Functional profiling methods have been extensively used in the context of high-throughput experiments and, in particular, in microarray data analysis. Such methods use available biological information to define different types of functional gene modules  whose representation in a pre-defined list of genes is further studied. In the most popular type of microarray experimental designs  or in other genomic experiments  these lists are composed by genes with a high degree of co-expression. Therefore, an implicit assumption in the application of functional profiling methods within this context is that the genes corresponding to the modules tested are effectively defining sets of co-expressing genes. Nevertheless not all the functional modules are biologically coherent entities in terms of co-expression, which will eventually hinder its detection with conventional methods of functional enrichment.Using a large collection of microarray data we have carried out a detailed survey of internal correlation in GO terms and KEGG pathways, providing a coherence index to be used for measuring functional module co-regulation. An unexpected low level of internal correlation was found among the modules studied. Only around 30% of the modules defined by GO terms and 57% of the modules defined by KEGG pathways display an internal correlation higher than the expected by chance.This information on the internal correlation of the genes within the functional modules can be used in the context of a logistic regression model in a simple way to improve their detection in gene expression experiments.For the first time, an exhaustive study on the internal co-expression of the most popular functional categories has been carried out. Interestingly, the real level of coexpression within many of them is lower than expected (or even inexistent), which will preclude its detection by means of most conventional functional profiling methods. If the gene-to-function correlation information is used in functional profiling methods, the results obtained improve the ones obtained by conventional enrichment methods. The popularisation of high-throughput technologies such as DNA microarrays has lead to a parallel demand of methods for data analysis. In particular, the necessity of providing a functional interpretation at molecular level that accounts for the macroscopic observations in high-throughput experiments has promoted the development of different methods for the functional profiling of this type of experiments during the last years ,2., etc.It is widely accepted that genes do not operate alone within the cell, but they carry out their functions through a complex interplay whose most obvious experimental evidence is the intricate network of protein interactions that we only just have started to decipher ,4. Most In an attempt to understand the functional basis of high-throughput experimental results different functional profiling methods have been proposed . DependiHowever, all these methods use functional modules as categorical variables. This fact, in the most typical microarray experimental designs  or in other types of genomic experiments , leads to the implicit assumption that such functional modules must be composed by sets of genes with a strong level of co-expression . Nevertheless this assumption might not be necessarily true for all these modules. In fact, early attempts to deduce gene functionality  from gene co-expression revealed that many functional modules did not even show a detectable degree of internal co-expression ,18. TherSurprisingly, there are no systematic studies on the extent of this lack of internal co-expression within the most commonly used functional module definitions. The aim of this study was to produce a detailed survey on the GO and KEGG functional module definitions so as to determine which ones among them can be considered coherent modules of co-expression across a wide range of representative human samples. In principle, such coherent functional modules will define the subset of GO and KEGG functional modules susceptible of being detected using common strategies for functional profiling. In order to do so, we have derived, for each functional module as defined in GO and in KEGG, a co-expression (or coherence) index which could be used to assess the strength of its internal correlation. This index has been further used to filter for functional modules with a weak internal degree of co-expression. The index was derived from a gene pairwise correlation matrix representing the overall correlation structure of the human transcriptome as estimated from microarray expression measurements of 3034 samples collected under the most diverse biological conditions. In addition, a second main aim of this work was to use this information to re-define the functional modules as non-discrete entities. Even in the case of the functional modules with a high degree of internal coherence, these cannot be considered as co-expression modules but rather as entities with a core of co-expressed genes along with a variable number of genes with lower correlation . In other words, not all the genes need to be expressed at the same time for the function to be activated. Then, for each gene annotated within a functional module, we estimate its degree of correlation with the main bulk of genes annotated under such module. In this way we provide an index which is useful for quantifying how essential each gene is in the activation of the functional module. At the same time, we introduce a framework within which functional modules can be treated as non-discrete entities. Under the prism of this new vision of gene function, we propose a simple modification of the functional profiling methodologies in order to enhance the use of biologically relevant information as described in the functional modules. Finally, we present some examples about how these modifications can enhance the detection of biologically meaningful functions which would have remained unnoticed using currently available techniques for functional enrichment.. When the median correlation between the transcripts of a functional module is compared to the empirical distribution of correlations, estimated over randomly sampled sets of genes, we are assessing how strong the departure of our estimate from the null hypothesis of module correlation is. In other words, we can test if the internal correlation of such module is significantly higher than the correlation observed in a similar number of functionally unrelated genes. The coherence index proposed is the percentile represented by the module correlation within the random distribution. This index accounts for the complement of the probability of observing, under the null hypothesis, a value as extreme as the observed median. The cut-off of 0.05 usually chosen to reject a null hypothesis when the observed p-value is lower would be represented, in this case, by the level 95 of our coherence index. We would reject the null hypothesis for a functional module when its estimated index is higher than such value.A coherence index that gives an idea of the internal correlation of the genes belonging to a functional module has been proposed and estimated for all the GO terms and KEGG pathways. This coherence index may have several interpretations but certainly the most direct one is its understanding as the complement of a p-value. We firstly calculate the all-against-all correlation matrix for all the 10866 transcripts across the 3034 arrays used , which is available as online supplementary material The application of the coherence index to the functional modules as defined by KEGG pathways showed that only 57% of them presented a correlation index greater than 95  tend to have a strong internal correlation whereas small modules show more variability  its estimated standard deviation was high  , we haveIn the following examples we show how to use this gene-to-function inter-dependence in order to incorporate the non-discrete nature of the membership of a gene to a functional module in the context of functional enrichment analysis.A study of 36 Head and Neck Squamous Cell Carcinoma (HNSCC) tumour samples, 8 of them corresponding to patients infected with Human Papillomavirus (HPV+) and the remaining 28 to non infected patients (HPV-)  was usedWe have systematically explored the GO and KEGG functional annotations of these 89 genes over-expressed in HPV+, testing for differences against the whole genome, that is, the remaining genes represented in the Affymetrix chip. A total 733 GO Biological Process terms and 161 KEGG pathways (with sizes comprised between 10 and 500 genes) were tested as described in the methods section.DNA replication initiation, p < 0.001, and DNA strand elongation, p < 0.001) were found. Also SRP-dependent cotranslational protein targeting to membrane (p < 0.001), probably accounting for the production of viral proteins is found. Finally, a term with no clear interpretation, regulation of smooth muscle contraction, was also found.A total of four GO terms were found as significantly over-represented in the group of over-expressed genes by the application of a standard, un-weighted test for functional enrichment with the permutation correction (see Table negative regulation of protein kinase activity (p < 0.001), while regulation of smooth muscle contraction disappears. It is long known the relationship between MAP kinase and growth factor activity, two terms descendant of negative regulation of protein kinase activity and HPV infection [The application of the alternative weighted analysis proposed here detects a new term: nfection   was found to be significantly over-represented in the genes over-expressed in HPV+ by the weighted test significant. It has recently been reported that Human Papillomavirus infection requires cell surface heparan sulfate . Urea cyin situ ductal carcinoma (DCIS) and invasive ductal carcinoma (IDC) [A second example on a matched-pair analysis of 24 breast tumours to study the transition between ma (IDC)  was analma (IDC)  under thWe have tested for enrichment in GO and KEGG terms in the up-regulated genes and in the down-regulated genes. A total of 733 GO terms of Biological Process and 161 KEGG pathways of sizes comprised between 10 and 500 genes where included in this study.Focal adhesion (p < 0.001) and ECM-receptor interaction (p < 0.0001), as well as two GO terms: transmembrane receptor protein tyrosine kinase signaling pathway (p < 0.001) and regulation of cell shape (p < 0.001), all of them related with the maintenance of cellular structures and cell motility, were found as differentially expressed. Again, the application of the alternative weighted analysis proposed here detects a new term: proteoglycan metabolism (p < 0.001). Proteoglycans are known to determine mitogenic responses of breast carcinoma cells to fibroblast growth factors, mediated by tyrosine kinase-signaling receptors [Using a standard, un-weighted test for functional enrichment with the permutation correction two KEGG pathways:, eceptors .Functional annotations, such as GO or KEGG pathways, have been used for the definition of modules of genes in functional enrichment methods ,9,10. ThWe have derived the correlation structure of the largest possible fraction of the human transcriptome, estimating its parameters from measurements from 3034 DNA microarrays stored in public data repositories. One of the strengths of the present study is, precisely, the big sample size  on whicThe correlation structure of the transcriptome has been used to derive a coherence score which measures the internal co-expression of 173 KEGG pathways and 2221 GO terms. Our estimations indicate that only 57% of the KEGG pathways and just 32% of the GO terms can be considered to have internal correlation stronger than random modules of functionally unrelated genes of the same size. We also provided separate estimates for each of the Gene Ontologies , showing that, in general, GO Biological Processes or Molecular Function have a weaker internal correlation than KEGG pathways or GO Cellular Component. Another interesting finding was the fact that many modules have high internal correlation but also high variability.Different reasons may account for these observations. In some cases there are functional modules defined in GO that are composed by independent or even antagonistic sub-modules and, consequently, their genes will never be found co-expressing in any experiment. Examples are transporters, which are composed by different independent types of sub-modules or any GO term starting by "regulation of", which usually has two antagonistic descendants called "positive regulation of" and "negative regulation of". In other cases, there are functional modules that require of a core of genes for properly carrying out the function and other genes of the module are only activated under particular physiological conditions, stresses, etc., displaying a lower degree of correlation. Modules composed by sub-modules can also exist, and many other situations can be imagined. In any case, the vision of a functional module as a discrete class, to which genes belong or do not belong to, is definitively not supported by the observations made. Thus, it is urgent to take a new approach that accounts for the non-discrete nature of the functional modules as defined by the most commonly used functional annotations (GO and KEGG).In addition we highlighted how the level of annotation of a GO term in the ontology structure may not be the most suitable indicator, at least in terms of co-regulation, of the described function, despite being often used as a measure of its specificity.Under the above mentioned considerations, most currently used functional profiling methods which model functional modules as groups of co-expressing genes, seem clearly inappropriate. The need of new methodologies for functional profiling and, above all, the essential requirement of a new notion of membership of a gene to a functional module is still an open issue. The proposed coherence score can be used in a first instance as a filtering criterion when the aim is to relate functionality to gene expression by discarding functional modules that will never be found as co-expressing units. Beyond this obvious use, this index can also be used to derive a weighting scheme that introduces the idea of non-discrete functional modules within the context of functional profiling methodologies in a straightforward manner. The proposed weighting scheme has the desirable property of using information on gene coexpression in the algorithm when such information is available but not introducing any bias when the information is missing. Relying on this new concept and using gene expression correlation information, we have shown with two examples how the proposed weighted approach discovers GO terms and pathways unnoticed under the equivalent standard un-weighted functional profiling method.The approach shown here is quite general and could easily be extended to any other species or different platforms just by calculating the corresponding correlation matrix in a straightforward manner. The methodology could also be easily extended to any other types of modules defined by functionality, regulatory motifs, etc. Obviously, the use of newer strategies for functional profiling such as the different versions of gene set enrichment analysis -14,16, wAlthough the weighting schema proposed is quite simple, it proves efficient in finding functional modules in a standard functional enrichment analysis framework ,10, as sThe aim of the manuscript was, on one hand to show the discrepancy between functional modules as defined in some popular repositories (GO and KEGG pathways) and real co-expressing modules and, on the other hand, to propose a new vision of such modules that combines the original definition of the function with the actual dynamics of co-expression. In this more realistic scenario, functional modules with a coherence index that makes them undistinguishable from functionally unrelated gene modules would be excluded from a functional analysis, thus increasing the power of any test in the process of adjustment for multiple testing. In the remaining functional modules to be tested, more importance will be given to the core of co-expressing genes while uncorrelated genes and negatively correlated genes  will be penalised in the analysis.Despite functional profiling of genome-scale experiments is an active field in which new proposals arise continuously ,2, the caffy library of Bioconductor [limma library of Bioconductor [All data used in this study was downloaded from the Gene Expression Omnibus (GEO), public repository of the NCBI . At the onductor  and finaonductor .The data covered an ample spectrum of biological conditions including different tissues, and diseases, male and female individuals as well as cell lines.Affymetrix probe-set identifiers were linked to their corresponding transcripts according to the Ensembl database, release 44 . Among t) which is assembled using data from public data repositories. 2221 GO terms  and 173 KEGG pathways  that had annotated at least two of the 10866 selected transcripts where used in this study. While KEGG pathways are conceptually considered as independent entities, GO terms are related among them by a hierarchical relationship . Terms closer to the root define more general concepts and terms towards the leaves define more specific terms. In the particular case of GO terms, the usual procedure is to consider that each gene annotated to a given level is automatically annotated to all its parents [GO and KEGG pathways annotation for the Affymetrix HG U133 Plus 2.0 array (the most abundant microarray in the databases) was taken from the Bioconductor meta-data package "hgu133plus2"  and to the list to which they belong to. Then some statistical test, like a chi-square, Fisher or other equivalent test, is used to find statistically significant over-representations of any functional annotation in the lists of genes compared. Here we use logistic regression models  to estimHere, we propose a very simple modification of the use of functional modules that can be applied within the context of functional enrichment analysis. The rationale for this modification is to give more importance to those genes that, being annotated in a functional module, are positively correlated to the main bulk of genes in the module. Likewise we seek to penalise the negative contribution to the detection of a functional module of those genes negatively correlated to this module. In order to achieve this, we have first to determine a measure of the internal correlation of genes within functional modules. Then, instead of using a discrete definition of functional modules, the correlations will be used to weight the membership of each gene to the module. When using the logistic model to test for each functional module, each gene was weighted depending on whether it was annotated or not within the module and whether it was positively or negatively correlated with it. Genes belonging to the functional module were given weight 2 if they were positively correlated to it and weighted by 0.5 if the correlation with the module was negative. The genes that were not in the functional module were given a neutral weight of 1. As in the classical functional enrichment test scenario, all computed p-values where corrected for multiple testing using the False Discovery Rate (FDR) method .DAG: Directed acyclic graph; FDR: False Discovery Rate; GEO: Gene Expression Omnibus; GO: Gene Ontology; KEGG: Kioto Encyclopaedia of Genes and Genomes.DM is the author of the algorithm and has participated in all the analyses, FAS and PM has participated in the analysis of the data and JD has conceived and coordinated the study and written the manuscript. All the authors have read and approved the final manuscript.Additional Figure 1. Coherence index values as a function of functional module size obtained for the three GO ontologies: Biological process (left), molecular function (center) and cellular component (right).Click here for fileAdditional Figure 2. Relationship between the coherence index and its standard deviation for KEGG (up left), GO biological process (up right), molecular function (down left) and cellular component (down right).Click here for file


Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood.Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III.Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV  but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different stages of subsequent evolution. These findings emphasize complex routes of viral evolution that become apparent through detailed phylogenomic analysis but not necessarily in reconstructions based on phyletic patterns of genes.This article was reviewed by: Patrick Forterre, George V. Shpakovski, and Igor B. Zhulin. Viruses are ubiquitous parasites of all cellular life forms. In recent years, extensive genome sequencing and comparative analysis of both viral and host genomes yielded unprecedented insights into the evolution of viruses. In particular, it has been shown that 4 diverse families of large DNA viruses of eukaryotes (NCLDV), namely, Poxviridae, Asfarviridae, Iridoviridae, and Phycodnaviridae, share a set of conserved genes with functions implicated in replication, transcription and virion morphogenesis, suggesting an origin from a single ancestral virus. This apparently monophyletic class of viruses was denoted Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) to emphasize the presence of a cytoplasmic stage in the reproduction of most if not all of these viruses . The exiOne of the most dramatic revelations of comparative genomics is that the set of universal genes (defined in terms of orthologous gene sets) in all life forms is much smaller than the set of universal molecular functions ,8. The pThe case of viral genes is especially complicated because many viral functions can be complemented and replaced by functionally analogous host proteins that may or may not be homologous to the respective viral proteins; additionally, many viruses integrate host genes into their own genomes, so the functional repertoire of viral genes evolves in an incessant, dynamic interaction with the host gene repertoire. For instance, the RNA polymerase holoenzyme is obviously essential for the expression of any DNA genome. However, many large DNA viruses including certain NCLDV (such as some of the phycodnaviruses) that replicate in the cell nucleus (as well as herpesviruses and baculoviruses) do not encode any RNA polymerase subunits and fully rely on the host transcription machinery . The DNAThe DNA ligases of the NCLDV seem to epitomize the haphazard aspect of viral genome evolution. Some of the NCLDV encode ATP-dependent ligases that, among cellular life forms, are ubiquitous and essential for replication in archaea and eukaryotes, but are only sporadically found in bacteria where they seem to contribute to distinct forms of DNA repair -16. FurtThe reconstruction of the NCLDV genome evolution tentatively placed the ATP-dependent ligase into the last common ancestor, with the implication that in some lineages the putative ancestral ligase was lost, whereas in others it was replaced with the NAD-dependent ligases . HoweverWe start with a census of DNA ligases encoded in the 45 currently available NCLDV genomes. Considering that some members of the ATP-dependent ligase family, particularly, in bacteria, show extreme sequence divergence , we searTo reconstruct the evolutionary scenario for viral ligases, we used multiple alignments of the NAD-dependent and ATP-dependent ligases that included the respective protein sequences from the NCLDV, other viruses, and representative archaea, bacteria, and eukaryotes , belonged to the eukaryotic ligase I branch  phyletic distribution of the ATP-dependent and NAD-dependent ligases among the NCLDV, ii) topologies of the phylogenetic trees for the NAD-dependent ligases Figure  and ATP-Emiliana huxlei virus and in chordopoxviruses. In particular, ligase III apparently was acquired by chordopoxviruses not only after the radiation of the entompoxviruses and chordopoxviruses  but some time into the evolution of chordopoxviruses themselves, given that the earliest branching group in this subfamily, crocodile pox virus, still has an NAD-dependent ligase  and eukaryotic ligase III possess a C-terminal BRCT  domain that mediates interactions with components of various protein complexes involved in repair and cell cycle control,28. The The phylogenomic analysis described here led to unexpected conclusions. Although ATP-dependent ligases are more common among the NCLDV than NAD-dependent ligases, it is the latter that can be traced back to the last common ancestor of the NCLDV whereas the ATP-dependent ligases apparently were acquired by several viral lineages at different stages of their evolution. The most general message brought about by these findings is that phyletic patterns alone, at least, in some cases, are insufficient for an accurate evolutionary reconstruction and can lead to substantially oversimplified or even false scenarios.More specifically, the study of the evolution of viral ligases has general implications for understanding the evolution of the NCLDV. The apparent acquisition of the NAD-dependent ligase gene at an early stage of evolution antedating the last common ancestor of the NCLDV is compatible with the scenario of the origin of eukaryotic viruses by assembly of genes from diverse sources including bacteriophages and bacteria in the course of eukaryogenesis . The appThe non-redundant protein sequence databases at the NCBI were searched using the BLASTP and PSI-BLAST programs with the expectation (E) value for sequence inclusion in PSI-BLASt iterations set at 0.005 . The seqMaximum Likelihood (ML) phylogenetic trees were constructed using the TreeFinder software , with thThe authors declare that they have no competing interests.EVK initiated and planned the study, and wrote the manuscript; NY analyzed data; the final manuscript was read and approved by both authors.The origin of viral genes is presently a hot topic of discussion. Some authors consider that all viral genes originated from cellular genes robbed by viruses whereas others emphasize that new genes can also appear in viral lineages and be transferred later on from viruses to cells. Phylogenies combining viral and cellular genes are indeed often difficult to interpret, especially the direction of transfer, and the interpretation can be strongly dependent of the prejudice of the author. This paper is an example of such case. Eugene Koonin has recognized in a previous paper the existence of specific viral genes  that, according to him, even originated before cells, in a primordial hydrothermal vent . HoweverI have a different prejudice. I think that most core genes of NCLDV are viral hallmark genes that originated directly in ancient viral lineages. Furthermore, I suspect that NCLDV proteins involved in replication might have played an important role in the origin of modern eukaryotic DNA genomes . I thus An important point, in my opinion, is to determine, if possible, which ligase (s) was (were) present in the last common ancestor of each of the three domains. This is missing in the manuscript. To my understanding, the ancestral bacterium should have contained an NAD-dependent DNA ligase. It should thus be important to present a more exhaustive tree of bacterial NAD ligase in Figure In the case of ATP-dependent ligases, it should be important to determine which ligases were present in the LECA (the Last Eukaryotic Common Ancestor), in the ancestor of Archaea, and possibly in the common ancestor of Archaea and Eukarya. The trees suggest for me that ATP-dependent ligases were in fact recruited several times independently in the three domains. It should be also important to indicate on the tree where are the hosts of respective viruses. This can sometimes helps to polarize the direction of transfer. For instance, if the host is not located at its expected position in the cellular tree, one can suspect a transfer from the virus to the host.bona fide ligase of the domain, or a ligase present in the genome of an integrated virus and/or plasmid [In these phylogenies, when the authors detect a DNA ligase gene in Bacteria or Archaea, it would be important to determine if this is the  plasmid . In otheFinally, it should be interesting to have an idea of the structural differences between the various ligase subfamilies studied here. In my opinion, cellular ligases (originating from the ancestor of a particular domain) should be very similar in terms of structure, as it is the case for instance for DNA gyrase in all Bacteria or Topo II in all Eukarya, or else for Topo IB in both Archaea and Eukarya ,38. In cI thus encourage the authors to consider the two alternative possibilities in the discussion of their results, either LGT from cells to viruses or from viruses to cells.We greatly appreciate this review that puts the rather limited and specific study described in this article into the much more general context of evolution of viruses and cells, and while doing so, offers a perspective on these general issues that is orthogonal to our own view in some important aspects while congruent in others. There is no need to discuss these concepts as this was done in several previous publications [However, a brief summary of the differences is due. The distinction between our position and that of Forterre that is of primary relevance for the conclusions of this study is the adoption of different scenarios for the origin of eukaryotes. Our position is that the first eukaryotic cells emerged as a result of engulfment of an alpha-proteobacterium, the future mitochondrion, by an archaeon. To the best of our understanding, this scenario is best compatible both with comparative-genomic results and with more general considerations stemming from the parsimony principle [This scenario, of course, has critical implications for the origin of viruses infecting eukaryotes: these viruses are thought to have evolved in a « second melting pot of viral evolution », concomitant with eukaryogenesis, through amalgamation of gene from viruses of prokaryotes, bacteria, archaea and the emerging eukaryote [Under this scenario, the possibility of the origin of any genes of the NCLDV directly from the primoridal pool of virus-like elements can be safely ruled out although some of the hallmarks could come from that pool through viruses of prokaryotes, having never been integral genomic components of cellular life forms. Acquisition of genes from the evolving NCLDV by the eukaryotic host remains a possibility but there seems to be no compelling evidence that this was a major route of eukaryote evolution.ications ,39,40. Hrinciple -44. Thisukaryote . Under tForterre propounds a different scenario of evolution that includes a primordial virus world as well but considers eukaryotes to be one of the primary lines of descent in the evolution of cells [It is further proposed that original bacterial, archaeal and eukaryotic cells might have possessed RNA genomes, whereas the DNA replication machineries were invented by viruses and independently grafted onto the 3 cell types [Other authors also have developed evolutionary scenarios under which eukaryotes represent a primordial cellular lineage, possibly, even the first type of cells to evolve [In our view, there is little evidence if any evidence in of the « primordial eukaryotes » scenarios , so we Having acknowledged this uncertainty, we would like to point out that the history of the NCLDV ligases elucidated in this study is poorly compatible with the contribution of viral genes to the host genomes that is hypothesized by Forterre. Indeed, we show that viral NAD-dependent ligases are monophyletic and by implication were probably present in the common ancestor of the extant NCLDV. However, this class of ligases is only sporadically represented in eukaryotic genomes . By contrast, ATP-dependent ligases are ubiquitous in eukaryotes but polyphyletic in the NCLDV, a finding that seems to effectively rule out the origin of eukaryotic ligases from viruses of this class. Moreover, there are two « smoking » guns of acquisition of ATP-dependent ligase genes from the hosts by distinct lineages of the NCLDV, namely, the ligase III homolog in poxviruses and the ligase I homolog in Emiliana huxlei virus.Finally, we should note that a more extensive (comprehensive) phylogenetic analysis suggested by Forterre is certainly of interest. However, in this paper, we focus specifically on the history of the NCLDV ligases; for this analysis, we used representative sets of ATP-dependent and NAD-dependent ligases from bacteria , archaea, and eukaryotes, so we do not expect that inclusion of more exhaustive sequence sets (which complicates the construction of ML trees) would affect the message.The authors have performed a detailed phylogenomic analysis and reconstructed the history of the DNA ligases in Nuclear-Cytoplasmic Large DNA Viruses of eukaryotes (NCLDV). The study reveals a quite complex evolutionary trajectory of the enzyme involved in NCLDV' genome replication: although ATP-dependent ligases are approx. 3 times more often present in different NCLDVs and previous studies (Ref. 1 & Ref. 4 in the paper) regarded this type of enzyme as the one which was present in the ancestral NCLDV genome , the last common ancestor of the NCLDV probably contained an NAD-dependent enzyme only Fig. , the preThe hypothesis that the ancestral NCLDV encoded no ligase was implicit in the original manuscript but in the revision we made it explicit. However, we also indicate that this scenario is not particularly plausible, given the monophyly of the NAD-dependent ligases of the NCLDV, because it would require multiple gene transfers between viruses infecting taxonomically distant hosts. Put another way, although the number of the events under this scenario indeed could be smaller than under the ancestral NAD-dependent ligase scenario that we favor, the nature of the inferred events also should be taken into account. As some classes of events (such as the well established gene loss) are more likely than other (such as the dubious gene transfer between viruses from distant hosts).This is a study of DNA ligases in Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV). Unlike other viruses, which exclusively use the host DNA replication machinery, many of NCLDV encode their own DNA replication machinery. This machinery proves useful when viruses inhabit the cytoplasm of eukaryotes, rather than the nucleus. NCLDV have been shown to encode two types of DNA ligases: NAD-dependent and ATP-dependent. ATP-dependent ligases were known to be more prevalent, but the details of their origins were unknown. Bacteria predominantly use NAD-dependent ligases, whereas Archaea and Eukaryotes primarily use ATP-dependent ligases. Here authors collected the NAD and ATP ligases from NCLDV genomes and examined their distribution patterns as well as their relationships to homologous enzymes in selected Bacteria, Archaea, and Eukaryotes. Phylogenetic analysis of the proteins shows that all viral NAD-dependent ligases are monophyletic, whereas viral ATP-dependent enzymes show sporadic distribution throughout the trees. This information along with distribution patterns mapped onto a viral species tree built from conserved NCLDV genes support that NAD-dependent ligases were present in the NCLDV common ancestor, but were displaced by ATP-dependent ligases on multiple independent occasions in viral evolution.This is a very clearly organized manuscript. It addresses a specific question, and the results support the conclusions. I'm not as excited about the results as are the authors, but that is because I do not know much about viruses.I have one minor question that is more of a personal curiosity than a criticism. The species tree Fig.  is a conThis issue is addressed elsewhere ,6. The dMy only issue with the paper is the methods section on sequence identification and alignment. It is too short and contains no detail whatsoever. There is nothing I can say that is not illustrated by simply copying it below:"Protein sequence databases at the NCBI were searched using the BLASTP and PSIBLAST programs . The seqWhat databases? What cutoffs and parameters? How were the poorly conserved positions identified?Providing the necessary methodology details is essential, especially for a computational paper.The details are included in the revised manuscript.Authors' response: Multiple sequence alignment of NAD-dependent ligases used for phylogenetic analysis.Click here for fileMultiple sequence alignment of ATP-dependent ligases used for phylogenetic analysis.Click here for fileA maximum-likelihood phylogenetic tree of fungal ATP-dependent DNA ligases.Click here for fileA maximum-likelihood phylogenetic tree of ATP-dependent DNA ligases including multiple vertebrate species.Click here for file


An adaptive coarse-grained (kinetic) Monte Carlo (ACGMC) simulation framework is applied to reaction and diffusion dynamics in inhomogeneous domains. The presented model is relevant to the diffusion and dimerization dynamics of epidermal growth factor receptor (EGFR) in the presence of plasma membrane heterogeneity and specifically receptor clustering. We perform simulations representing EGFR cluster dissipation in heterogeneous plasma membranes consisting of higher density clusters of receptors surrounded by low population areas using the ACGMC method. We further investigate the effect of key parameters on the cluster lifetime.Coarse-graining of dimerization, rather than of diffusion, may lead to computational error. It is shown that the ACGMC method is an effective technique to minimize error in diffusion-reaction processes and is superior to the microscopic kinetic Monte Carlo simulation in terms of computational cost while retaining accuracy. The low computational cost enables sensitivity analysis calculations. Sensitivity analysis indicates that it may be possible to retain clusters of receptors over the time scale of minutes under suitable conditions and the cluster lifetime may depend on both receptor density and cluster size.The ACGMC method is an ideal platform to resolve large length and time scales in heterogeneous biological systems well beyond the plasma membrane and the EGFR system studied here. Our results demonstrate that cluster size must be considered in conjunction with receptor density, as they synergistically affect EGFR cluster lifetime. Further, the cluster lifetime being of the order of several seconds suggests that any mechanisms responsible for EGFR aggregation must operate on shorter timescales (at most a fraction of a second), to overcome dissipation and produce stable clusters observed experimentally. The Epidermal Growth Factor (EGF) receptor (EGFR) is a well-studied member of the ErbB family of receptor tyrosine kinases (RTKs), which are involved in cell fate decisions and are implicated in numerous human cancers . Early sThe mechanisms that contribute to the localization of EGFR ,7 are coIn view of these complexities, theoretical approaches can aid in understanding the mechanisms involved in these processes and hold great potential to assist with the design of cancer related pharmaceuticals . To thisThe models just discussed do not take into account spatial inhomogeneities in the cell membrane. Yet, the presence of the membrane skeleton partitions the membrane area into compartments, between which the receptor particles hop. Leitner et al.  have devIn the present work, we simulate membrane inhomogeneities that pertain to membrane rafts or clathrin pits. We present a simple biological model that includes receptor diffusion, reversible dimer formation, and the dynamics of cluster dissipation. Membrane diffusion barriers are assumed to separate high- from low-receptor-density areas. The barrier is assumed to be higher for dimers than monomers, thereby allowing monomers to diffuse faster out of the high-density-area. The justification for this choice is that the cell would need to keep the activated dimeric repressor into the pit to stimulate endocytocis, but the monomers could be left to diffuse out of the high-density-area. Such a mechanism is not known yet, and it is difficult to experimentally isolate the effect of dimerization, since inhibitors of dimerization also affect kinase activity . On the We use the KMC method due to its ability to resolve microscopic and mesoscopic spatial  Monte Carlo (CGMC) method , which gS occupies site i of type ϕ; otherwise, it returns 0. For our simulations, a species may be a receptor monomer (M) or a dimer (D). The empty site is represented by an asterisk. For example, if i is unoccupied. Two types of sites are utilized to represent the high- and low-density regions of the cell membrane and the hops between them.A stochastic lattice model for EGFR dynamics is simulated, in which a membrane domain is represented as a grid of sites that may be occupied by a species. The state of the system is represented by the occupancy function The processes that are considered are shown in Tables i1 of type ϕ1 to site i2 of type ϕ2 will be:The stochastic rates of occurrence (propensities) of the processes just noted are given as functions of the occupancy vector i1 of type ϕ1 will be:The dimer dissociation (monomerization) at site i1 and i2 (of types type ϕ1 and ϕ2) will be:The dimer formation (association) between monomers existing at sites Once all propensities have been calculated, the random time at which the next event will occur is calculated as:a0 is the sum of all propensities and u is a uniformly distributed random number. The event m to be realized at time tcurrent + τ is randomly chosen from the list of all possible events, which consists of all microscopic processes  at each site. The higher the propensity of event m, the higher the probability that this event will be realized at time tcurrent + τ. This simulation scheme is similar to the Gillespie algorithm [where lgorithm ,31 but flgorithm ,33 to ha2) were also simulated and showed comparable results (not shown).The plasma membrane often consists of small areas of high-density receptors in large areas of low-density receptors. In order to simulate such a system, in all simulations (unless otherwise noted) a single rectangular domain of side length equal to 48 nm was placed in the simulation space. This side length is within the generally accepted membrane raft size of 10-200 nm ,35. ThisTo initialize the simulation, a high number of EGFR monomers are placed within one high-density region (or within multiple regions where applicable), the latter being surrounded by an area of low-density of receptors. Consequently, most reactions will happen at short time scales in the high-density region, whereas few will occur at the low-density region.For the simulations of this work three frameworks were used and compared: KMC, uniform mesh CGMC (hereafter abbreviated as UCGMC), and ACGMC. Exact KMC simulations fully resolve microscopic events, and can thus be extremely computationally intensive when simulating fundamental EGFR processes , for long times and in treating diffusion barriers from high to low-density regions. The need for long simulations arises from the necessity of simulating long-lived structures, such as clusters of receptors. To overcome these time scale issues, one has to resort to approximate methods in order to reduce the computational cost with minimal loss of accuracy.Uniform mesh CGMC (UCGMC) simulations have been shown to accelerate simulations of systems with a wide range of time and space scales . These mϕ for coarse-cell Ck is:We propose that the adaptive CGMC (ACGMC) method  is one pqϕ,k is the number of sites of type ϕ existing in coarse cell Ck. If qϕ,k= 1 then the microscopic events are resolved. The coarse-grained propensities are given as in the following examples. For dimer dissociation:Here For dimer formation:k = k' and ϕ1 ≠ ϕ2 , and the concentration of monomers is expected to be low (fraction of occupied sites is <0.01). Under these conditions, spatial detail in the low-density region is unimportant  and any level of coarse graining is expected to return a reasonably accurate rate of reaction.Simulations (not shown) indicate that the error depends on the diffusion and reaction time scales  as well as the density of receptors. The rate of diffusion is roughly 3 orders of magnitude faster than monomer dimerization  . Howeverested in  and is gmicro and Γbarrier are the microscopic and barrier hopping rates, respectively, for a single microscopic site jump. LCG is the coarse cell center-to-center distance of the two relevant coarse-grained cells perpendicular to the cell boundary over which diffusion occurs. Eq. (8) was applied to inhomogeneous membrane EGFR simulations to test the accuracy of UCGMC for the case of pure diffusion . At this point the number of molecules and vacancies on the fine-grid, high-density region is equal, leading to the maximum frequency of expensive microscopic diffusion events. As receptors leave the central domain and the coverage fraction of occupied high-density sites falls below 0.5, the ACGMC cost drops by an order of magnitude. This is because there are fewer receptors in the expensive fine-grid central domain, and more receptors in the coarse-grained outer domain.The previous section demonstrated that UCGMC simulations provide the same results as the KMC simulation at a much reduced cost for a diffusion only system. We expect similar CPU savings when extended to reacting systems, but the accuracy of the simulation comes into question. In the following, we investigate the performance and accuracy of the CGMC method for diffusion-reaction systems.The reaction-diffusion model for the EGFR system is shown in Table This lack of sensitivity can be explained by identifying the rate limiting mechanism for cluster dissipation in our problem. Since the diffusivities of the receptor monomers and dimers in the inner and outer domain are much higher than the hopping rates, the rate limiting mechanism is the hopping of the monomer and the dimer across the different domains. The diffusion of monomers over the barrier separating high- and low-density regions is three orders of magnitude slower compared to the microscopic monomer diffusion inside the two regions. On the other hand, dimer diffusion over a barrier is four orders of magnitude slower than the microscopic dimer diffusion. Thus, as soon as a receptor hops out of the central domain, it quickly diffuses away, without introducing any crowding effects. Further, the values of different barrier hopping rates were chosen in accordance to intuition that once the receptors dimerize, they will be held into a clathrin pit or caveola and eventually be internalized through the corresponding endocytocis pathways. Monomeric receptors are inactive and therefore need not be internalized; instead, they are expected to diffuse easily out of the high-density domain. In the simulation, all receptors are seeded inside the high-density region in monomer form. This initial condition corresponds to the worst case scenario for the receptor cluster lifetime, namely it will give the shortest lifetimes.Typical results for the diffusion-reaction model are shown in Figure In these simulations, clusters of receptors are maintained for a while by stabilizing the dimer form inside. Initially, all receptors are in the monomer form and within a short time (<0.01 s), they either leave the high-density region or dimerize. Dimers have such a reduced rate of hopping over the barrier that the dimerized receptors are essentially locked inside the high-density area. The cluster thus formed slowly dissipates via two mechanisms: i) dimers dissociate and the resulting monomers hop through the diffusional barrier before associating again; ii) dimers hop through the diffusional barrier. Counting of the jumping events shows that the contributions of both mechanisms are of the same order of magnitude; yet, the contribution of mechanism (i) is more significant under our conditions.The short-time simulations of Figure KMC is expensive to run at this timescale; as a result, KMC comparisons with the coarse-grained simulations were done up to only 20 s. These comparisons reveal that the ACGMC and UCGMC methods produce results that are in excellent agreement with those of the KMC simulation Figure , and areThe time courses of receptor density simulated with the ACGMC and UCGMC methods show that the monomer coverage reaches quasi-steady-state for <1 s, due to monomer diffusion between high-density and low-density regions. On the other hand, dimer hopping over the diffusional barrier and dimer dissociation is slow, and thus, the dimers are kinetically held in the high-density area and do not relax to a uniform density until ~30 s. Due to this effect, the ACGMC method becomes more efficient as the kinetically held clusters dissipate  around 1 s Figure . These sIn order to investigate the dominant mechanisms controlling the properties of receptor clusters, a sensitivity analysis of the diffusion-reaction model of Table HDR/ρoverall), which remains constant, vs. time. These metrics are: i) initial cluster density and ii) cluster lifetime. The initial cluster density is defined as ρHDR/ρoverall at 0.1 s. At this time, the initial monomers have either dimerized or left the high-density area . The cluster lifetime illustrates how effective the diffusional barrier is at stabilizing receptors in the dimer form.To facilitate our analysis, we used two metrics, noted in Figure a Figure . This meThese metrics were calculated for a range of values of the following variables: size of the high-density region, overall receptor density, and dimer barrier hopping propensity. The sizes (48 nm and 24 nm) were chosen within the observed 10-200 nm range of membrane rafts on living cells . The ove2) exhibit more pronounced initial clustering relative to low-density simulations. This result can be attributed to the higher receptor density increasing the number of dimerization partners adjacent to any given monomer (higher dimerization rate). Consequently, a higher proportion of receptors remain in the dimer form as the density of receptors in the central domain increases.The results of the sensitivity analysis on these metrics appear in Figure Smaller domain sizes have a higher circumference to area ratio and therefore result in a higher likelihood for a monomer to border an edge (increased monomer barrier hopping rate). The diffusion time from the center of the domain to the circumference also drops. As the above logic would suggest, smaller domains have a lower initial cluster density at the same overall densities as larger domains.Disabling dimer barrier diffusion Figure  has veryDisabling dimer diffusion extends cluster lifetimes Figure  by half It appears that for large clusters, the receptor density plays a secondary role in the cluster lifetime Figure . On the M, kD, and DM-Barrier, respectively), we defined a new nominal case with an overall receptor density of ~833 receptors/μm2, disabled dimer barrier hopping, and use a side length of 48 nm for the rectangular high-density region  has little influence, which is to be expected, since at short times the primary events taking place are dimerization reactions and barrier hopping.greater effect on cluster lifetime than proportional changes to the rate of dimerization.Moreover, all kinetic rate constants affect the cluster lifetime. Changes to the rate of monomerization and dimerization influence the lifetime more than changes to the monomer barrier diffusion rate. Increasing the dimerization rate by an order of magnitude increases the kinetic lifetime of the cluster by about half an order of magnitude and well into the range of minutes. Increasing the rate of monomerization by an order of magnitude shortens the cluster lifetime by approximately an order of magnitude. The increase in the rate of monomerization seems to have a relatively These results can be explained as follows. More time spent in monomeric form directly correlates with faster cluster dissolution. This is because two monomers are only capable of associating to form a dimer if they both reside in the high-density region long enough for a dimerization event (a bimolecular reaction) to occur. If either monomer resulting from dimer disassociation (a monomolecular reaction) leaves the high-density region, the remaining monomer will most likely leave this region as well. This causes the overall probability of dimerization of two monomers to be a function of both the rate of monomer barrier hopping and the dimerization reaction rate constant, whereas the rate of monomerization is only a function of the monomerization reaction rate constant.Given the uncertainty in kinetic and diffusion rate constants, it is quite possible that regions of high concentration of receptors could kinetically lock clusters over the period of minutes. Eventually, though, without a thermodynamic stabilization or regeneration mechanism, no long-term (beyond seconds to minutes) clustering will be observed with this model. Stabilization of these clusters would be necessary in order to effectively trigger endocytocis. The results of our simulations postulate that any stabilization or regeneration mechanism must operate on shorter timescales (possibly fraction of seconds or even shorter), to overcome dissipation and result in stable clusters seen experimentally.Adaptive Coarse-Grained kinetic Monte Carlo (ACGMC), a multiscale spatial stochastic simulation, was applied to the EGFR diffusion-reaction system. The ACGMC method properly captures the detailed spatial reactions in high receptor density regions of the membrane while efficiently and accurately simulating the low-density areas of the membrane with a low resolution grid. A sensitivity analysis of the density and longevity of these clusters was carried out.Given the uncertainty in the kinetic parameters of receptor chemistry, kinetic stabilization of EGFR clusters on the order of minutes is quite plausible. EGFR clusters would only need to exist on the order of seconds or less to successfully pass on signals, or for the formation of endosomes. Our work demonstrates that diffusion barriers provide a plausible mechanism for locking clusters for short times. Any stabilization or regeneration mechanism must operate on shorter timescales (possibly fractions of seconds or even shorter), to overcome cluster dissipation. In view of our results, methods to break, strengthen, reorganize, or otherwise manipulate membrane barriers that localize trans-membrane receptors will be valuable to develop.At relatively high receptor densities, smaller domains exhibit stronger clustering than large ones, whereas at low receptor densities clustering is weaker in smaller domains. Our results demonstrate that cluster size must be considered in conjunction with receptor density, as they synergistically affect EGFR clustering. It would thus be inappropriate to compare the behavior of cells of similar receptor densities but with different high-density region sizes or dispersions.The ACGMC method is promising for a wide variety of multiscale and spatially heterogeneous problems. For example, it could also be conceivably expanded to three dimensions and applied to a whole new class of systems. Using the EGFR system as an example, the lifetime of an endosome, including receptor clustering, budding, and cytoplasmic transport may be simulated using 'thin' coarse cells for the membrane and coarse 3D cells representing the cytoplasm. ACGMC would allow detailed spatial resolution for high-reaction locations , with coarse and computationally cheap cells for diffusion-heavy processes (cytoplasmic transport).SC developed the algorithm, set up and performed the simulations and drafted the manuscript. MS revised the manuscript, wrote the majority of the methods sections, and assessed the sensitivity of the results. DGV conceived the study, and participated in its design and coordination and helped refine the manuscript. All authors read and approved the final manuscript.Supplementary material. Simulation results showing that the conclusions of this study are not sensitive to the diffusivities of the monomer and the dimer in the central and outer domains of the interval.Click here for file


The aim of this study was to examine the feasibility of performing a cohort study on health risks from occupational exposure to radiofrequency electromagnetic fields (RF-EMF) in Germany.A set of criteria was developed to evaluate the feasibility of such a cohort study. The criteria aimed at conditions of exposure and exposure assessment , the possibility to assemble a cohort and the feasibility of ascertaining various disease endpoints.Twenty occupational settings with workers potentially exposed to RF-EMF and, in addition, a cohort of amateur radio operators were considered. Based on expert ratings, literature reviews and our set of predefined criteria, three of the cohorts were identified as promising for further evaluation: the personnel (technicians) of medium/short wave broadcasting stations, amateur radio operators, and workers on dielectric heat sealers. After further analyses, the cohort of workers on dielectric heat sealers seems not to be feasible due to the small number of exposed workers available and to the difficulty of assessing exposure , although exposure was highest in this occupational setting. The advantage of the cohort of amateur radio operators was the large number of persons it includes, while the advantage of the cohort of personnel working at broadcasting stations was the quality of retrospective exposure assessment. However, in the cohort of amateur radio operators the exposure assessment was limited, and the cohort of technicians was hampered by the small number of persons working in this profession.The majority of occupational groups exposed to RF-EMF are not practicable for setting up an occupational cohort study due to the small numbers of exposed subjects or due to exposure levels being only marginally higher than those of the general public. The widespread use of cordless and cellular phones led to a rapid increase in the number of persons exposed to radiofrequency electromagnetic fields (RF-EMF), accompanied by concerns and fears concerning possible adverse health effects of RF-EMF exposure being widely raised. In total, 48% of citizens of the Europe Union (25 member states) are very much or fairly concerned about potential health risks of electromagnetic fields . The conAlthough there are currently many studies available on the health risks of mobile phone use, the evidence is inconclusive -9. A serIt is of public health interest to get valid estimates of RF-EMF disease risks, emitted from mobile phone handsets and masts as soon as possible. An alternative would be to investigate other RF-EMF exposures with longer exposure times, for example occupational RF-EMF exposure. So far, only a few epidemiological cohort studies have investigated the effects of RF-EMF on health in occupational settings or during leisure time (amateur radio operators) -22. In tAs case-control studies generally focus on fewer numbers of subjects than cohort studies, the assessment of occupational RF-EMF exposure seems to be more feasible. Hence, the analysis of the association between RF-EMF exposure and health risks might be more appropriately done using case-control study designs. So far, two case-control studies on brain tumour and occupational exposure of RF-EMF were conducted. In one of them, a nested case-control study, the complete occupational histories were obtained from personnel records of each study subject including job title and starting and ending dates . In a seGiven the well-known problems in occupational case-control studies, the aim of the present study was to examine the feasibility of a cohort study on health risks due to radiofrequency exposure except mobile phone use. This study design was chosen due to the advantage of a cohort study: prospective assessment of exposure and several diseases as outcome.A major objective for the feasibility study was to investigate health effects in a group of persons in which a reasonable exposure assessment was feasible, and in which a potential for some high and long-lasting exposure was present.Firstly, different occupational groups and amateur radio operators were considered as potential cohorts with high and long-lasting exposure to RF-EMF. . Information about possibly exposed occupational groups was obtained from different professional associations, visits of industry sites and contacts with committees and administrative bodies. We performed a literature review including published papers and technical reports. We searched for studies on the subject "health risk by exposure to radiofrequency electromagnetic fields". The search was performed using the literature data bases PubMed and CancerLit. Different search terms were used, among others: "electromagnetic fields", "radiation", "radiation/adverse effects", "cancer" and "cohort study". In PubMed the filter "human" was set. In CancerLit, the filter "non Medline" was additionally used. Furthermore, a search with given medical subject headings (MeSH) was carried out in PubMed, i.e. "electromagnetic fields/adverse effects", "radiation non-ionizing/adverse effects". For this search the methodical filter "cohort study" was additionally used. All searches were restricted to publications in the English and the German language. Further information is given in . In addiPossible outcomes of the cohort study were identified by reviewing the literature. In a first step, we compiled a list of all diseases mentioned as outcomes in these papers. Morbidity studies as well as mortality studies were considered. In a further step it was decided that mainly mortality should be considered as an outcome, because then a retrospective part of the cohort study would be possible in Germany. Thus, mortality from cancer, cardiovascular diseases, and neurodegenerative diseases were determined as possible outcomes for the cohort study.After identifying potential occupational cohorts, we checked whether each of them fulfilled our pre-defined set of criteria.Four criteria were used:Subjects in an occupational cohort have to be exposed continuously as well as over a long period of time of ten years or more, and the exposure has to be higher than that in the general population. In addition, the duration of employment as well as information about the affiliation to an occupational group must be available for at least 90 to 95% of all members of the cohort.Prospective exposure estimates should be possible on an individual level. A retrospective estimate of exposure should be possible at least on an aggregated level by using a job-exposure-matrix.A well defined group of persons needs to be available. It must be possible to choose an unselected cohort from personnel records of the employers, public authorities or companies. Demographic variables must be available from documents of the employers, and should retrospectively be available for at least 5 or 10 years. Cohorts in large companies or public authorities are of special interest, as it would be difficult to include a large number of small factories or companies for the study. Representatives of employees and the management of the company must be willing to endorse the study. Important parameters were also the total numbers of persons employed in positions exposed to RF-EMF.A follow-up of preferably all members of the cohort is needed. As a cohort study with mortality as outcome was planned, follow-up can be easily carried out if the addresses (even former) of the cohort members are available. In this case the follow-up can be done through registration offices and public health authorities. Therefore cohorts could only be eligible if the addresses of cohort members were available.To calculate the power of a planned cohort, assumptions have to be made about the size of the cohort in terms of person years, the age distribution and the expected relative risk for exposed persons. The following assumptions were used for the power calculation: lag period = 5 years, loss to follow-up = 5%, age of study population 20 to 59 years with a uniform age distribution. Table For example, if we are interested in brain cancer, we need 10,000 subjects and a 30-year follow-up to reject a RR = 1.0 (number of cases >= 9), if the true RR is greater or equal 2.0, which is given analyzing brain cancer in females (n = 9) and males (n = 12).The numbers in table Twenty occupational settings and amateur radio operators were considered as potentially exposed to RF-EMF table . ExposurEighteen of the twenty-one situations described in table 1. Exposure to RF-EMF rare or at very low level: e.g. for captains and boat personnel and persons working in a lock, exposure to RF emitted by radio communication does not play a major role. An exposure to microwave emitted by radar is probably very low if present at all, as the position of radar equipment is on the roof of the ship and rather far away from the working position.2. Small number of exposed subjects: e.g. firms where gluing presses are used. Generally only very few persons are highly exposed in these firms. Assembling a cohort seems to be impracticable.3. Automated and shielded working processes: e. g. blister packaging, is an automated and shielded process. Maintenance of the devices only takes place when the machine is switched off (see table Comments and conclusions for exclusion are presented in table At broadcasting stations about 200 to 250 employees (technicians) are potentially exposed to radiofrequencies from the antennas . Personnel are employed only on stations with a transmitting power of ≥ 100 kW. In Germany 20 of 29 broadcasting stations operate at this power level. The employees in these workplaces  are continuously exposed and the duration of the exposure corresponds to the total working time.The exact determination of the current individual exposure can be obtained by measurements. In addition, it is possible to obtain an estimation of past exposure from computer simulations basing on current exposure. This is important because the antennas were previously operated partly with a higher transmitting power and other modulation procedures. The operating conditions (transmitting power and field-strength) of the amplitude modulated medium wave antennas can be traced back quite well over approximately the last 20 years.The strengths of this cohort would be the readiness for co-operation and support of the project, the measurable and/or valid estimation of exposure, the almost daily exposure over a long time of the working life, the relatively high constancy of the cohort and the good accessibility. Furthermore personnel data are retrospectively available for a period of at least 10 years.The limitations of the study design include the rather low levels of exposure , the small cohort size  and the fact that such a cohort consists exclusively of persons in technical occupations (highly selective group).Altogether, 80,000 amateur radio operators are registered in Germany. It is estimated that only two-thirds of them are active.Radio equipment with a transmitting power of > 10 W is notifiable. The permissible frequencies for amateur radios lie between 2 MHz and about 300 GHz. However, less than 5% of amateur radio operators transmit in the frequency range 900 – 2.200 MHz and above. This is because most amateur radio operators do not possess the necessary technical equipment to transmit within this frequency range.The average exposure time of an amateur radio operator will rarely exceed the value of 10 h/week, with large individual variation. A high exposure to RF-EMF arises as a result of the adjustment of the antenna or other work on the radio transmitters and radio traffic with an antenna which is installed in the house. Measured exposure values are not available. The whole body exposure, amongst others from antennas installed within the house, can however be comparably high.A cohort with a large size can easily be ascertained and, due to the structure of the organization of amateur radio operators, is also retrospectively available for many years. Demographic data of the members are present, also for the past. Long-term exposure is common and varies widely among the individual members. The fluctuation of the membership is small due to the high expenditure involved .Amateur radio operators are most likely not exposed to RF-EMF on a daily basis. This cohort was comprised of quite a specific study population (technicians and handicapped persons) which can not be easily compared with the general population.High frequency dielectric heat sealers are mainly used for welding of plastic products, operate with the industrial frequency of 27.12 MHz and have been used in Germany since the 1960's. At dielectric heat sealers, the workers are mainly occupied with the introduction and removal of the product to be welded. Depending upon the shielding of the electrodes, exposure to different levels of RF-EMF can occur.Measurements done at the request of the Lower Saxony Ministry of Social Affairs  and the Regional Office for Ecology (Niedersächsisches Landesamt für Ökologie) in 1996 showed that the majority of plants exceeded the licit exposure range. Discussions held with the Lower Saxony Regional Office for Ecology and the Trade Association for Precision Mechanics and Electro-Technology also confirmed that workers on dielectric heat sealers are highly exposed to RF-EMF.The availability of information about the number of exposed workers per company was scarce. Interviewed experts of different professional associations and the Lower Saxony Regional Office for Ecology stated that in general these workers are employed for a long time. They also believed that protective clothing was rarely used. The workers are employed in small and medium sized companies.The disadvantage of this cohort is the small number of exposed workers per company, as this would necessitate contacting a large number of companies. It was also unclear whether appropriate measurement of exposure could be obtained in all these firms. Additionally, several other occupational exposures  can arise. It was also not possible to obtain an estimation of the number of exposed employees due to an incomplete view of the company structures.Measurements of the Lower Saxony Social Department and of the Trade Association of the Chemical Industry showed that workers on dielectric heat sealers are exposed to considerably higher levels of RF-EMF than the general population. The fact that workers are continuously employed at the same company for long periods of time and that they work on a regular daily basis on these machines support the classification of the long exposure duration as suitable  and different operational procedures, depending on the current manufacturing process  because they could not comply with the German labour protection regulations, which are stricter than the ICNIRP's  reference exposure levels ,33. HencThe aim of our study was to identify cohorts of subjects occupationally exposed to RF-EMF and to investigate the feasibility of conducting such a cohort study in Germany. We identified three cohorts , in which the investigation of RF-EMF-associated health risks is, in principle, feasible. The conduction of a cohort study with persons exposed to RF-EMF poses a number of methodological problems that seem difficult to overcome. In the meantime, prospective cohort studies of mobile phone users became an option and have been started in some countries.A/m: ampere per meter; GHz: gigahertz; h/week: hours per week; kHz: kilohertz; kW: kilowatt; V/m: volt per meter; MeSH: medical subjects headings; MHz: megahertz; RF: radiofrequencies; RF-EMF: radiofrequency electromagnetic fields; W: watt.The authors declare that they have no competing interests.MB, GBB, BS, JW and JS conceptualised the study and developed the study protocol. JB and EM were responsible for the conduction of the study. GBB and JB wrote the initial draft of the paper, which was subsequently modified in discussions with all authors. JB is the guarantor of the work. All authors read and approved the final manuscript.Summary of results. The table summarizes the advantages and disadvantages of the three selected potential cohorts.Click here for file


A neural field model is presented that captures the essential non-linear characteristics of activity dynamics across several millimeters of visual cortex in response to local flashed and moving stimuli. We account for physiological data obtained by voltage-sensitive dye (VSD) imaging which reports mesoscopic population activity at high spatio-temporal resolution. Stimulation included a single flashed square, a single flashed bar, the line-motion paradigm – for which psychophysical studies showed that flashing a square briefly before a bar produces sensation of illusory motion within the bar – and moving squares controls. We consider a two-layer neural field (NF) model describing an excitatory and an inhibitory layer of neurons as a coupled system of non-linear integro-differential equations. Under the assumption that the aggregated activity of both layers is reflected by VSD imaging, our phenomenological model quantitatively accounts for the observed spatio-temporal activity patterns. Moreover, the model generalizes to novel similar stimuli as it matches activity evoked by moving squares of different speeds. Our results indicate that feedback from higher brain areas is not required to produce motion patterns in the case of the illusory line-motion paradigm. Physiological interpretation of the model suggests that a considerable fraction of the VSD signal may be due to inhibitory activity, supporting the notion that balanced intra-layer cortical interactions between inhibitory and excitatory populations play a major role in shaping dynamic stimulus representations in the early visual cortex. Understanding the functioning of the primary visual cortex requires characterization of the non-linear dynamics that underlie visual perception and of how the cortical architecture gives rise to these dynamics. Recent advances in real-time voltage-sensitive dye (VSD) imaging permit recording of cortical population activity with high spatial and temporal resolution. This wealth of data can be related to cortical function, dynamics, and architecture by computational modeling. Here we used a mesoscopic neural field model to describe brain dynamics at the population level as measured by VSD imaging. Introduced in 1972 by Wilson and Cowan, these models are derived from statistical mechanics to analyze the collective properties of large numbers of neurons. For simplicity, the cortical planar tissue is assumed to contain only two types of homogeneously distributed neurons (excitatory and inhibitory) that interact via recurrent lateral connections. This study shows 1) how a concise neural field model can simulate VSD data quantitatively in space and time by identifying the underlying non-linear dynamics, 2) how such a model can support hypotheses about visual information processing, and 3) how the model can be linked to the neuronal architecture. Visual cortical activity does not exclusively mirror visual input but rather reflects the contribution of additional recurrent processes involving lateral and local feedback couplings. Understanding cortical processing requires a theoretical understanding of the underlying activity dynamics, which can be attained by modeling at various levels of abstraction. Naturally, the chosen level should match the level at which neuronal recordings are made The imaging data that we model showed: i) Two stationary stimuli (a square and an elongated bar) presented in rapid succession produce a pattern that signals propagation of activity across the bar's retinotopic representation in early visual cortex. ii) The obtained pattern was different from activity when the bar was flashed alone, and did not match the simple superposition of activities evoked by individually-presented square and bar stimuli. iii) Rather, we observed propagation of a wave front of activity that was also found when a square stimulus moved physically in visual space Based on the VSD imaging data in vivo cortical activity at sub- as well as suprathreshold level with at least 10 ms temporal resolution and a spatial resolution of 50 Voltage sensitive dye imaging measures relative fluorescence changes that are linearly correlated to changes in membrane potentials ty e.g., . We expeIn the following, we first describe the underlying data and its preprocessing, our model structure, and our parameter identification procedure. Then we present the results including the model fit in comparison to further simplified models, the model prediction regarding similar yet novel stimuli, and the results of a standard linear stability analysis of the homogeneous solution of the model. We then follow with a discussion of the findings in relation to alternative modeling approaches, the physiological interpretation of our model, and the role of excitation and inhibition in the model. Finally, we consider our results in the context of hypotheses concerning the cortical representation of motion and the origin of the line-motion illusion.The data underlying this study were recorded by Jancke et al. at the Department of Neurobiology at the Weizmann Institute of Science in Israel using VSD imaging of cat visual cortex The imaged signals . The model should be at the same level of abstraction as the neuronal data. As our data reflect the dynamics of neuronal populations and describe the spread of activity across the cortical sheet, NF models are an appropriate choice The model is one-dimensional as the dynamics of interest evolve in one dimension along the (apparent) movement direction as described above. Our two-layer NF is governed by the following system of integro-differential equations:There are several reasons for the local coupling from the inhibitory to the excitatory layer. From a technical point of view, we want our model to have as few parameters as possible. Equations 1 and 2 can be viewed as the minimal extension of the original Amari neural field model The presented NF model is parameterized by 15 values. However, because all model neurons communicate only through the transfer function The VSD data reflect the activity of both excitatory and inhibitory populations of neurons. Accordingly, we define the modeled VSD signal The time delay between the stimulus presentation and the response onset in the VSD data is a sum of the time the neuronal signal needs to travel from the retina to the primary visual cortex and the time the neuronal populations in the primary visual cortex need to build up a detectable activity. A fixed retino-cortical time delay of two time frames (19.2 ms) was used in our model to align the response onsets of the model and the VSD data.In our numerical experiments, we iterated the dynamical systems defined by Eqns 1 and 2 starting from the initial conditions In order to explore the system parameter space we adopted a grid search procedure on a reduced parameter set. The reduced set contained 10 parameters After we have simulated our NF model with a given set of model parameters and obtained the spatio-temporal patterns of the excitatory and inhibitory layer in response to the stimuli used for system identification, we can compute the values We selected parameter configurations from the sets produced by the grid search procedure according to the following criteria: (1) the system with given parameters is stable ; (2) after stimulus presentation, the simulated activity eventually decays to zero; (3) the correlation coefficient between the dye and the simulated data is larger than 0.8; (4) the rate of activity change during the onset period . Our method of choice for such problems is the covariance matrix adaptation evolution strategy (CMA-ES) The imaged visual cortical area included the complete retinotopic representation of the applied stimuli. The NF parameters found by grid search are summarized in For the flashed square , activitWe next analyzed whether nonlinear interactions are required to explain our findings to explain the space-time responses of the LM condition. The superposition of the responses to the single square and the bar alone differed from the response to their combined presentation in the LM stimulus . Note thhe model  showed bIn order to verify that the different components of our model are necessary to reproduce the VSD-recorded dynamics, we tested several simplified models. We asked whether the spatio-temporal patterns could be simulated by a simple feed-forward model without lateral interactions, in which Gaussian smoothing of the input stimulus and low pass filtering produce the spread of activity  were fed into the model. The results, summarized in For a better understanding of the dynamics of our model, we performed a standard linear stability analysis in the absence of afferent input . In this context, we suggest a significant contribution of inhibitory activity to the VSD dynamics.Finally, the here presented modeling of V1 implies that feedback from higher brain areas is not necessary to produce activity patterns resembling the percept of illusory motion.First, we compare our model to other in silico simulations that addressed the same experimental findings. Rangan et al. Comparing the simulations in Rangan et al. In the large-scale model, the response following the bar arises about 20 ms earlier than the VSD-recorded responses. In contrast, the response to the square stimulus was tely see .The model responses to both the flashed bar and LM condition fitted the observed VSD measurements. In contrast, for the single flashed and the moving squares the model revealed a discrepancy to the VSD data in the extent of lateral spread. One reason for this effect is our simple Gaussian smoothing that we used as a model for the retino-cortical processing. Increasing the kernel width For the flashed square, prolonged activity was observed compared to the data. Importantly, tuning the gains ancy see .Finally, the flashed and moving square stimuli evoked model responses that were lower in amplitude than that measured. As stated in “Relation to alternative large-scale model”, using a normalization method Our model is functional in the sense that it characterizes the dynamics of the VSD signal. Still, the model must also be plausible from a physiological point of view in the sense that it can be implemented by the underlying brain structures. In the following, we therefore discuss the ability of the cortical architecture to give rise to the model dynamics.The NF model is a graded response mean-field approximation. As shown in the work of Eggert and van Hemmen The parameters The lateral connectivity is determined in the model by the Gaussian interaction kernels In contrast, inhibitory interneurons in the primary visual cortex act mostly locally We considered a mixing ratio of For example, as summarized in their review article, Ferster and Miller Different amplitudes of the dye signal correspond to different levels of the degree of depolarization across the observed neural populations. Thus, with higher amplitudes, the probability rises that the signal reflects supra-threshold activity in vivo VSD imaging (as well as in vivo intracellular recordings) must measure weak deviations from baseline levels to capture the earliest input and are therefore confronted with low signal-to-noise-ratios. These initially very low activity levels observed in the VSD experiments  Covariance matrix adaptation evolution strategy; B) Linear stability analysis of the homogeneous solution.(0.97 MB PDF)Click here for additional data file.