that one expects to find in a database search by chance. Thus, the lower the E-value, the more significant is the score. Typically, matches with an E-value lower than 1e-20 are assumed to be relevant, while those sequence pairs with an E-value higher than 0.01 need further experimental evidence to be accepted as being distantly related. Values in between belong to the so called twilight zone, and a clear statement about relatedness cannot be made for them. All sequence pairs whose E-value was worse than 0.05 were assumed to be unrelated and their distance was set to infinity. We are aware that we may miss distantly related sequences with this E-value threshold in a single sequence search. However, by using each sequence in the data set as query in a database search and combining all results we hope to overcome this problem. The resulting symmetric distance matrix D contains all pairwise distances d for each pair of protein sequences si and sj, 1 ≤ i, j ≤ n, for which d < ∞.We model the remaining protein space as a weighted undirected graph with pairwise distances attached to the edges. We decided on using E-values computed from pairwise local sequence alignments  as distaD can be represented by an undirected weighted graph G, which we call the distance graph. G =  is defined as follows: V = {vi | vi = {si}, i ∈ {1, ..., n}} and E = { | i, j, ∈ {1, ..., n}, i ≠ j, d < ∞}. The weight w of an edge  ∈ E is given by w = d.The distance matrix G in an agglomerative manner. The algorithm starts with a forest (collection of trees) F where each sequence corresponds to a distinct tree. As long as there are edges in the graph G, the edge with the smallest weight is selected and the adjacent nodes in G are merged. Edges linking this newly created node to adjacent ones in the graph receive the weight of the smaller of the two original edges. The two corresponding trees in F are collected together in a new tree rooted by a parental node labeled with the connecting edge weight. Finally, to allow for a better handling of the data, the resulting unconnected trees are rooted by connecting their roots to an artificial overall root node with weight infinity.The single linkage tree is built based on the distance graph p and a child q, we compute the quantityDifferent protein superfamilies display a different degree of conservation. Therefore, for each superfamily, the twilight zone starts at a different cutoff. A crucial problem thus lies in the determination of an appropriate E-value threshold for each superfamily. To this end we have devised the following procedure. For an edge of the tree linking, say, a parent J represents the ratio between the size of all the subtrees below p without the child q and the size of the subtree below q. Watching the development of J as one walks up the tree from a leaf towards the root, one can observe that J tends to increase dramatically as one leaves the superfamily to which the leaf belongs, and then decreases again. This intuition is captured by our algorithm. For each leaf, we determine the maximum J as one proceeds from the leaf to the root of the single linkage tree. This strategy is applied to all leaves in the tree, assigning a superfamily to each leaf. In the end, inclusions are resolved by keeping the largest superfamilies. We call the internal node induced by a superfamily the superfamily root. The E-value linked to this node is called the superfamily cutoff. Refer to Algorithm 1 in Figure SF be the set of sequences belonging to the superfamily sf and c the corresponding superfamily cutoff. We call the connected weighted graph G =  with V = {vi | vi = {si}, si ∈ SF} and E = { | w = d, si, sj ∈ SF, i ≠ j, d ≤ c} the superfamily distance graph of sf.Stepping down the hierarchy of the single linkage tree starting at a superfamily root usually splits off one sequence after another, but does not lead to a meaningful partitioning into families. Since the single linkage tree is built using only the best (lowest) E-values connecting subtrees, information about the connectivity within these subtrees is lost in the hierarchy. For each superfamily we construct a distance graph that includes only those nodes labeled with sequences belonging to the respective superfamily and those of the induced edges which are labeled with a distance better than or equal to the superfamily cutoff. Let et al. [edge-connectivity k(G) of a graph G is the minimum number k of edges whose removal results in a disconnected graph. A cut in a graph is a set of edges C whose removal disconnects the graph into two disjoint components H1 and H2. A minimal cut is a cut with a minimum number of edges. The length p of the shortest path between nodes u and v in G is the minimum length of a path from u to v, if such a path exists; otherwise p = ∞. The diameter of a connected graph G is the maximum shortest path length p over all pairs of nodes u and v in G.To split a superfamily distance graph into family clusters, we use an algorithm that can be seen as a weighted version of a method presented by Hartuv et al. . First, G with n > 1 nodes is called highly connected, if k(G) >. A highly connected subgraph (HCS) is an induced subgraph H ⊆ G, such that H is highly connected. In an unweighted graph this definition results in the following property: The diameter of every highly connected subgraph is at most two. Thus, these subgraphs are compact clusters which need not meet the constraint of being fully connected.The key definition of the algorithm in  is the fhighly connected subclusters is reached. For our purposes we had to modify the algorithm to be able to handle a weighted graph. Precisely, in our weighted HCS algorithm, if the edge weights covered by the minimal cut are approximately the same as in the remaining graph, the graph is assumed to be already highly connected and is not further split into subclusters , where N is the number of clusters found and f is the time complexity of computing a minimum cut in a graph with n nodes and m edges. We use the implementation of the "mincut" algorithm given in the LEDA [(nm + n2 log n).The E-values in our data set range from 0  to 0.05. To be able to find a minimal cut in our graph, edge labels should be positive values with a low value representing a weak connection and a high value representing a strong connection. Instead of using the raw E-values we label the edges in our graph with the negative logarithm of the corresponding E-value each. Since the logarithm of 0 is not defined, we use an arbitrary value  for these edges instead of the logarithm. The running time of both HCS algorithms is bounded by 2the LEDA  distribuTo apply this algorithm to our data set we added a preprocessing as well as a postprocessing step as shown in Algorithm 3 in Figure Sequences being connected with the remaining graph by only one edge are either fragmental or are the so far sole representative of a protein family in the sequence database. The underlying data of our clustering is known to contain lots of fragmental sequences. Before applying the HCS algorithm to our graph, we repeatedly merge all nodes connected to the remaining graph with only one edge with their respective adjacent node.singleton adoption), if there is no contradiction. When there are several minimum cuts in a graph, the original as well as our weighted HCS algorithm might choose a minimum cut which, from the clustering point of view, is not optimal. In many cases this process will break clusters into singletons. In the original algorithm in [Nevertheless, the HCS algorithm may split off single sequences as subclusters. Thus, in a postprocessing step, sequences which ended up after the subclustering as a single sequence cluster are assigned to their closest neighboring cluster . This data set consists of 5,724 single domain families assigned to 733,830 sequences. Since our aim is not to cluster single domains but full-length sequences, we define a "true" cluster consisting of all sequences having the same domain composition. Fragmental sequences will cause a problem in our analyses by showing a different domain composition than complete sequences. We restrict our analyses to sequences not annotated as being fragmental in the Swiss-Prot or TrEMBL databases. The resulting "true" cluster set thus consists of 442,872 sequences sorted into 16,990 distinct families.The ENZYME database  stores dA : type of reaction catalyzed (at present 6 classes)B : subclass, information about type of compound or group involvedC : sub-subclass, further specifies the nature of the reactionD : serial number to identify individual enzyme within sub-subclassAlthough several distinct proteins may catalyze the same reaction, they are all ascribed the same EC number, since the naming system is based upon the reaction catalyzed. Thus, sequences given the same EC number do not necessarily show sequence similarity.For our analyses we used all sequences from the Swiss-Prot and TrEMBL databases annotated with a unique EC number. We define two different "true" cluster sets representing different levels of granularity as follow: (1) sequences having A, B, C, and D in common build a cluster, (2) sequences having A, B, and C in common build a cluster. The data set consists of 84,405 sequences.Assuming we have a well defined cluster set, we can compare our cluster set with this "true" cluster set based on the following numbers:Number of sequence pairs clustered together ina: both cluster sets ("true positives").b: the "true" cluster set, but not in our cluster set .c: our cluster set, but not in the "true" cluster set .. A perfect clustering which is identical to the "true" cluster set would result in S = 1.As similarity measure we decided on the Jaccard similarity  defined ) and the specificity (the ability to reject non-related sequences: ) for all cluster sets.Additionally we calculated the sensitivity  choice of a cutoff (data not shown).TribeMCL  is a metFor the Pfam data set we were not able to perform TribeMCL clustering due to memory allocation problems while executing the program.MV had the initial ideas for SYSTERS. JS developed the superfamily determination. AK developed the subfamily determination, implemented the workflow from the raw sequence databases to a working web-server and made the validations. All authors read and approved the final manuscript.


Epidemiologic Perspectives & Innovations. Epidemiology  is a critically important field in informing decisions about the health of individuals and populations. But the desire for new information means that the health science literature is overwhelmingly devoted to reporting new findings, leaving little opportunity to improve the quality of the science. By creating a journal dedicated to all topics of and about epidemiology, except standard research reports, we hope to encourage authors to write more on the neglected aspects of the field. The journal will publish articles that analyze policy implications of health research, present new research methods and better communicate existing methods, reassess previous results and dogma, and provide other innovations in and perspectives on the field. Online publishing will permit articles of whatever length is required for the work, speed the time to publication and allow free access to the full content.This editorial introduces the new online, open-access, peer-reviewed journal,  Epidemiologic Perspectives & Innovations (EP&I) was created to provide a forum for efforts to improve the quality of health science research and its applications.Epidemiology is a critically important field in informing decisions about the health of individuals and populations. It is also a young field, with the potential for seeing fundamental improvements in the conduct of the science every year. But the desire for new information means that the health science literature is overwhelmingly devoted to reporting new findings, leaving little opportunity to improve the quality of the science. Successful enterprises know they must devote a substantial portion of their resources – at least a few percent and often ten percent or more – to assessing whether the rest of their resources are being optimally directed. Such efforts include research and development, which improves the quality of products, and outcomes research, which assesses the impact of those products. In an applied science like epidemiology, these efforts should be devoted to designing new methods for conducting studies and interpreting results, translation of research into effective policy recommendations, critical review of past findings and current practice, and improvement of teaching the next generation of scientists.Given the resources expended by the health science research enterprise, epidemiology  is characterized by remarkably little innovation, let alone critical review of existing dogma. A well-educated epidemiologist transported forward in time from 1980 would probably be able to read (and participate in) most current research and would find few surprises other than a few specific study results. Yet a substantial portion of all the epidemiology ever conducted has been carried out since 1980 – probably more than half, even including in the count all medical literature back to the dawn of literacy. The outputs of the science have increased to a torrent; research to improve the quality of the science is a trickle.It can hardly be argued that this slow innovation and lack of perspective is because current approaches offer little room for improvement. Current discussions of advanced statistical methods, the nature of random error, sensitivity analysis and uncertainty quantification, and proper interpretation of results, to name just a few, show that most current epidemiologic research uses methodology in need of improvement. Granting that many problems would be eliminated if health researchers – who often have minimal training in epidemiology – just followed the dictates of a good basic epidemiology textbook, there are still major problems that lack simple solutions. It is troubling that we plow ahead with billions of dollars worth of research every year while making minimal effort to answer fundamental questions about what that research is really telling us. Epidemiology is far too important to our society to be treated as an exercise in uncritically following existing formulae.The limitations of the field become even more apparent after the research is conducted. Results are cast out into the world as if they speak for themselves, forcing policy makers, clinicians, and interested lay people to interpret them, despite their lack of expertise in the topic area and analytic methods, and lack of necessary context.These problems leave plenty of blame to go around, but a fair amount of it rests with the lack of opportunity to publish scholarly analytic work aimed at solving the problems. While some health researchers might be guilty of not giving the field's limitations a second thought, most probably can envision contributions they would like to make to improve it. Every conclusions section containing a single paragraph of policy discussion suggests that researchers would like to contribute to policy analysis, but have exhausted their paltry word limit. Every dissertation that reflects upon and challenges standard methods shows fresh analytic thinking, but the innovations might be read by just the half dozen people who view the actual dissertation, since the resulting publications will likely be limited to a brief recounting of the methods and results (narrowly defined). Every time a professor explains to her students that something they learned in a previous class or from reading the literature is wrong, there is a lesson that should be getting out to everyone in the field, not just the ten students in that room.These scenarios call for full-length, analytically complete presentations (see endnote 1). Such analysis cannot be grafted onto a paper primarily focused on presenting numerical results from a study, given the severe length limitations in print journals in the health sciences. Indeed, such analyses must usually be longer than typical word limits allow, even without the study results or applications that are needed to illustrate them or provide background. A researcher who writes such a paper has a very difficult time getting it published. Moreover, it is easy to anticipate that difficulty and never even try to write the paper. The founding editors of this journal were inspired by their own experiences with these difficulties.There are journals that cover some of these areas, but to a remarkably limited extent. "Health policy"  journals focus on the financial side of health care, rather than more general health policy or economic issues. Statistics journals, and even the research methodology slots available in the health research journals, favor mathematically complicated advances over more practical advice. With the exception of occasional relevant entries in medical journal education series, articles devoted to improving the teaching or understanding of epidemiologic research have no clear venue. Even with some niches for certain methods, policy, overview, or perspectives articles, it is difficult to be enthusiastic about writing in these areas with no clear idea of where the results are likely to be published.Epidemiologic Perspectives & Innovations (EP&I) was created to provide this forum, taking advantage of the greater visibility and article length offered by open-access online publishing.EP&I will publish. , rather than commentary.Methodologic Innovation and Communication: EP&I welcomes submissions in all areas of epidemiologic research methodology, from study design to data analysis and reporting, including new tools, simple but important observations, and widely understandable applications of existing tools. The strength of our editorial board in this area means that submissions will be reviewed by experts who understand and appreciate new methods. Unlike most other journals publishing methods articles, EP&I welcomes submissions that are not necessarily at the technological cutting edge , but that contain lessons that are not widely known. We will spare authors the all-too-common experience of being told "everyone already knows that" when they submit a paper that calls for the use of methods or practices that are widely overlooked. Research articles are needed to translate methodological findings that are "known" , to make them known (in the sense of being understood and usable by most researchers in the field).Ethics, Philosophy, and Critical Analysis of the Field: In most of the health research literature, any discussion of philosophical points or assessments of the quality of research is labeled "commentary" and restricted to the opinions of a few luminaries. But carefully reasoned ethical analysis, epistemology, analysis of quality, and the like are not mere commentary, and often come from junior researchers or outsiders. Our accompanying "wish list" editorial provides some examples of these types of analysis.Re-analyses: The deluge of research results in health science means that few study results are ever carefully re-analyzed, even when their implications are quite important. When such re-analyses do occur, they are often limited to letters or perfunctory assessments in systematic reviews. EP&I offers a forum for publishing full-length re-analyses  of important previous research.Teaching Methods and Innovations: Many fields have a dedicated teaching section in one or more journals. EP&I will include articles that provide teaching tools, innovations, and methods. Online publishing allows authors to include computer code, spreadsheets, datasets, and other tools that will allow readers to make use of the teaching tools. Teaching articles will be peer reviewed by experienced teachers and at least one current student at the appropriate level to judge the material. The ideal teaching articles will present an approach or method, the specific tools necessary for a reader to implement it, and a report of the authors' experience in using the material. Review will be based primarily on the apparent usefulness of the presented approach.Multidisciplinary Research: This category is somewhat redundant, given that many of the aforementioned article types necessarily draw upon knowledge from multiple disciplines. But it is worth mentioning specifically because it is often difficult to publish work that is based in multiple fields of inquiry and thus does not fit easily into any one of them, or that is squarely in another discipline but is intended for an audience of health scientists. EP&I encourages such submissions and will review them based on their analytic merits in the fields in which they are based and their potential usefulness to health researchers.EP&I is a home for all articles of and about epidemiology, with the exception of standard research reports.  This includes many types of papers that are not themselves epidemiologic analysis, but inform epidemiology or are about epidemiology. We suspect that many papers of the above types exist on paper or in researchers' heads, but have previously been difficult to get published. Many more will be written when they are appreciated as analytic work that is central to the field.More generally, EP&I to be this article type, which allows authors to create a structure that fits their analysis  and is labeled to emphasize that the article is analytic (as opposed to commentary).To provide maximum flexibility for these kinds of articles, we worked with BioMed Central to create the "Analytic Perspectives" article type. We expect most submissions to We are also taking the unusual  step of encouraging the use of endnotes. We believe that the lack of substantive endnotes or footnotes – to provide important asides, definitions, or clarifications, to note exceptions to general rules, or provide other elaboration – is a major detriment to the content of health research papers, making it difficult to present certain analytic points. For example, an interesting statistical claim or policy observation that is almost always true can either be made without further elaboration (in which case the exceptions make the claim incorrect), can include a paragraph of caveats (which is awkward and distracting), or left out (see endnote 2). Often the latter is the author's choice, which impoverishes the literature. An endnote could solve the problem. In other cases, endnotes could include short derivations of calculations that will be obvious to some readers and uninteresting to others, but may be of interest to some. Anyone familiar with the social science literature or many other fields will understand the beneficial uses to which such notes can be put (see endnote 3). Authors should consult the instructions for submissions for mechanical details of endnote use.EP&I from virtual cover to virtual cover. Most readers of most health science journals scan the table of contents and read the one or two articles that report results in their subspecialty, or never even see a table of contents, but merely a list of search hits on PubMed. Most articles in EP&I should be of some interest to researchers who are serious about understanding analytic health research and its implications.We hope that many readers will read We welcome submissions from researchers with all levels of experience in the field, and from experts in other fields writing for health researchers. Great innovations and critical analysis often come from senior scholars in a field, but they also often come from graduate students, outsiders, and others who are not heavily invested in the status quo of a field of inquiry.Science published the controversial article, "Epidemiology Faces its Limits" [EP&I hopes to encourage the pursuit of breakthroughs  by providing a ready home for publishing a broad collection of such material.In 1995,  Limits" , which s1. "Analytic" should not be confused with quantitative calculations and results, which seldom contain much actual analysis. Analysis can be thought of as the intellectual process of systematic inquiry aimed at understanding, explaining, or characterizing phenomena or concepts.2. For example, authors might want to point out that nondifferential misclassification error that they suspect exists most likely biased a result toward the null. However, an unqualified statement to this effect will likely generate criticism that such error is not always toward the null and that believing so is a sure sign of methodological naivete. An endnote in which the authors point out that there are exceptions, but the bias is still usually toward the null, would allow them to make their point without a long awkward caveat breaking up the main text.3. The literature in most of these fields uses footnotes, conveniently located at the bottom of the page. Online publishing leaves us without a bottom of the page, but allows for convenient opening of multiple windows, offering the opportunity to have the endnotes open in a separate browser window. Readers of printed PDF versions will, alas, have to flip to the end.


Drug therapy has become increasingly efficient, with more drugs available for treatment of an ever-growing number of conditions. Yet, drug use is reported to be sub optimal in several aspects, such as dosage, patient's adherence and outcome of therapy. The aim of the current study was to investigate the possibility to optimize drug therapy using computer programs, available on the Internet.One hundred and ten officially endorsed text documents, published between 1996 and 2004, containing guidelines for drug therapy in 246 disorders, were analyzed with regard to information about patient-, disease- and drug-related factors and relationships between these factors. This information was used to construct algorithms for identifying optimum treatment in each of the studied disorders. These algorithms were categorized in order to define as few models as possible that still could accommodate the identified factors and the relationships between them. The resulting program prototypes were implemented in HTML (user interface) and JavaScript (program logic).Three types of algorithms were sufficient for the intended purpose. The simplest type is a list of factors, each of which implies that the particular patient should or should not receive treatment. This is adequate in situations where only one treatment exists. The second type, a more elaborate model, is required when treatment can by provided using drugs from different pharmacological classes and the selection of drug class is dependent on patient characteristics. An easily implemented set of if-then statements was able to manage the identified information in such instances. The third type was needed in the few situations where the selection and dosage of drugs were depending on the degree to which one or more patient-specific factors were present. In these cases the implementation of an established decision model based on fuzzy sets was required. Computer programs based on one of these three models could be constructed regarding all but one of the studied disorders. The single exception was depression, where reliable relationships between patient characteristics, drug classes and outcome of therapy remain to be defined.Algorithms for optimizing drug therapy can, with presumably rare exceptions, be developed for any disorder, using standard Internet programming methods. During the last decades the possibility of computer support for optimum management of various disorders has attracted interest -3. RegarIn the current study guidelines for 246 diseases were analyzed regarding the content of facts, and relationships between these facts, influencing the selection and dosage of therapeutic drugs. The aim of study was to identify one or more algorithms (descriptions of how to solve a problem in a definite number of steps), which could be used to support optimum drug therapy in any disease.The studied material consisted of 110 officially endorsed text documents, published between 1996 and 2004. Forty-four of these documents were published by the Swedish Medical Products Agency , 13 by tThese documents were analyzed as follows: All statements in the particular document regarding patient-, disease- and drug-related factors were recorded. In a second step the statements in the texts describing the relationship between these factors were identified. After that the obtained information was rewritten in a format similar to a generic computer language, using if-then statements and logical operators as needed. These rewritten documents were grouped according to similarity in structure, based on the number of if-then statements, the need for branching or calculations in the code and the occurrence of 'left-overs', i.e. information in the text that could not be explicitly expressed using if-then statements and logical operators. In theory this procedure could result in an intractable combinatorial explosion. This was observed only in one situation, regarding side effects of anti-hypertensive medication.Figure In situations, where treatment can by provided using drugs from different pharmacological classes, a more elaborate model is required. An example is heart failure. A 25-page summary of 69 pages of background documentation described six physiological states and 11 available drug classes. This knowledge was modelled using an ordered set of it-then statements, see figure In some diseases the selection and dosage of drugs depend on the degree to which one or more particular factors are present. In such cases a specific program implementation of an established decision model based on fuzzy set theory  could beThe pharmaceutical information about all therapeutics drugs, approved in Sweden, is available on the Internet at a site managed by the association of pharmaceutical industries . By linkThe main findings in the current study are that simple algorithms can support drug therapy and that such algorithms can be easily implemented using standard Internet programming techniques. Problems in the implementation of information system designed to support actual care are discussed in a study by Bates et. al. . The autOne disorder, depression, was identified for which relevant computer program support could not be constructed. Initially, the program model with the above-mentioned decision algorithm, based on fuzzy sets, was thought to be applicable, since the number of relevant facts is well within manageable range; four major classes of drugs, slightly more than ten diagnostic variables, some ten receptors and seven neurotransmitters. However, the information in the official consensus document , did notProblems in pharmacotherapy related to the simultaneous occurrence of multiple disorders in the same patient are well studied and presented in the pharmacological information as contraindications or recommendations of "caution in patients with disease ...". This information is used as output from the programs in the current study.The information in the analyzed text documents was not always sufficiently detailed and logically consistent to allow the construction of computer code. Additional information could usually be gathered from other sources, but such procedures may induce errors and should be avoided. Methods are currently available to support logical structure in text documents, e.g. the Guideline Elements Model . This moThe introduction of a new drug for treatment of a particular disease, or the results from new scientific studies may have considerable influence on guidelines. Yet only a few documents were changed during the study period and the lifespan of pharmacotherapeutic guidelines can be assumed to be 2–5 years. However, new information may appear unexpectedly, and a reasonably complete system for pharmacotherapy requires frequent updates. The approach taken in the current study, with the programs stored on a website as scripted HTML pages, facilitates the maintenance and update of the system and ensures that all users always have access to the latest version. An additional way to increase relevance over time is to link from the programs to recently published studies, e.g. by providing automatic searches in the PubMed database .® are helpful. The use of Internet implies that only one version of the programs is in use, that upgrading is very convenient, and that no interference occurs with existing systems. No confidential patient data has to be communicated and the programs are always available from any Internet connected computer.In the current study based on guidelines regarding two hundred and forty-six diseases, three basic program models were found to be sufficient for computerized decision-support in pharmacotherapy. The programs could be developed using HTML and JavaScript, i.e. simple and widespread programming techniques. No specific development tools are needed although standard programming tools such as Macromedia DreamweaverIn summary, algorithms for optimizing drug therapy can, with presumably rare exceptions, be developed for any disorder using standard Internet programming methods.None declared.PW conceived and designed the study, collected data, supervised data analysis, did most of the JavaScript and some of the HTML programming and contributed to the writing of the paper.LM did most of the HTML and some of the JavaScript programming and contributed to the data analysis and the writing of the paper.Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


This study sought to identify the extent to which employee level and work stressors were associated with mental health problems experienced by Australian government employees, and with their use of primary care services.806 government employees aged between 40 and 44 years were surveyed as part of an epidemiological study conducted in Australia. Data collected from participants included sociodemographic attributes, physical health, psychological measures and work stressors relating to job control, job demands, job security and skills discretion at work. For 88% of these participants, information on visits made to general practitioners (GPs) for the six months before and after their survey interview was obtained from health insurance records.When work stress and personal factors were taken into account, men at more junior levels reported better mental health, more positive affect and used fewer GP services. Women at middle-management levels obtained less GP care than their more senior counterparts. Both men and women who reported higher levels of work stress were found to have poorer mental health and well-being. The impact of such stressors on GP service use, however, differed for men and women.Measures of work stress and not employee level affect the mental health and well-being of government employees. For governments with responsibility for funding health care services, reducing work stress experienced by their own employees offers potential benefits by improving the health of their workforce and reducing outlays for such services. In 1999, the World Health Organization reported that workers continued to suffer high levels of work-related injuries and deaths ,3. Such The applicability of this model for the government sector is well supported by cross-sectional and longitudinal studies drawing on the Whitehall II study of a large cohort of 10,308 London-based government employees. Again, such studies have found that those in lower grades report that they have less job control, less variety in their work, and less job satisfaction . Those rThere has been little research undertaken on the health impact of job level and work stressors for government employees in Australia. An earlier study that explored the relationships between work stressors and blood pressure in Australian government employees, found chronic perceived work stress to be associated with blood pressure change . The impWe have been able to explore these issues using data collected from 806 government employees who participated in the PATH Through Life Project, a large community-based study being conducted by the Centre for Mental Health Research in Canberra, Australian Capital Territory (ACT). Survey participants provided information on sociodemographic measures, mental and physical health, employment level and work-related stress. For 88% of these participants, independently collected information on their use of general practitioner services was also available. These data have allowed us to examine the impact of employment level and work-related stress on Australian government employees' mental and physical health, their psychological well-being, and also their use of general practitioner care. We hypothesised that those working in lower level government positions would report higher levels of work-related stress, that they would be found to have more mental and physical health problems and that they would use higher numbers of primary medical services.The PATH Through Life Project is a longitudinal study of individuals living in the community with participants being drawn from three age groups: 20–24, 40–44 and 60–64 years. Those in the age group of interest for this study were aged from 40 to 44 years on 1 January 2000 and drawn from the Australian Electoral Rolls for Canberra in the Australian Capital Territory and adjacent town of Queanbeyan in New South Wales. Enrolment on these rolls is compulsory for all Australians aged 18 and over. Potential participants were drawn from a 10-year age range, the minimum range then released for research purposes by the Australian Electoral Commission. The number of potential participants found, and in the required age range, was 3919, of whom 2530 participated in the survey, giving a response rate of 64.4%.Canberra is the national capital of Australia and many Australian Government entities are based in the ACT, including both houses of parliament, and the 16 major agencies that currently comprise the Australian Public Service. In this study, 806 respondents aged between 40 and 44 reported that they worked in office-based government administrative positions, developing and implementing government policy. As well as providing information on labour force status and the type of position held, respondents who worked in government positions were specifically asked to provide details on the level of the position they occupied. Five mutually exclusive employee categories were formed by grouping together those whose levels of employment were broadly comparable as follows. Employees who occupied positions at Australian Public Service (APS) Levels One to Four were grouped together as Junior employees and those at both the APS Levels 5 and 6 were classified as belonging to the Intermediate category of employees. Employees in the next two classifications of the APS (Executive Levels 1 and 2) were allocated to separate groups, Senior 1 and Senior 2. While those in Executive Level 1 positions develop policy and implement government programs, those at the Executive 2 level are primarily managers with direct responsibility for managing a number of employees from APS Level 1 to Executive Level 1 . FinallySurvey participants completed a questionnaire that included socio-demographic characteristics and measures of physical and mental health, and well-being. Participants in the workforce were asked 22 questions relating to their work situation. These questions matched those used in the Whitehall II study ,13. NineSocio-demographic measures used in these analyses included sex, age, years of education, level of household responsibilities, and experience of any of six life events during the previous six months. Since each of these factors has the potential to modify an individual's mental health independently of their work stress, we adjusted for these in our final analyses. Scores for level of household responsibility were drawn from participants' responses to questions concerning the extent to which, in their household, they were responsible for four areas: household tasks, childcare, financial management, and providing money. Comparable scores for participants who did not have children in their households were then derived by calculating the mean of their measures for household tasks, financial management and providing money, and adding this to their total household responsibility score. Scores for these measures could range from zero to 16 with higher sores representing more household responsibilities.Health measures obtained from participants and used in these analyses included: scores on Goldberg's depression and anxiety scales  and statR2 for linear regressions and Incidence Rate Ratios when the Poisson or negative binomial regression model was used. Incidence Rate Ratios (IRRs) are interpreted in a similar manner to odds ratios and represent the expected change in the dependent variable in response to one unit change in the predictor variable. The contribution of employee level and work stress measures in explaining variation in health measures was also obtained. This contribution was measured using change in R2 for linear regressions and the change in the Chi-square estimate of the fit of model for the negative binomial and the Poisson regression analyses. A final analysis examined the impact of employee category and work stressors on use of GP services, taking into account demographic, lifestyle and health measures. Analyses were undertaken using SPSS 11.5 and STATA 7 [Analyses of variance (ANOVAs) were first undertaken to examine the extent to which sociodemographic measures and work stress measures changed with level of employment. Similar analyses, conducted separately for men and women, then compared mean mental health measures across the five employee levels. Finally, regression analyses were used to examine the contribution of employee category and work attributes in explaining participants' health and health service measures, whilst controlling for the following possible modifying factors: participant's age, years of education, level of household responsibilities and life events experienced in the past six months. For these analyses, categorical variables identifying each of the five government employee categories were created and the first four of these included in the regression equations, taking the most senior category, Executive, as reference group. After initial testing indicated that two dependent variables, negative affect and use of GP services, were not normally distributed and more closely fitted the negative binomial and Poisson distributions respectively, analyses of these two measures used negative binomial and Poisson regressions respectively. Strength of associations between dependent variables and predictor variables were measured using  STATA 7 .Across the five categories of government employees, there was no significant difference in education level or in numbers of life events experienced in the past six months. Employees working at higher levels, however, reported lower levels of household responsibility and had more opportunities to develop and use different skills in their work, reported more job control and felt more secure in their current jobs. As expected, those in more senior positions also had less manageable job demands and worked longer hours.Analyses were then performed, separately for men and women, to examine differences in measures of mental health, well-being and GP service use across the five government employee categories. Level of physical health, as measured by the SF-12, was also examined for comparative purposes. The only measure to differ significantly across employee categories was level of positive affect in the past four weeks Table . For botWe next used regression analyses to examine the impact of employee category and work stress on participants' measures of health and well-being and on their use of GP services. In preliminary testing, we found that the two measures – job control and skill discretion – both contributed significantly and independently to mental health measures, hence these measures were not combined but included separately in the analyses. The results of these analyses for men are in Table For women, we found no effect of employee level on their measures of mental or physical health. However, employment level was associated with GP service use with those in middle management positions being less likely to have obtained GP care, compared with those at the executive level. Women's levels of mental health and well-being were better when they worked in a job that offered higher levels of skill discretion. Manageability of job demands impacted on all health measures considered while those who worked longer hours were more likely to have obtained GP services.Finally, we explored the contributions of employment level and work stress factors in explaining participants' use of GP services, after including in the model SF-12 measures of mental and physical health that could also contribute to explaining such service use. Again, men and women were considered separately. Overall, controlling for mental and physical health in addition to sociodemographic measures had little impact on our findings. Men in lower employee categories continued to use significantly fewer GP services compared with their counterparts in executive positions while those with less job control and less job security again obtained more care. After controlling for mental and physical health, women in middle management levels again used fewer GP services compared with those at the executive level. Similarly, women with more manageable job demands and those working longer hours continued to be higher users of GP care.This paper has reported on the associations between categories of employee levels, work stressors and mental health measures of 806 government employees aged between 40 and 44 who participated in the PATH Through Life Project being conducted in Canberra, Australia's national capital.Our first hypothesis, that government employees working at lower levels would report higher levels of work-related stress, is supported in the main by our research. Overall, participants at more senior levels reported that they had more control over aspects of their work, greater opportunities to do interesting work using a range of skills, more job security but also that they were subject to higher job demands. These results closely match those reported by Marmot and colleagues in their 1991 study of over 10,000 British civil servants aged between 35 and 55 .per se, is not automatically associated with poorer mental or physical health for Australian government employees. While these findings are unusual, they do align with some previous research undertaken in the UK showing that those working in lower grades had better mental health [We had hypothesized that those in lower level positions would have poorer physical and mental health compared with more senior staff. None of our results supports this hypothesis. Although both male and female participants in the higher grades reported better well-being as measured by higher positive affect, we found no mental health benefit associated with having a more senior position. Furthermore, for men, we found this result to be reversed when the analyses controlled for work stress factors. Men in lower level positions reported higher levels of positive affect and better mental health, as measured by the SF-12. Women's mental health and negative affect were not affected by their employee level both when this factor was considered alone and when work stress factors were taken into account. We were unable to replicate the finding by Marmot and colleagues that those in higher positions had significantly better physical health compared with their subordinates. This difference in our results might be explained by our having a smaller sample. However, it also indicates that while those working at lower levels are more likely to experience some types of work stressors, working at those lower levels, l health . One posWe found government employee level affected men's use of GP services but the direction of this effect was the reverse of that hypothesised. After taking into account education, other personal measures and levels of work stress, men at lower levels of the public service used fewer GP services than their superiors. This aligns with the previous finding that men at lower level positions also had better mental health  and suggWork stress factors experienced at all levels of employment played a more significant role in affecting participants' health and well-being. For male and female participants, those whose job demands were more manageable had significantly better mental health. This measure of work stress was particularly important for women, for whom manageability of demands was significantly associated with all health measures. Correspondingly, level of skill discretion had a greater impact on men's mental health. This last finding concurs with a recent Australian study of government employees which reported that, for men, having interesting work was an important reason for their staying in the government sector whereas women saw their relative job security as the advantage of this form of employment .Since previous research has linked job insecurity and poorer mental health ,21, we eA number of work stress factors contributed significantly to explaining men's and women's use of GP care. Women who used more services had less job control while men were more likely to visit a GP if they reported less skill discretion, job control or felt more insecure about their job. Research reported in the 1980's also found that factory workers with higher job insecurity used more health services . This fiWomen who worked longer hours used slightly more GP services and also had more manageable job demands. Men who worked fewer hours, however, obtained more GP care. These findings indicate that time spent at work is unlikely to have deterred women from addressing their health care needs. For men, on the other hand, working fewer hours per week might have given them needed, additional opportunity to set aside time for a GP visit. In Australia, many GP surgeries are open during standard working hours and offer only limited access outside of these times .This study is limited by having access only to cross-sectional data, since the direction of relationships between psychological stress and work stressors cannot be confirmed. It may be that those with fewer symptoms of depression or anxiety, for example, also have more positive views about the current or potential benefits of their positions. A number of issues raised here may become clearer when data from the next wave of the PATH project are collected.These findings have implications for governments in their role as major employers. Large organisations in the public and private sectors inevitably develop hierarchical systems of employment as efficient mechanisms for allocating work functions and responsibilities. This study suggests that employees' health is less affected by their position within this structural hierarchy and is more associated with various work stressors that can be experienced by individuals across all levels of employment. For a large employer, reducing the impact of work stress on its workforce may be beneficial, not only for individual employees, but also for the productivity of the organisation as a whole. These findings also have implications specific to the Australia setting where GP services are provided and subsidised through the universal health insurance system, Medicare. This component of health care is the financial responsibility of the Australian Government, which is also the employer of the majority of participants in this current study. Initiatives aimed at reducing work stress experienced by government employees, and correspondingly, the numbers of GPs services obtained by that workforce, might prove to be a judicious use of Australian Government resources. Such potential benefits may well apply to other governments that have responsibility for funding health care services.The authors declare that they have no competing interests.ACT Australian Capital TerritoryAPS Australian Public ServiceGP General practitionerIRR Incidence rate ratioPANAS Positive and Negative Affect ScaleSF-12 12-item Short Form Health SurveySPSS Statistical Package for Social ScientistsRP contributed to the conception of this study, and was responsible for all analyses of data, interpretation of results and writing of successive drafts of the paper. AFJ was leader in designing and running the PATH Through Life Project, contributed to interpretation of findings and edited successive drafts of the paper. HC was co-leader in designing and running the PATH Through Life Project, contributed to interpretation of findings and edited successive drafts of the paper. DB, LS and RD contributed to the conception of the study and editing successive drafts of the paper. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Rotational thromboelastography (ROTEG) has been proposed as a monitoring tool that can be used to monitor treatment of hemophilia with recombinant factor VIIa (rFVIIa). In these studies special non-standard reagents were used as activators of the coagulation. The aim of this study was to evaluate if standard ROTEG analysis could be used for monitoring of effects of recombinant factor VIIa (rFVIIa) on Hydroxy Ethyl Starch-induced dilutional coagulopathy.The study was performed in vitro on healthy volunteers. Prothrombin time (PT) and ROTEG analysis were performed after dilution with 33% hydroxy ethyl starch and also after addition of rFVIIa to the diluted blood.PT was impaired with INR changing from 0.9 before dilution to 1.2 after dilution while addition of rFVIIa to diluted blood lead to an overcorrection of the PT to an International Normalized Ratio (INR) value of 0.6 (p = 0.01). ROTEG activated with the contact activator ellagic acid was impaired by hemodilution (p = 0.01) while addition of rFVIIa had no further effects. ROTEG activated with tissue factor (TF) was also impaired by hemodilution (p = 0.01) while addition of rFVIIa lead to further impairment of the coagulation (p = 0.01).The parameters affected in the ROTEG analysis were Clot Formation Time and Amplitude after 15 minutes while the Clotting Time was unaffected. We believe these effects to be due to methodological problems when using standard activators of the coagulation in the ROTEG analysis in combination with rFVIIa. Patients undergoing massive hemorrhage experience dilutional coagulopathy with crystalloid and/or colloid resuscitation. If hemorrhage progresses, packed red cells (RBC) are transfused together with crystalloids and/or colloids. Regarding the coagulation this is not optimal, and the patients often develop a dilutional coagulopathy, sometimes worsened by hypothermia. In addition a coagulopathy caused by the administration of dextrane or hydroxy ethyl starch (HES) may be induced. The common approach to this is transfusion of fresh frozen plasma (FFP) and platelets, but bleeding might continue, with often fatal outcome. Prophylactic use of fresh frozen plasma (FFP) or platelet transfusion is not proven beneficial to prevent hemorrhage in massively transfused patients. HemorrhA novel approach to treat these patients is the use of recombinant factor VIIa (rFVIIa) to improve the coagulation.. RecentlDilutional coagulopathy can be detected by the use of thromboelastographic measurements -11. RotaPrevious studies have shown that hemodilution with HES impairs the coagulation already at a dilution of 33% while crystalloid hemodilution does not appear to give a readily detectable impairment of the coagulation until the level of hemodilution reaches 50% ,12. In tThe local ethics committee of the University of Lund approved this study on healthy volunteers. Volunteers were not allowed to take any medication 14 days prior to the study. Informed consent was obtained from the participants and a total of eight were recruited. All participants had an indwelling intravenous catheter placed into the brachiocephalic vein, from which the blood samples were drawn with sterile disposable 5-ml syringes . A first 5-ml blood sample was discarded before every blood sample for the experiment described below. No tourniquet was used on the arm when samples were drawn.®, Fresenius Co., Bad Homburg, Germany), the HES preparation found to cause the least pronounced coagulopathy after hemodilution [Dilution of the blood samples was performed with HES 130/0.4 , Prothrombin time (PT) and ROTEG analysis. All tests were performed at normal body temperature (37°C). For the Hb measurements a Hemocue  was used. PT measurements were performed with a Rapidpoint Coag Analyzer  with PT-ONE test cards. The ROTEG analyses were performed on a Rotational Thromboelastograph  and the samples were analysed 120 seconds after the blood was drawn from the intravenous catheter. Both INTEG and EXTEG analyses were performed according to standard procedure recommended by the manufacturer. In INTEG analysis the coagulation is initiated with the addition of 20 μl of the contact activator ellagic acid  to 320 μl of blood pipetted from the test tube to a reaction cup used in the ROTEG. In EXTEG analysis the coagulation is activated by the addition of 20 μl of a preparation containing tissue factor (TF)  to 320 μl of blood pipetted from the test tube to the reaction cup. TF activates the coagulation through binding to Factor VIIa and this is believed to be the important interaction when in vivo coagulation occurs. The parameters obtained from the ROTEG analysis were Clotting Time (CT) reflecting the initiation of the coagulation, Clot Formation Time (CFT) reflecting the rate of clot formation once the formation is initiated and A15 describing the strength of the clot 15 minutes after initiation of the coagulation . A p value of < 0.05 was considered statistically significant.The insertion of venous catheters and the blood sampling were performed uneventfully. The Hb values decreased as an expected sign of dilution table . PT valuIn neither INTEG nor EXTEG analysis we found any change in the CT between normal and dilution groups, while CFT and A15 were impaired in the dilution group tables  and 3. TTreatment of dilutional coagulopathy is challenging and the primary monitoring tools are measurement of PT, activated partial thromboplastin time (APTT), platelet count and fibrinogen -16. ThesIn our study we found that hemodilution in vitro with HES lead to augmentation of the PT and the addition of rFVIIa results in a prompt decrease of the PT. This is in line with previous studies, which have shown that dilution with HES lead to readily detectable changes in the coagulation system,12,20. Fin vitro, worsening both these parameters according to EXTEG analysis, suggested that platelets or fibrinogen became dysfunctional in contrast to the clinical effect of administration of rFVIIa, where rFVIIa has been found successful in case stories of bleeding patients [When analysing the ROTEG parameters we found that the 33% dilution with HES resulted in a prolongation of CFT values and impairment in A15 values in accordance with previous studies,11,12,20patients ,7,13,23,The lack of effect in the INTEG analysis when adding rFVIIa to the diluted blood is most likely due to the fact that a contact activator is used to activate the coagulation in the INTEG and therefore insensitive to rFVIIa as rFVIIa initiates coagulation through interaction with TF.The impairment of CFT and A15 in the EXTEG analysis after addition of rFVIIa to the diluted blood is harder to explain. We expected addition of rFVIIa to the diluted blood to result in an improvement of the TEG parameters measured in the EXTEG analysis. This was expected partly because TF is used as an activator of the coagulation in the EXTEG analysis and the first step in the initiation of the coagulation system is the interaction between TF and FVIIa. ,18,24. IThe previously reported improvements of coagulation in hemophilia and liver transplant patients as evaluated with ROTEG after administration of rFVIIa also lead us to believe that ROTEG parameters would be improved after addition of rFVIIa to the diluted blood,18,24. IIn conclusion we found that 33% dilution of blood with HES 130/0.4 lead to impairment of the coagulation when evaluated with ROTEG or PT. Addition of rFVIIa lead to overcorrection of the prolonged PT. ROTEG analysis revealed that INTEG analysis was insensitive to effects of addition of rFVIIa and that EXTEG analysis was dramatically impaired by the addition of rFVIIa. It may well be that rFVIIa is not effective in improving the coagulopathy induced by HES hemodilution, but the further impairment of the coagulation seen in the EXTEG analysis is likely due to methodological problems. These problems make the commercially available EXTEG analyses inappropriate for monitoring of rFVIIa effects under circumstances of HES dilution.The author(s) declare that they have no competing interests.ME contributed to the design of the study, performed the analyses and drafted the manuscript. PR contributed to the design of the study, to data interpretation and to preparing the manuscript. US contributed to the design of the study, performed the analyses and participated in manuscript preparation. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Virtual Reality (VR) offers a blend of attractive attributes for rehabilitation. The most exploited is its ability to create a 3D simulation of reality that can be explored by patients under the supervision of a therapist. In fact, VR can be defined as an advanced communication interface based on interactive 3D visualization, able to collect and integrate different inputs and data sets in a single real-like experience.However, "treatment is not just fixing what is broken; it is nurturing what is best" . For rehabilitators, this statement supports the growing interest in the influence of positive psychological state on objective health care outcomes.transformation of flow. In this view, VR can be used to trigger a broad empowerment process within the flow experience induced by a high sense of presence.This paper introduces a bio-cultural theory of presence linking the state of optimal experience defined as "flow" to a virtual reality experience. This suggests the possibility of using VR for a new breed of rehabilitative applications focused on a strategy defined as The link between its experiential and simulative capabilities may transform VR into the ultimate rehabilitative device. Nevertheless, further research is required to explore more in depth the link between cognitive processes, motor activities, presence and flow. What is virtual reality (VR)? For many health care professionals, VR is first of all a technology. Since 1986, when Jaron Lamier used the term for the first time, VR has been usually described as a collection of technological devices: a computer capable of interactive 3D visualization, a head-mounted display and data gloves equipped with one or more position trackers.But is this definition enough to describe the potential of VR for rehabilitation? If we look at its actual applications in rehabilitation, the answer is probably not .VR can be considered the leading edge of a general evolution of present communication interfaces like television, computer and telephone . The maiIn fact, VR is used in rehabilitation as "an advanced form of human-computer interface that allows the user to interact with and become immersed in a computer-generated environment in a naturalistic fashion" .et al. [Following this vision Rizzo et al.  identify• The capacity to systematically deliver and control dynamic, interactive 3D stimuli within an immersive environment that would be difficult to present using other means.ecologically valid assessment and rehabilitation scenarios.• The capacity to create more • The delivery of immediate performance feedbacks in a variety of forms and sensory modalities.• The provision of "cueing" stimuli or visualization tactics designed to help guide successful performance to support an error-free learning approach.• The capacity for complete performance capture and the availability of a more naturalistic/intuitive performance record for review and analysis.• The capacity to pause assessment, treatment and training for discussion and/or integration of other methods.• The design of safe testing and training environments that minimize the risks due to errors.• The capacity to improve availability of assessment and rehabilitation by persons with sensorimotor impairments via the use of adapted interface devices and tailored sensory modality presentations built into VE scenario design.• The introduction of "gaming" features into VR rehabilitation scenarios as a means to enhance motivation.• The integration of virtual human representations (avatars) for systematic applications addressing social interaction.• The potential availability of low cost libraries of VEs that could be easily accessed by professionals.• The option for self-guided independent testing and training by clients when deemed appropriate.In summary, VR provides a new human-computer interaction paradigm in which users are no longer simply external observers of images on a computer screen but are active participants within a computer-generated three-dimensional virtual world -8: in thThis outline better clarifies the possible role of VR in rehabilitation: a communication interface based on interactive 3D visualization, able to collect and integrate different inputs and data sets in a single real-like experience ,11.presence [This is possible because the key characteristic of VR, differentiating it from other media or communication systems, is the sense of presence , usuallypresence , or the presence .This feeling is theorized to contribute to the efficacy of VR as rehabilitation tool: the successful use of VR exposure therapy for phobias -18, postThanks to presence, not only knowledge acquisition is possible in VR, but also this acquired knowledge can be transferred in a real environment ,27. ThisNow, the challenge within this area is the creation of new paradigms . As cleaWithin this context we propose to investigate the impact of VR on rehabilitation and subjective experience from a theoretical perspective that stresses the active role of individuals in interacting with their natural and cultural environment ,30. In tWhat is presence? Answering to this question is not a simple task ,31,32. Iperceptual illusion of non-mediation [A first group of researchers describes the sense of presence as a function of our experience of a given medium -41. The ediation , produceThis approach, however, does not address some broader questions: What is presence for? Is it a specific cognitive process? What is its role in our daily experience?To answer these questions a second group of researchers considers presence as a neuropsychological phenomenon, evolved from the interplay of our biological and cultural inheritance, whose goal is the control of agency ,12,42-49Within this paper, we will support the second vision, trying to link it with the outcome of the first one . Here, pTo fully understand the key ideas behind this vision, three points are critical:– Presence has a simple but relevant role in our everyday experience: the control of agency through the unconscious separation of "internal" and "external". The meaning of "internal" and "external" is not related only to the body but also to the social and cultural space (situation) in which the self is in;– The presence-as-process (the separation mechanism) produces, but it is different from the presence-as-feeling (the experienced level of presence);– The presence-as-feeling is experienced indirectly by the self through the characteristics of action and experience. In fact the self perceives directly only the variations in level of presence-as-feeling: breakdowns and optimal experiences.distal attribution – the referencing of our perception to an external space beyond our boundaries – and effectively control its agency.First, presence is described here as a defining feature of self and it is related to the evolution of a key feature of any central nervous system: the embedding of sensory-referred properties into an internal functional space. As noted by Waterworth and Waterworth , the appIn this vision it is important to distinguish between presence-as-process and presence-as-feeling. The presence-as-process is the continuous activity of the brain, organized around the three functionally and phylogenetically different layers discussed in the next paragraph, in separating "internal" and "external" within different kinds of afferent and efferent signals.A critical point here is to explain why we need to introduce a new cognitive process – presence-as-process – to monitor our activity. The answer comes from a recent paper by de Vignemont and Fourneret . These aHowever, both the neuroscience of action and the neuropsychology of schizophrenia are countering his position . For insAs clarified by Russell : "Actionbeing-in-the-world" In fact, a higher level of presence-as-feeling is experienced by the self as a better quality of action and experience [being-in-the-world when its level is modified. More in detail, the self perceives directly only the variations in the level of presence-as-feeling: breakdowns and optimal experiences.For this reason, the presence-as-feeling (level of presence) is not separated from the experience of the subject but it is related to the quality of our actions. It corresponds to what Heidegger  defined perience ,56. HoweThe process of adaptation to the natural environment provided humans with specific biological features, such as the upright position, the opposing thumb and the increase in brain mass that allowed survival and reproduction in any environmental niche. At the same time, by means of the differential investment of attention and psychic resources, the individual selects and organizes the information acquired from his/her context according to an emergent, autonomous criterion: the quality of experience ,58. In oOn one side we have optimal experiences. According to Csikszentmihalyi ,60, indibreakdowns: a breakdown occurres when, during our activity, an object or an environment becomes part of our consciousness. If this happens, we shift our attention from action to the object/environment to cope with it: e.g., when a wall stops our movement.On the other side we have breakdowns. Winograd and Flores  refer toWhy do we experience these breakdowns? Our hypothesis is that breakdowns are a sophisticated evolutionary tool used by the presence-as-process to control the quality of experience: the more the breakdown, the less is the level of presence-as-feeling, the less is the quality of experience, and the less is the possibility of surviving in the environment.The importance of breakdowns for understanding presence is well reflected by Slater's concept of "break in presence" (BIP) : a breaktotal response  to being in a place, and to being in a place with other people. The 'sense of being there' is just one of many signs of presence – and to use it as a definition or a starting point is a category error: somewhat like defining humor in terms of a smile" (p. 7).The answers to these questions are related to another important point of our vision: the meaning of "internal" and "external". In our vision, the boundaries are not only physical and related to our body (being there), but also social and cultural (making sense there). As underlined by Slater , PresencIf, in relatively simple organisms, this separation involves only a correct coupling between perceptions and movements, in humans it also implies the relation of the subject with a social and cultural space ,64. In f– it is also "external" to the subject what is not related to his/her activities, interests and values.– to be more "present" in the situation  defined by a symbolic system, the user has to be aware of its meaning. Only "making sense there", the user really experiences a full sense of presence. In giving sense to a situation an important role is usually played by narratives ,65.To make these concepts clearer an example may help. I'm in a restaurant for a formal dinner with my boss and some colleagues, but I don't know how to use the many different strange forks I have around my dish. In this situation I'm physically there, but the lack of knowledge puts me outside, at least partially, from the social and cultural space of the "formal dinner". The result is a limitation in my agency: I don't use the forks to avoid mistakes. This example shows clearly how both physical boundaries  and social and cultural boundaries have a strong influence on the possibility of action and the quality of experience of the subject.At this point our conclusions are:In the real world, the feeling of presence is not the same in all the situations but can be different in relation to the characteristics of the social and cultural space the subject is in. For instance, if I'm attending a lesson in university, my level of presence can be lower or higher in relation to the interest I have in the topic discussed. If the lesson is totally boring I can be "absent" : in absence attention is mostly directed towards internally-generated scenarios (in imagination) which are not currently present in the world [• he world . The rol• There are some exceptional situations in real life in which the activity of the subject is characterized by a higher level of presence. In these situations the subject experiences a full sense of control and immersion. When this experience is associated to a positive emotional state, it can create an optimal experience, usually defined "flow". An example of flow is the case where a professional athlete is playing exceptionally well (positive emotion) and achieves a state of mind where nothing else matters but the game (high level of presence).At this point we have defined what presence is and its role in the human experience. However, there is yet another open question: how can we achieve high level of presence-as-feeling? To answer this question we have to analyze the neuropsychological nature of presence-as-process.Even if presence is a unitary feeling, on the process side it can be divided in three layers/subprocesses (for a broader and more in-depth description see ). These The proto self: a coherent collection of neural patterns that map, moment by moment, the physical state of the organism;• core self: a transient entity which is continuously generated through encounters with objects• The extended self: a systematic record of the more invariant properties that the organism has discovered about itself.• The proto presence (self vs. non self), core presence , and extended presence .Each layer of presence solves a particular facet of the internal/external world separation and it is characterized by specific properties. In particular we can make conceptual distinctions between proto presence as an embodied presence related to the level of perception-action coupling (self vs. non-self). The more the organism is able to couple correctly perceptions and movements, the more it differentiates itself from the external world, thus increasing its probability of surviving. Proto presence is based on proprioception and other ways of knowing bodily orientation in the world. In a virtual world this is sometimes known as "spatial presence" and requires the tracking of body parts and appropriate updating of displays.More precisely we can define Core presence can be described as the activity of selective attention made by the self on perceptions : the more the organism is able to focus on its sensorial experience by leaving in the background the remaining neural processes, the more it is able to identify the present moment and its current tasks, increasing its probability of surviving. Core presence is based largely on vividness of perceptible displays. This is equivalent to "sensory presence" (e.g. in non-immersive VR) and requires good quality, preferably stereographic, graphics and other displays features.extended presence is to verify the significance to the self of the events experienced in the external world . The more the self is present in significant experiences, the more it will be able to reach its goals, increasing the possibility of surviving. Extended presence requires intellectually and/or emotionally significant content.Finally, the role of In humans the sense of presence-as-feeling is a direct function of these three layers: the more they are able to separate between "internal" and "external", the more is the feeling of presence, the better is the quality of action and experience.A corollary of the proposed vision is critical for our goals: it is possible to design mediated situations that elicit exceptionally high presence -69.In particular, here we argue that virtual reality is the medium able to support the highest level of presence because it can trigger at the same time all the three layers discussed before. To understand this point and in particular the difference between VR and other media, here are some examples :• In reading an engrossing book while sitting in a comfortable, safe place, extended presence will be engaged by media (engagement) but the other layers will not be involved.• In looking at a movie, you can activate a high level of core presence (vividness), a high level of extended presence (engagement) but no proto presence .• In experiencing an interesting immersive VR experience, proto , core presence (vividness) and extended presence (engagement) will be activated by the medium.• In an immersive VR experience, if you are pre-occupied with personal worries and the mediated content is not very engaging, proto  and core presence (vividness) will be invoked by the medium, but not extended presence.perceptual illusion of non-mediation [The possibility of activating all the three layers at the same time reduces the occurrence of breakdowns. As suggested by Marsh and colleagues , one of ediation , produceTo achieve an optimal experience, however, a next step is required: the highest level of presence has to be linked to a positive emotional experience.Csikszentmihalyi ,70 definStarting from this definition, different authors tried to define flow in an operational way. For Ghani and Deshpande  the two In this paper we suggest that VR is the preferred medium for the activation of the flow experience. A number of recent experimental results might be considered to foster this vision. A first support comes from the work of Hoffman and his group in the treatment of chronic pain -25. Few A second set of results comes from the work of Gaggioli ,30. GaggA final result, is the preference of phobic patients for VR vs. traditional treatments as showed by two studies from García-Palacios and colleagues ,76. In tAuthentic rehabilitation implies the active participation of the patient in the cultural context, their exposure to opportunities for action and development, their freedom to select opportunities they perceive as the most challenging and meaningful ones for the subject ,77. Follcultivation; it fosters the growth of complexity not only in the performance of flow activities but in individual behavior as a whole." (p. 28).Optimal experiences promote individual development. As underlined by Massimini and Delle Fave,  "To repltransformation of flow. Where possible, they keep cultivating former flow activities. Otherwise, as often happens, they manage to identify new and unexpected sources of concentration and involvement, sometimes in areas very different from their previous interests.This process can be activated also after a major trauma. As noted by Delle Fave , to copeThe vision behind the concept of transformation of flow is the one from "Positive Psychology" . AccordiIn this area VR may offer a critical advantage: the possibility for the patient to manage successfully in a VE a problematic situation related to his/her disturbance. Using VR in this way, the patient is more likely not only to gain an awareness of his/her need to do something to create change but also to experience a greater sense of personal efficacy . This apIn another case study, Riva  tested tempowerment is considered a multi-faceted construct reflecting the different dimensions of being psychologically enabled, and is conceived of as a positive additive function of the following three dimensions [Generally, these techniques can be used as triggers for a broader empowerment process within the flow experience induced by a high sense of presence. In psychological literature mensions :perceived control: includes beliefs about authority, decision-making skills, availability of resources, autonomy in the scheduling and performance of work, etc;– perceived competence: reflects role-mastery, which besides requiring the skillful accomplishment of one or more assigned tasks, also requires successful coping with non-routine role-related situations;– goal internalization: this dimension captures the energizing property of a worthy cause or exciting vision provided by the organizational leadership.– Virtual reality may be considered the preferred environment for the empowerment process, since it is a special, sheltered setting where patients can start to explore and act without feeling threatened. In this sense the virtual experience can be described as an "empowering environment" that rehabilitation provides to patients: nothing the patient fears can "really" happen to them in VR. With such assurance, they can freely explore, experiment, feel, live, and experience feelings and/or thoughts. VR thus becomes a most useful intermediate step between the therapist's office and the real world. Within this frame, therapists are encouraged to explore whether and how VR induced optimal experiences may facilitate recovery .The use of VR as an empowerment tool was recently tested in the support of HIV-AIDS patients . The sysIn terms of emotional impact, the participants found their experience with the system mostly encouraging, particularly the narratives relating to adjustment and coping.VR is an advanced communicative interface based on interactive 3D visualization. Its simulative capabilities allow for the precise presentation and control of dynamic multi-sensory 3D stimulus environments, as well as providing advanced methods for recording behavioral responses.presence [However VR, differently from other media, can induce a high sense of presence , usuallypresence , or the presence . Thanks This paper introduces a bio-cultural theory of presence linking the state of optimal experience, defined as "flow", to a virtual reality experience.The key ideas behind the proposed vision of presence are:– Presence has a simple but critical role in our everyday experience: the control of agency through the unconscious separation of "internal" and "external". The meaning of "internal" and "external" is not related only to the body but also to the social and cultural space (situation) in which the self is in.– The presence-as-process (the separation mechanism) produces, but is different from, the presence-as-feeling (the experienced level of presence);– The presence-as-feeling is experienced indirectly by the self through the characteristics of action and experience. In fact the self perceives directly only the variations in level of presence-as-feeling: breakdowns and optimal experiences;proto presence (self vs. non self), core presence , and extended presence .– The presence-as-process can be divided in three different layers/subprocesses. They are phylogenetically different, and strictly related to the three levels of self identified by Damasio: A corollary of the proposed vision is the possibility to design mediated situations that elicit exceptionally high presence. In particular, here we argued that virtual reality is the medium able to support the highest level of presence because it can activate at the same time all the three layers. To achieve an optimal experience (flow), however, a next step is required: the highest level of presence has to be linked to a positive emotional experience.transformation of flow. In this view, VR can be used to trigger a broad empowerment process within the flow experience induced by a high sense of presence. Linking this possibility to its simulative capabilities may transform VR in the ultimate rehabilitative device.The link between presence, flow and VR suggests the possibility of using it for a new breed of rehabilitative applications focused on a strategy defined as Applications of VR in rehabilitation include the following disturbances: memory disorders, planning and motor disabilities, executive functions and spatial knowledge disabilities. For a full review, see Morganti . HoweverEven if the significant advances in computer and graphic technology drastically improved the characteristics of a typical VE, VR is still limited by the maturity of the systems available. Today, no off-the-shelf solutions are available. So, the set up of a VR system usually requires much patience for dealing with conflicting hardware or lacking drivers. Nearly every VR system requires a dedicated staff or at least a computer technician to keep the system running smoothly.Moreover, introduction of patients and clinicians to VEs raises particular safety and ethical issues. In fact, despite developments in VR technology, some users still experience health and safety problems associated with the use of immersive headsets. Generally, for a large proportion of VR users these effects are mild and subside quickly.Further, even if the clinical rationale behind the use of VR in rehabilitation is now clear, much of this research growth has been as feasibility studies and pilot trials. Hence, there is still limited convincing evidence coming from controlled studies.Why there are so few controlled trials in VR research? The possible answers are two.First, there is a lack of standardization in VR devices and software. To date, very few of the various VR systems available are interoperable. This makes difficult their use in contexts other than those in which they were developed. Second, there is a lack of standardized protocols that can be shared by the community of researchers.Clearly, building new and additional virtual environments is important so therapists will continue to investigate applying these tools in their day-to-day clinical practice. In fact, in most circumstances, the clinical skills of the rehabilitator remain the key factor in the successful use of VR systems.Future research should explore how to develop VR environments able to provide the degree of challenge required to elicit the optimal experience. Further, research should also deepen the analysis of the link between cognitive processes, motor activities, presence and flow. This will allow a new generation of VEs in which the added value of VR is not only simulation and control.