Calochroi is one of the most species-rich lineages in the genus Cortinarius  and is widely distributed across boreo-nemoral areas, with some extensions into meridional zones. Previous phylogenetic studies of Calochroi (incl. section Fulvi) have been geographically restricted; therefore, phylogenetic and biogeographic relationships within this lineage at a global scale have been largely unknown. In this study, we obtained DNA sequences from a nearly complete taxon sampling of known species from Europe, Central America and North America. We inferred intra- and interspecific phylogenetic relationships as well as major morphological evolutionary trends within section Calochroi based on 576 ITS sequences, 230 ITS + 5.8S + D1/D2 sequences, and a combined dataset of ITS + 5.8S + D1/D2 and RPB1 sequences of a representative subsampling of 58 species.Section Cortinarius section Calochroi was consistently resolved with high branch support into at least seven major lineages: Calochroi, Caroviolacei, Dibaphi, Elegantiores, Napi, Pseudoglaucopodes and Splendentes; whereas Rufoolivacei and Sulfurini appeared polyphyletic. A close relationship between Dibaphi, Elegantiores, Napi and Splendentes was consistently supported. Combinations of specific morphological, pigmentation and molecular characters appear useful in circumscribing clades.More than 100 species were identified by integrating DNA sequences with morphological, macrochemical and ecological data. Calochroi is an exclusively northern hemispheric lineage, where species follow their host trees throughout their natural ranges within and across continents. Results of this study contribute substantially to defining European species in this group and will help to either identify or to name new species occurring across the northern hemisphere. Major groupings are in partial agreement with earlier morphology-based and molecular phylogenetic hypotheses, but some relationships were unexpected, based on external morphology. In such cases, their true affinities appear to have been obscured by the repeated appearance of similar features among distantly related species. Therefore, further taxonomic studies are needed to evaluate the consistency of species concepts and interpretations of morphological features in a more global context. Reconstruction of ancestral states yielded two major evolutionary trends within section Calochroi: (1) the development of bright pigments evolved independently multiple times, and (2) the evolution of abruptly marginate to flattened stipe bulbs represents an autapomorphy of the Calochroi clade.Our analyses demonstrate that  Calochroi is one of the most diverse lineages within the Cortinarius radiation, with estimates of between 90 and 170 species recognised so far from Europe , as wisations . Most reisations  studied a et al.  analysedr et al.  and Garnr et al.  used a mr et al. . Frøslevr et al.  used 54  America  and Noth America ,20. Furt America -25]. TheCalochroi sensu M.M. Moser using nuclear DNA sequence data from the internal transcribed spacers , the D1/D2 regions of the nucLSU rDNA and from the gene coding for domains A-C of RPB1 from a representative subsample. We use these phylogenies to address the following main objectives. First, we compared our own sequences with available ITS sequences from GenBank and UNITE databases to establish congruence and uniformity of species concepts for European calochroid species. Second, we estimated intra- and interspecific sequence divergences of closely related taxa by comparing the internal transcribed spacers  alone or including D1/D2 regions. Third, we inferred phylogenetic relationships and major morphological evolutionary trends within the section Calochroi by integrating molecular phylogenetic analyses of single and combined DNA sequence datasets with morphological, chemical and ecological features. This study provides the first insights into the geographical structure of species and populations occurring in parts of Central America, North America and Europe within a diverse and complex lineage of ectomycorrhizal fungi.Here, we present phylogenetic hypotheses including nearly all of the known species of Calochroi appeared as a well-supported monophyletic clade within the genus Cortinarius in our preliminary neighbor-joining analyses (tree not shown). These analyses also showed that C. inexpectatus from Europe, and C. rhodophyllus, C. chlorophanus, C. stephanopus and C. vaginatus from South America, which are currently classified in Calochroi and Fulvi, cluster outside of the section Calochroi as conceived here. Thus, all species assignable to section Calochroi (incl. section Fulvi) in this study are from the northern hemisphere. A total of 576 sequences including ITS and 5.8S, 230 sequences comprising ITS, 5.8S and nucLSU D1/D2 rDNA, and 56 RPB1 sequences including the conserved domains A-C were analysed in three separate datasets: dataset 1 including ITS and 5.8S sequences , C. eufulmineus (= C. quercus-ilicis), C. majusculus , C. alnobetulae (= C. moseri) and C. olivascentium (= C. xanthochlorus).Comparative intraspecific ITS-5.8S sequence analyses between our collections and those of Frøslev et al. ,16 reveaC. albobrunnoides var. violaceovelatus and C. napus; C. albobrunnoides var. violaceovelatus and C. subpurpureophyllus var. sulphureovelatus; and between different collections of C. aureocalceolatus, C. aureofulvus and Cortinarius sp. USA 7. Collections of C. aurantiorufus and C. haasii, C. elotoides and C. pseudoglaucopus, and C. flavaurora and C. pseudoglaucopus showed a 0.2% sequence divergence. There was a 0.2% sequence divergence within collections of C. dibaphus and C. lilacinovelatus, respectively. New World collections of C. arcuatorum clustered within three distinctive lineages designated as groups A-C in Figures C. arcuatorum. New World and European Elegantiores collections were distributed into three monophyletic subgroups labeled as D-F in Figures C. elegantior var. americanus JFA 11452 from Wyoming with an isolated position; group E comprises the C. elegantior collections JFA 12438 from Wyoming and JFA 11693 from Oregon with a sequence divergence of 0.3%; and group F includes only European collections with identical sequences. Sequence divergences ranged from 0.2% to 0.5% among the groups F and D, and 0.6% to 0.7% between the groups D-E and D. Sequences from the following specimens were identical: C. albobrunnoides JFA 12426, C. napus and C. subpurpureophyllus var. sulphureovelatus; C. claroflavus and C. xanthophyllus; C. elotoides (IB 19870060 Holotypus), Cortinarius sp. JFA 11618 and JFA 11619 and C. flavaurora; C. elotus IB 1999/0192 and C. pseudoglaucopus; and C. olivellus and C. humolens. Collections of C. aurora, C. corrosus  and C. rufoolivaceus differed by single nucleotide indels in the ITS1 region, the collections TUB 012714 and TUB 012715 of C. haasii differed by a single nucleotide indel in the ITS2 region. Collections of Cortinarius sp. JFA 11833, JFA 11845 and C. prasinus had identical sequences and inclusively shared a polymorphism at the same position (Y = C/T).Many collections of the same species from different, but geographically restricted, localities  shared identical sequences. Generally, sequence variation within different collections of the same species was caused by obvious polymorphic sites and/or single nucleotide indels distributed mainly across the ITS1 and ITS2 regions. There was a 0.1% sequence divergence between Calochroi, Caroviolacei, Dibaphi, Elegantiores, Splendentes and Pseudoglaucopodes. ML bootstrap support for the monophyly of Napi, Rufoolivacei and Sulfurini was below 50%; these groups appear polyphyletic in the best tree found in heuristic ML analysis .Maximum likelihood and maximum parsimony analyses of dataset 3 revealed at least seven major lineages in the section Calochroi clade. The acquisition of bright flesh pigments appears as a synapomorphy for Rufoolivacei, Pseudoglaucopodes and Sulfurini and probably evolved independently in C. citrinus and C. fulvocitrinus as well as in Splendentes and Elegantiores.Maximum parsimony reconstruction of ancestral states on the tree with the highest likelihood found for dataset 3 suggests that the bright pigmentation of the basidiomes and an abruptly marginate to flattened stipe bulb base evolved independently several times . In general, our morphological and phylogenetic concepts for European calochroid species fit with those applied by Frøslev et al. . Altst trees . TherefoCalochroi clade roughly matches the distribution of several broadleaf trees throughout boreo-nemoral areas  and Calochroi into seven major clades , whereas Rufoolivacei and Sulfurini were not supported as monophyletic entities. Major phylogenetic groups agree partially with morphology-based and earlier molecular phylogenetic studies. Some phylogenetic relationships within these clades are unexpected based on phenotypic features, which may be explained by convergent evolution of similar morphologies within phylogenetically divergent lineages in Calochroi. This study shows that combinations of features such as colour and colouration patterns, colour reactions with KOH, pigment contents, veil, and pileipellis colouration of basidiomes are useful for circumscribing clades, whereas characters related to size and shape of the basidiomes, stipe shape, basidiospore size and shape, and pileipellis structure display high levels of homoplasy and/or phenotypic plasticity. Bright pigments evolved independently multiple times in the section Calochroi, and abruptly marginate to flattened stipe bulbs represents an autapomorphy for the clade Calochroi.This study demonstrates the great potential of comparative DNA sequence analyses of multiple populations from different geographical origins for understanding patterns of genetic and morphological variation at the species/population level within a biogeographical and taxonomic context. Our phylogenetic analyses support the division of the section Calochroi, Dibaphi, Elegantiores, Napi and Pseudoglaucopodes. Main geographical distributional patterns for Calochroi species include clinal, regional and disjunct intercontinental distributions across Europe, Central and North America, where species/populations seem to follow the natural distribution of their host trees. Species in the section Calochroi show different degrees of host fidelity: some are associated exclusively either with coniferous or frondose trees; while others show relatively high host fidelity at least at the host genus level, suggesting potential co-evolutionary/co-migratory patterns.Furthermore, our study suggests the need for future phylogenetic analyses using multiple molecular markers of those lineages with poorly understood phylogenies as well as the critical re-evaluation of species concepts especially within the clades Cortinarius, subgenus Phlegmacium, as a phylogenetic framework to filter out calochroid species which do not belong to our focal group, the monophyletic section Calochroi sensu Frøslev et al. RAxML ML are from ; EUXXXXXClick here for fileDistribution of macro- and microscopical features among calochroid species. Macro- and microscopical features referring to habit, basidiome and veil coloration, macrochemical reaction with 40% KOH, pileipellis structure, hyphal coloration in 3% KOH and spore morphology were documented.Click here for fileSpecimens used in this study and their respective collection sites, host tree(s), herbarium numbers and GenBank accession numbers. Herbarium abbreviations: Arangu-Cort = Herbarium Sociedad Micológica Aranguren, Spain; IB = Herbarium Innsbruck, Austria; JFA = J. F. Ammirati, MES = Matthew Smith; MTS = Michelle Seidl, ST = Steve Trudell, BW = Ben Wood, University of Washington Herbarium (WTU), USA; KS-CO = private herbarium of Karl Soop; O = Herbarium Oslo, Norway; TUB = Herbarium Tubingense, University of Tübingen, Germany; OSC = Oregon State University; S = Herbarium Stockholm, Sweden; SCR = Sierra Research Center (Matthew Smith), University of California Berkeley (UCB), USA. aSequence from [bSequence from [cSequence from [nce from , bSequennce from , cSequennce from .Click here for file


Pseudomonas and to infer the metabolic networks of the ancestral pseudomonads. The results suggest that pathway maps that are conserved across the Pseudomonas phylogeny have a stronger neighborhood structure than those which have a variable distribution of reactions across the phylogeny, and that some Pseudomonas lineages are going through genome reduction resulting in the loss of a number of reactions from their metabolic networks.The availability of genomes of many closely related bacteria with diverse metabolic capabilities offers the possibility of tracing metabolic evolution on a phylogeny relating the genomes to understand the evolutionary processes and constraints that affect the evolution of metabolic networks. Using simple (independent loss/gain of reactions) or complex (incorporating dependencies among reactions) stochastic models of metabolic evolution, it is possible to study how metabolic networks evolve over time. Here, we describe a model that takes the reaction neighborhood into account when modeling metabolic evolution. The model also allows estimation of the strength of the neighborhood effect during the course of evolution. We present Gibbs samplers for sampling networks at the internal node of a phylogeny and for estimating the parameters of evolution over a phylogeny without exploring the whole search space by iteratively sampling from the conditional distributions of the internal networks and parameters. The samplers are used to estimate the parameters of evolution of metabolic networks of bacteria in the genus  Pseudomonas are going through genome reduction resulting in the loss of metabolic functionalities. We also show the potential of stochastic approaches to infer the networks present at ancestral levels of a given phylogeny compared to deterministic methods such as parsimony.Metabolic networks correspond to one of the most complex cellular processes. Most organisms have a common set of reactions as a part of their metabolic networks that relate to essential processes such as generation of energy and the synthesis of important biological molecules, which are required for their survival. However, a large proportion of the reactions present in different organisms are specific to the needs of individual organisms. The regions of metabolic networks corresponding to these non-essential reactions are under continuous evolution. Using different models of evolution, we can ask important biological questions about the ways in which the metabolic networks of different organisms enable them to be well-adapted to the environments in which they live, and how these metabolic adaptations have evolved. We use a stochastic approach to study the evolution of metabolic networks and show that evolutionary inferences can be made using the structure of these networks. Our results indicate that plant pathogenic  The evolution of metabolic networks is characterized by gain and loss of reactions (or enzymes) connecting two or more metabolites and can be described as a discrete space continuous time Markov process where at each step of the network evolution a reaction is either added or deleted until the desired network is obtained Here, we present an extended model called the hybrid model that combines an independent edge model, where edges are gained or lost independently, and a neighbor-dependent model of network evolution et al.et al.Pseudomonas. The Gibbs sampler can also be used to infer the ancestral networks of a given phylogeny. This is shown by inferring the metabolic networks of Pseudomonas spp. ancestors.We use the hybrid model to study the evolution of a set of metabolic networks connected over a phylogeny. Previous attempts to study the evolution of metabolic networks in a phylogenetic context include Dandekar Reference Network which contains all these hyperedges. If the hyperedges in the reference network are labeled 1 to In the neighbor-dependent for the evolution of metabolic networks The neighborhood component Although the neighbor-dependent model summarized above produces a biologically relevant behavior whereby highly connected reactions are toggled more frequently than the poorly connected counterparts, it does not allow one to determine the strength of the neighborhood structure effecting the evolution of metabolic networks. To overcome this limitation, a parameter can be introduced in the model that corresponds to the neighborhood effect during the course of metabolic network evolution.Consider two networks It can been seen from (4) that the model behaves under the independent edge model when Biological networks are connected over a phylogenetic tree which is known through sequence analysis. Calculating the likelihood over a phylogeny requires a sum, over all possible networks that may have existed at the interior nodes of the tree, of the probabilities of each scenario of events. For example, Here In general, the likelihood of a tree with more than three networks can be calculated using the recursion described by Felsenstein et al.Evaluating Equations 5 and 7 requires an algorithm to systematically and efficiently sample networks at the internal nodes of a tree and a method to calculate the pairwise likelihood of network evolution. A Metropolis-Hastings algorithm to calculate the pairwise likelihood based on sampling paths between network pairs was described by Mithani Given a set of networks related by a phylogenetic tree, the networks at the internal nodes of the tree can be sampled using a Gibbs sampler. The general idea is to sample each internal network by conditioning on its three neighbors (one parent and two children). This approach for sampling internal networks is similar to the one used by Holmes and Bruno Consider a network For each hyperedge Calculate, for each neighbor Sample the new state Example Consider the network Once the transition probability matrices have been obtained, the sample for the new network The samples for hyperedges labeled 2 to 10 can be drawn in a similar fashion to obtain the new network.The Gibbs sampler described above samples the internal networks on a phylogenetic tree for given parameter values. This can be extended to estimate the parameters Choose initial values for the parameters Generate Use Repeat et al.The samples for parameters can be drawn using a Metropolis-Hastings algorithm For a given tree Starting from root, calculate the proportion of insertion events The calculation of hyper-parameters The hybrid model for metabolic network evolution described above allows estimation of the neighborhood effect shaping the evolution of given set of networks. The proposal for the parameter Calculate the average number of neighbors present in the networks present at the leaves of the phylogeny. For example, if the network The shape parameter The proposal probability The proposal probability et al.The Metropolis-Hastings procedure described above to sample parameters requires the likelihood of the tree when moving in the parameter space. The likelihood can be calculated using Equation 5 which in turn requires calculation of the pairwise likelihood between network pairs. The pairwise likelihood can be calculated using the Metropolis-Hastings algorithm described in Mithani Let Initialize Select the hyperedge Add the top Remove the hyperedges present in Repeat steps 2–4 until Increment et al.An example is given in network   for diffPseudomonas. The results show that the hybrid model fits the metabolic data better than the neighbor-dependent model. For example, consider the metabolic networks in Pseudomonas fluorescens Pf0-1. The maximum likelihood estimates (MLEs) for the evolution of glycolysis/gluconeogenesis map Pseudomonas fluorescens Pf-5 to P. fluorescens Pf0-1 obtained using the Gibbs sampler described by Mithani et al.et al.P. fluorescens Pf0-1 used in this analysis are listed in Pseudomonas strains used in this analysiss showed similar results (data not shown).To see if the hybrid model fitted the metabolic network data better than the neighbor-dependent model, a likelihood ratio test was performed using the metabolic data for the bacteria belonging to the genus P. fluorescens Pf0-1, the parameter values listed in P. fluorescens Pf0-1 as an example which suggest a better fit for the hybrid model than the neighbor-dependent model. Similar results (data not shown) were obtained for the other genome sequenced Pseudomonas strains used in this analysis.The fit of the data was further tested by comparing the degree distributions of the nodes obtained by simulating network evolution under the neighbor-dependent and hybrid models. The MLEs for the evolution of networks obtained under the two models were used as the simulation parameters. For example, when evolving the pathway maps in To test the Gibbs sampler described in Section ‘Sampling internal nodes’, the three network phylogeny shown in We also ran the Gibbs sampler for parameter estimation for the toy networks. The sampler was run from a random starting value for 60,000 iterations with the first 10,000 iterations regarded as burn-in period. The samples were collected every ntiation .Pseudomonas. The diversity of pseudomonads, and the availability of genome-sequence data for multiple plant-associated Pseudomonas fluorescens, Pseudomonas mendocina, Pseudomonas putida, Pseudomonas stutzeri and Pseudomonas syringae strains, along with genome data for clinical isolates of Pseudomonas aeruginosa and for the insect pathogen Pseudomonas entomophila provide an excellent opportunity to use comparative genomic approaches to develop insight into the evolution of metabolic networks. The phylogeny connecting the seventeen genome-sequenced strains of Pseudomonas is shown in gltA, gapA, rpoD, gyrB) Pseudomonas strains shown in Pseudomonas species: P. fluorescens, a saprotroph that colonizes the soil environment, and P. syringae, a plant-pathogen that is found on leaf surfaces and in plant tissues. The phylogenetic relationships between these species is shown in Pseudomonas strains used in this analysis. The basic information for each network across the seventeen Pseudomonas strains is given in To study the metabolic evolution in bacteria, we used the Gibbs sampler to estimate the evolution parameters for the metabolic networks of bacteria belonging to the genus Pseudomonas strains were defined as core edges and the hyperedges corresponding to the reactions not present in any of these seventeen species were defined as prohibited edges. Three independent replicates of the sampler were run from random starting values for 60,000 iterations for P. fluorescens and P. syringae phylogenies, and 110,000 iterations for the phylogeny connecting the seventeen Pseudomonas strains with the first 10,000 iterations regarded as burn-in period in each case. The samples were collected every P. fluorescens phylogeny  and that many Pseudomonas strains are able to use histidine as sole carbon and nitrogen source Pseudomonas strains, i.e. glycolysis/gluconeogenesis map, pentose phosphate pathway map, pyruvate metabolism map and histidine metabolism map, also have higher neighbor dependence probabilities compared to the other two pathway maps, which have variable reaction distribution across the Pseudomonas phylogeny. This might suggest a relationship between the neighborhood structure and the conservation of networks.The high insertion to deletion ratio  for all P. fluorescens and P. syringae provides interesting insights into the evolution of the metabolic networks of these bacteria. For example, the insertion and deletion rates are generally higher in P. fluorescens than those in P. syringae suggesting a higher number of insertion and deletion events in P. fluorescens networks compared to P. syringae networks. This was expected since the evolutionary distance between the P. fluorescens strains is greater as compared to P. syringae strains . In addition, a very low insertion to deletion ratio , is reported to have high likelihood values of being present in all internal networks of the phylogeny.The ancestral network reconstruction using the Gibbs sampler reported high likelihood values for reactions which are present in all the networks down a lineage and low likelihood values for reactions which show variable distributions across the hway map , the reaPseudomonas common ancestor using parsimony analysis are nearly identical to predictions generated for the P. aeruginosa common ancestor, which would be expected as parsimony assumes minimum evolution. In addition, parsimony generated a conservative model of network evolution in which a minimum number of events occur, but the stochastic approach takes network information into account when predicting ancestral networks. For example, in the case of the lysine degradation map  in an approach similar to the one used by Holmes and Bruno de novo from metabolic networks A Gibbs sampler to estimate the evolution parameters was also presented. Standard distributions were used to generate proposals for parameters. The standard distributions provide satisfactory mixing of the MCMC sampler with appropriate scaling Pseudomonas using the Gibbs sampler. The likelihood values for reactions to be present at various levels of the Pseudomonas phylogeny were also calculated using the networks visited by the Gibbs sampler and the results were compared to those obtained using parsimony. The stochastic assignment of reactions in ancestral networks offers an edge over deterministic approaches like parsimony which provides the minimum number of transformations required to explain the evolution of a reaction of the tree and can, therefore, underestimate the total number of changes. In addition, using the MCMC approach based on neighbor dependence takes network structure into account, and may be particularly useful in resolving ancestral predictions at the root of phylogenies, or in situations where parsimony is unable to assign states unambiguously Click here for additional data file.Figure S21 shown in 1 but present in the sub-network are shown in gray.Sub-networks of the toy network H(0.05 MB PDF)Click here for additional data file.Figure S3Likelihood surfaces calculated by matrix exponentiation using all 1024 networks (True Likelihood) and using the networks visited by the Gibbs sampler (Estimated Likelihood) for different insertion and deletion rates for the toy networks phylogeny shown in (0.11 MB PDF)Click here for additional data file.Figure S4An example MCMC trace showing the rate parameters for the first 1,000 iterations of the Gibbs sampler for the toy networks phylogeny shown in (0.04 MB PDF)Click here for additional data file.Figure S5Pseudomonas fluorescens phylogeny shown in Example MCMC traces showing the rate parameters for the first 1,000 iterations of the Gibbs sampler initiated from different starting values. The sampler was run on the (0.22 MB PDF)Click here for additional data file.Figure S6Pseudomonas strains shown in th iteration.Number of insertion and deletion events for the alterable reactions, that is the reactions which were neither defined as core nor were defined as prohibited in the network obtained using the Gibbs sampler run under the hybrid model. The sampler was run for the six pathway maps used in this analysis over the phylogeny connecting the seventeen (0.02 MB PDF)Click here for additional data file.Figure S7Pseudomonas strains shown in th iteration. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Number of insertion and deletion events at each branch of the phylogeny connecting the seventeen (0.02 MB PDF)Click here for additional data file.Figure S8Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.04 MB PDF)Click here for additional data file.Figure S9Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.07 MB PDF)Click here for additional data file.Figure S10Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.08 MB PDF)Click here for additional data file.Figure S11Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.04 MB PDF)Click here for additional data file.Figure S12Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.03 MB PDF)Click here for additional data file.Figure S13Pseudomonas phylogney shown in Pseudomonas strains are shown in red. Strain abbreviations: pae: P. aeruginosa PAO1, pap: P. aeruginosa PA7, pau: P. aeruginosa PA14, pag: P. aeruginosa LESB58, pen: P. entomophila L48, pfl: P. fluorescens Pf-5, pfo: P. fluorescens Pf0-1, pfs: P. fluorescens SBW25, pmy: P. mendocina ymp, ppf: P. putida F1, ppg: P. putida GB-1 ppu: P. putida KT2440, ppw: P. putida W619, psa: P. stutzeri A1501, psb: P. syringae pv. syringae B728a, psp: P. syringae pv. phaseolicola 1448A, and pst: P. syringae pv. tomato DC3000.Degree distributions of nodes at the ancestral levels of the (0.08 MB PDF)Click here for additional data file.Table S1*(H2|H1) conditioned on H for the toy networks shown in et al.Pseudo-likelihood P(0.03 MB PDF)Click here for additional data file.Table S2Pseudomonas used in this study. A reversible reaction was represented by two hyperedges (one in either direction) in this study. The codes MAPxxxxx correspond to the respective KEGG pathway codes Basic information of the metabolic networks for the seventeen genome-sequenced strains of (0.04 MB PDF)Click here for additional data file.Table S3Pseudomonas strains  for the estimated parameters  using the Gibbs sampler run under the hybrid model for the evolution of metabolic networks over the phylogeny connecting different  strains . Hypered(0.03 MB PDF)Click here for additional data file.Table S4Pseudomonas strains  run under the hybrid model for different metabolic networks over the phylogeny connecting different  strains . Hypered(0.03 MB PDF)Click here for additional data file.Text S1Testing of the Gibbs sampler for parameter estimation.(0.04 MB PDF)Click here for additional data file.


Plasmodium knowlesi is a cause of symptomatic and potentially fatal infections in humans. There are no studies assessing the detailed parasitological response to treatment of knowlesi malaria infections in man and whether antimalarial resistance occurs.P. knowlesi infections. These patients were given oral chloroquine for three days, and at 24 hours oral primaquine was administered for two consecutive days, primarily as a gametocidal agent. Clinical and parasitological responses were recorded at 6-hourly intervals during the first 24 hours, daily until discharge and then weekly to day 28. Vivax malaria patients were studied as a comparator group.A prospective observational study of oral chloroquine and primaquine therapy was conducted in consecutive patients admitted to Kapit Hospital, Sarawak, Malaysian Borneo with PCR-confirmed single 50) and 90% (PCT90) parasite clearance were 3.1  hours and 10.3 (9.4-11.4) hours. These were more rapid than in a group of 23 patients with vivax malaria 6.3 (5.3-7.8) hours and 20.9 (17.6-25.9) hours; P = 0.02). It was difficult to assess the effect of primaquine on P. knowlesi parasites, due to the rapid anti-malarial properties of chloroquine and since primaquine was administered 24 hours after chloroquine. No P. knowlesi recrudescences or re-infections were detected by PCR.Of 96 knowlesi malaria patients who met the study criteria, 73 were recruited to an assessment of the acute response to treatment and 60 completed follow-up over 28 days. On admission, the mean parasite stage distributions were 49.5%, 41.5%, 4.0% and 5.6% for early trophozoites, late trophozoites, schizonts and gametocytes respectively. The median fever clearance time was 26.5 [inter-quartile range 16-34] hours. The mean times to 50% (PCTP. knowlesi.Chloroquine plus primaqine is an inexpensive and highly effective treatment for uncomplicated knowlesi malaria infections in humans and there is no evidence of drug resistance. Further studies using alternative anti-malarial drugs, including artemisinin derivatives, would be desirable to define optimal management strategies for  Plasmodium knowlesi causes symptomatic infections in humans throughout South-east Asia in areas inhabited by its natural macaque hosts (Macaca fascicularis and M. nemestrina) and mosquito vectors of the Anopheles leucosphyrus group .To explore 50 and PCT90 for late trophozoites to be shorter (PCT50 2.5 [95% CI: 2.3-2.9]) than other asexual stages, while gametocytes took longer to clear .Three hourly slides performed in nine selected patients to determine stage specific clearance times found the PCTP < 0.01). The fever and parasites cleared at a similar time after treatment in knowlesi infections, while the fever cleared sooner than parasites in the vivax group.The calculated parasitaemia at the time of fever clearance was significantly lower in the knowlesi group . This suggests a low level of knowlesi malaria transmission and, therefore, low background levels of immunity in the population. The successful response to treatment with chloroquine and primaquine also implies that these infections are chloroquine and primaquine naïve, supporting a zoonotic mode of transmission.P. knowlesi is difficult to quantify. Using primaquine would seem unnecessary since gametocytes appeared sensitive to chloroquine in the first 24 hours. P. knowlesi is not known to have a hypnozoite stage [FCTs and PCTs were similar in the knowlesi group, while fever cleared before parasitaemia in the vivax group. This suggests that knowlesi malaria has a higher pyrogenic density than vivax malaria but it may also reflect differences in immune status between the two groups. Under Malaysian Ministry of Health treatment guidelines, oral primaquine was administered to knowlesi malaria patients for two days in this study as a gametocidal drug. The effect of this on both asexual and sexual forms of te stage . If perste stage ,22,23.P. knowlesi is sensitive to most antimalarial drugs including clindamycin [P. knowlesi W1 strain, while proguanil and pyrimethamine resistance were inducible in experiments with the P. knowlesi Nuri strain [Besides chloroquine, other anti-malarial agents have been used with apparent success for knowlesi malaria. These include mefloquine , quininendamycin , naphthondamycin , sulphonndamycin , tetracyndamycin , trimethndamycin  and erytndamycin . In Rhesi strain -35. Althi strain ,36,37.P. falciparum or P. vivax co-infection had been excluded. Further studies using alternative anti-malarial drugs, including artemisinin derivatives, would be desirable to define optimal management strategies for P. knowlesi.In the 1980's, oral chloroquine was thought to be more effective than oral quinine as treatment for chloroquine sensitive falciparum malaria . ChloroqP. knowlesi, reflecting a zoonotic mode of transmission and thus an absence of prior drug pressure. Further carefully-designed studies are required to determine whether chloroquine is effective in severe knowlesi malaria.Chloroquine is an inexpensive, highly effective and well-tolerated anti-malarial drug for the treatment of naturally-acquired uncomplicated knowlesi malaria in humans. There is currently no evidence of drug-resistant FCT: fever clearance time; PCT: parasite clearance time; 95% CI: 95% confidence interval; PCR: polymerase chain reaction.The authors declare that they have no competing interests.BS and TMED designed this study and wrote the paper with CD and JC-S. CD and MZR recruited and managed the patients. CD was responsible for collection and entry of clinical and laboratory data, and together with TMED, analysed the data. SKZ and PCSD, supervised by JC-S, conducted molecular detection assays. All authors read and approved the final manuscript.


It is well known that abnormal lipid profil is related to atherosclerosis and the measurement of carotid-intima media thickness (CIMT) is one of the surrogate marker of atherosclerosis. The serum concentration of high-density lipoprotein (HDL-C) has been known to have an inverse correlation with the development of atherosclerosis. Paraoxonase-1 (PON1) is a major anti-atherosclerotic component of HDL-C. PON1 activity is related to lipid peroxidation and prospective cardiovascular risk. The aim of this study was to investigate CIMT and serum PON1 activities along with lipid parameters in H. pylori positive and negative subjects.The role of inflammation in the pathogenesis and progression of atherosclerosis has been increasingly discussed. Although the seroepidemiological studies have suggested a relationship between Helicobacter H. pylori positive subjects and thirty-one negative subjects were enrolled. H. pylori infection was diagnosed by the presence of positivity of stool H. pylori antigen test or Carbon 14 labeled urea breath test. Serum PON1 activity was measured spectrophotometrically. Traditional cardiovascular risk factors were investigated and laboratory analysis included measurement of serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein cholesterol (LDL-C). We assessed CIMT by high-resolution ultrasound of both common carotid arteries.Thirty H. pylori positive subjects were significantly thicker than those of H. pylori negative subjects. There was no significant differences in serum HDL-C, LDL-C, TC levels and TC/HDL-C ratios between two groups. Serum TG levels of H. pylori positive subjects were significantly higher than those of H. pylori negative subjects (p = 0.014). We found that PON1 activities were significantly lower in H. pylori positive subjects compared with negative subjects. No significantly correlation was observed between PON1 and CIMT values.We found that the mean and maximum values of right and overall CIMT in H. pylori positive compared to H. pylori negative subjects. PON1 activity decrease significantly in H. pylori positive subjects. However, an association between PON1 and CIMT was not found. These data indicated that H. pylori may have a role in atherosclerotic processes, however, further studies are needed to evaluate the exact mechanisms.There is an increase in CIMT values in patients with  Severclerosis . Atherosclerosis . Infecticlerosis . It has clerosis ,6.A low concentration of HDL-C is a well-known risk factor for coronary heart disease . There aExisting cardiovascular diseases (CVDs) are found to be correlated with carotid artery intima-media thickness (CIMT) measured by ultrasound and are predictive of CVD in individuals without clinically evident disease. CIMT is now widely used as a early marker for atherosclerotic disease .H. pylori. For this purpose, we measured the CIMT and we compared the serum PON-1 activity and blood lipids between H. pylori positive and negative subjects.The aim of the present study was to evaluate the association between serum PON-1 activities and CIMT values in patients with H. pylori infection in Akdeniz University Hospital, between August and November 2009. H. pylori infection was identified by the presence of positivity of H. pylori stool antigen test or Carbon 14 labeled urea breath test. We excluded patients who had acute infectious, rheumatologic and cardiovascular disease. For the study we included 30 H. pylori infected individuals . 31 healthy noninfected, , age and sex matched individuals were enrolled in this study for a control. All subjects were informed about the study and the written consent was obtained. We obtained detailed medical history about smoking habits, the presence of diabetes mellitus, hypertension, hyperlipidemia, family history of cardiovascular.disease and medication including antihypertensive and antihyperlipidemic drugs. Blood pressure was measured with manual sphygmomanometer. Body mass index  was calculated by dividing the body weight (kg) with squared height (m2). Their routine laboratory tests which contain complete blood count, serum glucose, creatinine and alanin aminotransferase (ALT) levels were recorded.This study was approved by institutional ethical committee of Akdeniz University, Faculty of Medicine. The subjects were selected among adults who visited the Department of Gastroenterology because of dyspeptic symptoms and were tested for Venous blood samples were obtained following an overnight fasting state. Serum samples were separated and stored at -80°C until the analysis. Serum total cholesterol (TC), HDL cholesterol (HDL-C) and triglyceride (TG) levels were measured with the enzymatic colorimetric method by using commercial kits on a Modular PPP auto-analyzer (Roche Diagnostics). Serum LDL-C levels were calculated by using Friedewald formula . Serum T2 and 5.5 mmol/L paraoxon . The rate of generation of p-nitrophenol was determined at 405 nm. according to MacKness B et al. [U/L.Serum PON1 activity was measured by adding serum to Tris buffer  containing 2 mmol/L CaClB et al. . The metSubjects were evaluated for carotid intima media thickness (CIMT) and plaque occurrence by using high resolution grey-scale Doppler ultrasonography. In a semi-dark room, all subject lay supine with their necks slightly hyperextended and rotated away from the imaging transducer. Both carotid arteries were scanned. CIMT was defined as the distance between the leading edge of the lumen intimal interface and the leading edge of the media adventitia interface of the far wall .t-test. Correlation analyses were performed using Pearson's correlation test. A p-value of < 0.05 was considered as significant.Data analysis was done with a statistical software package . Quantitative data were expressed as mean (± SD) or as medians. The comparisons of parameters were performed using Student's H. pylori and 31 subjects  without H. pylori infection. The demographic and clinical characteristics of study population are shown in Table We included 30 subjects  infected with H. pylori positive subjects than H. pylori negative subjects . There were no statistically significant differences in TC, HDL-C, LDL-C levels and TC/HDL-C ratios between the two groups. Serum PON1 activity was significantly lower in H. pylori positive subjects when compared with negative subjects .As shown in Table H. pylori positive and negative subjects at enrollment are shown in Table H. pylori positive patients and only 0.3% (1 of 30) of control subjects. The mean and maximum values of right CIMT were significantly increased in H. pylori positive subjects compared with negative subjects (p < 0.05). The mean and maximum values of left CIMT were tend to be higher in subjects with H. pylori but the differences were not statistically significant between two groups. We calculated mean and maximum overall CIMT by using left and right CIMT measurements. The mean and maximum values of overall CIMT were significantly higher in H. pylori positive subjects than negative subjects (p < 0.05) , with TG  and TC/HDL-C ratio . Serum HDL-C levels were significantly correlated with mean  and maximum  right CIMT, mean  and maximum  overall CIMT values while no correlation was found with mean and maximum left CIMT values (p > 0.05). We did not found any significant correlation between PON1 and CIMT values.In H. pylori negative subjects, serum PON1 activities were significantly correlated with TG , HDL-C  and TC/HDL-C ratio . We did not find any significant correlation between serum PON1 activities and CIMT values .In H. pylori infection was found in epidemiological studies [H. pylori infection in patients with CAD than in controls (77% vs 59%) [H. pylori infection and coronary heart disease; some of the underlying mechanisms still need to be discovered. It has been reported that chronic H. pylori infection results in decreased HDL-C levels, and these lipid alterations could, partially contribute to the initiation and development of coronary atherosclerosis [H. pylori positive and negative subjects. However, H. pylori positive subjects had significantly higher plasma triglyceride levels than negative subjects. Laurila et al. found significantly increased triglyceride (1.17 vs 1.00 mmol/L) and total cholesterol (6.34 vs 5.87 mmol/L) levels in 460 H. pylori positive subjects compared with 269 H. pylori negative subjects but HDL-C levels were found to be similar in both groups [A relation between atherosclerosis and chronic  studies -19. Pell vs 59%) . Althougclerosis ,22. Infeclerosis . In the h groups .H. pylori infection and PON1 has been reported in only one study [H. pylori positive patients [H. pylori positive subjects when compared with negative subjects.Major risk factors of atherosclerosis may explain only 50% of its etiology. Therefore, looking for new risk factors of atherosclerosis is necessary. HDL-C is a well known parameter inversely related to the risk for CVD. It plays a key role in the reverse cholesterol transport, protects LDL against oxidation and reduces lipoprotein associated peroxides. The antioxidant characteristics of HDL-C have been attributed to PON 1. In recent years, authors have suggested that low PON1 activity and concentration were important determinants of the presence of coronary artery disease . Severalne study . Aslan epatients . In our H. pylori infection. Some researchers have reported no relationship between H. pylori and CIMT [H. pylori positive subjects compared with negative subjects. The mean and maximum values of left CIMT tended to be higher in H. pylori infected subjects but the differences were not significant between the two groups. We determined the correlations between PON1, HDL-C and CIMT respectively. While CIMT was negatively correlated to HDL-C, it showed no correlation with PON1. As is reported by Chen et al., HDL-C is a complex particle populated by multiple proteins that play a critical role in determining the overall effects of the lipoprotein [There is considerable evidence suggesting that ultrasonic measurements of early atherosclerosis are clinically significant. In prospective studies increased IMT has been related to an increased risk of cardiovascular diseases ,29. Therand CIMT ,30,31. Hand CIMT . In our oprotein . Our resH. pylori.The current study has certain limitations. Our study group was limited. We did not perform endoscopy and determine the grade of gastric inflammation or virulence factors of H. pylori positive subjects while right and overall CIMT were significantly higher. Although we found no correlation between CIMT and PON1, these results implied that decreased PON1 activity may have an important role on vascular structural changes induced by H. pylori infection. Further studies with larger populations are needed to explore whether there is a strong relationship between PON1 activity and atherosclerosis in H. pylori infection.In conclusion; serum PON1 activities were lower in The authors declare that they have no competing interests.SHA participated in the design of study and performed the biochemical analysis. FD performed the statistical analysis. SB, IS, GS participated in the coordination of study. DK, SK, ID performed CIMT measurements. All authors read and approved the final manuscript.


Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus faecalis) by disc diffusion method. Ethanol extract of Zingiber officinale and Punica granatum showed strong antibacterial activity against Escherichia coli. Ethanol extracts of Terminalia chebula and Ocimum sanctum exhibited antibacterial activity against Klebsiella pneumoniae. Ethanol extract of Cinnamomum cassia showed maximum antibacterial activity against Pseudomonas aeruginosa while ethanol extract of Azadirachta indica and Ocimum sanctum exhibited antibacterial activity against Enterococcus faecalis. The results support the folkloric use of these plants in the treatment of urinary tract infections by the tribals of Mahakoshal region of central India.Seventeen Indian folklore medicinal plants were investigated to evaluate antibacterial activity of aqueous, ethanol and acetone extracts against 66 multidrug resistant isolates of major urinary tract pathogens ( Escherichia coli, Candida albicans, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis[Urinary tract infections (UTI) are most common form of bacterial infections, affecting people throughout their lifespan2. The pamirabilis. The incmirabilis. Despitemirabilis. These dPlant parts were collected on the basis of the information provided in the ethanobotanical survey of India and local medicine men of Mahakoshal region (MP), India. Each specimen/plant material was labeled, numbered, annoted with the date of collection, locality and their medicinal uses were recorded . Fresh pet al.[Plant material (10 g) was extracted with 100 ml distilled water by subjecting it slow heat for 6 h; filtered through muslin cloth and centrifuged at 5000×g for 15 min. The supernatant was collected and concentrated to one-fourth of the total volume. Organicet al. with somE. faecalis (7 isolates) and gram-negative E. coli (34 isolates), K. pneumoniae (15 isolates), and P. aeruginosa (10 isolates). All the 66 UTI isolates were obtained from the bacterial germplasm culture collection, Bacteriology laboratory, R. D. University, Jabalpur, India. The bacteria were grown in the nutrient broth at 37° and maintained on nutrient agar slants at 4°.The test organisms included the gram-positive In vitro antibacterial activity was examined for aqueous, ethanol and acetone extracts from 17 different traditionally used medicinal plants. Antimicrobial activities of these extracts were evaluated by Agar disc diffusion method[6 CFU/ml for inoculation of the agar plates[on method. For allar plates. A lawn ar plates was madeZ. officinale, P. granatum and acetone extract of T. chebula against E. coli with the inhibition of 14 mm zone size (T. chebula and O. sanctum were found to be active with the inhibition of 16 mm against K. pneumoniae (C. cassia showed significant activity with 18 mm of clear inhibition zone against P. aeruginosa (C. cassia such as the fruit, oil, inner bark and leafy twigs possessed good antimicrobial activity. The ethanol extract of C. asiatica and O. sanctum show broad-spectrum activity against E. faecalis with the inhibition zone of 19 mm (Z. officinale, P. granatum, T. chebula, O. sanctum, C. cassia, C. asiatica and acetone extract of T. chebula against multidrug resistant UTI pathogens. This probably explains the use of these plants by indigenous people against a number of infections since generations. The plants studied here had shown that they are potentially rich in antimicrobial compounds and have also been extensively used by the tribals. The millenarian use of these plants in folk medicine suggests that they represent an economic and safe alternative for treatment of Urinary Tract Infections.Fifty-one aqueous, ethanol and acetone extracts of the plant belonging to 17 families were tested against gram-positive and gram-negative urinary tract pathogenic bacteria. Ethanolic extract showed considerably more activity than the acetone and aqueous extract. Maximum antibacterial activity was shown by ethanol extract of one size . Similareumoniae  while etruginosa . Previouof 19 mm . Best an


Artemisia nilagirica were screened for the potential antimicrobial activity against phytopathogens and clinically important standard reference bacterial strains.The six organic solvent extracts of A. nilagirica extracts against 15 bacterial strains. The Minimum Inhibitory Concentration (MIC) of the plant extracts were tested using two fold agar dilution method at concentrations ranging from 32 to 512 μg/ml. The phytochemical screening of extracts was carried out for major phytochemical derivatives in A. nilagirica.The agar disk diffusion method was used to study the antibacterial activity of Klebsiella pneumoniae, Enterococcus faecalis and Staphylococcus aureus. The hexane extract was found to be effective against all phytopathogens with low MIC of 32 μg/ml and the methanol extract exhibited a higher inhibition activity against Escherichia coli, Yersinia enterocolitica, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa (32 μg/ml), Bacillus subtilis (64 μg/ml) and Shigella flaxneri (128 μg/ml). The phytochemical screening of extracts answered for the major derivative of alkaloids, amino acids, flavonoids, phenol, quinines, tannins and terpenoids.All the extracts showed inhibitory activity for gram-positive and gram-negative bacteria except for A. nilagirica extracts. Hence, the isolation and purification of therapeutic potential compounds from A. nilagirica could be used as an effective source against bacterial diseases in human and plants.All the extracts showed antibacterial activity against the tested strains. Of all, methanol and hexane extracts showed high inhibition against clinical and phytopathogens, respectively. The results also indicate the presence of major phytochemical derivatives in the  Artemisia is one of the diverse genera of Asteraceae family with many important medicinally valuable essential oils and secondary metabolites. Essential oils of Artemisia spp. have been widely used for a variety of medicinal purposes for many years. Artemisia nilagirica (Clarke) pamp commonly called Indian wormwood, is widely found in the hilly areas of India. A. nilagirica has been reported to exhibit insecticidal activities [A. nilagirica which showed an inhibitory activity on Phytophthora capsici, causing "foot rot" in pepper [tivities . Around n pepper .Artemisia have been characterized for their biological activities. It is considered to produce most medicinally important secondary metabolites [Artemisia spp. showed a series of antimicrobial and antioxidant activities [Artemisia spp. were detected by HPLC, GC-MS and NMR [Artemisia spp. were used as natural pesticide and also in the treatment of few human diseases [Artemisia nilagirica extracts could be more informative for the future use in controlling phytopathogens and also in clinical treatment as natural antimicrobial agents.Various species of abolites ,4. Severtivities -9. The q and NMR ,11. Few  and NMR ,13. Furtdiseases -17. The Escherichia, Enterobacter, Klebsiella, Proteus, Shigella and Staphylococcus species are implicated to cause severe infections in human, as they are found in multiple environmental habitats [Erwinia spp., Clavibacter michiganense, Pseudomonas syringae and Xanthomonas campestris were reported to be severe phytopathogens, causing damage in carrot, potato, tomato, leafy greens, onion, green pepper, squash and other cucurbits. Furthermore, these phytopathogens cause disease in any plant tissue it invades [The organisms like habitats ,19. Erwi invades .Artemisia nilagirica was investigated. The antibacterial activity was determined by disk diffusion method and minimum inhibitory concentration (MIC) test. Four plant pathogens and 11 clinically important CLSI [In the present study, the antimicrobial potency of chloroform, diethyl ether, ethanol, hexane, methanol and petroleum ether extracts of ant CLSI  referencArtemisia nilagirica were collected from Nilgiri district, Tamil Nadu, India. Plant leaves were cleaned with deionized water and dried at shade for a week. Blotted leaves were grounded and filtered using four layers of gauss cloth. The plant powder was stored in air tight container and maintained at 4°C until use.Fresh leaves of Solvent systems used for the extractions were chloroform, diethyl ether, ethanol, hexane, methanol and petroleum ether. Soxhlet and flask extraction procedures were adapted for extraction. Ten grams of the powered samples were packed in muslin cloth and used for extraction by soxhlet apparatus at a temperature below the boiling temperature of each solvent. A portion of the powdered plant samples was soaked in the conical flask containing solvent, wrapped with aluminum foil and placed in shaker for 48 hours at 120-130 rpm.After 48 hours, the extracts were filtered using Whatman filter paper No: 1. The solvent was evaporated and the residue was dissolved in sterile dimethylsulfoxide (DMSO-9:1) in 50 mg/ml concentration. The extract was filtered using 0.22 micro filter (Type GV- Millipore) and stored at 4°C for further antimicrobial activity study.Erwinia sp. (MTCC 2760), Xanthomonas campestris (MTCC 2286), Pseudomonas syringae (ATCC 7386), Clavibacter michiganense (ATCC 27822), Escherichia coli (ATCC 25922), Yersinia enterocolitica (MTCC 840), Klebsiella pneumoniae (ATCC 15380), Salmonella typhi (SSFP 4S), Enterobacter aerogenes (MTCC 111), Proteus vulgaris (MTCC 1771), Pseudomonas aeruginosa (ATCC 27853), Shigella flaxneri (MTCC 1457), Bacillus subtilis (MTCC 441), Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (MTCC 29212). The stock cultures were maintained in nutrient agar (NA) slant at 4°C and sub-cultured monthly. Working cultures were prepared by inoculating a loopful of each test microorganism in 3 ml of nutrient broth (NB) from NA slants. Broths were incubated at 37°C for 12 hours. The suspension was diluted with sterile distilled water to obtain approximately 106 CFU/ml.The 15 bacterial cultures of both gram-positive and gram-negative bacterial strains used for screening are: Disk diffusion: 5 mm of sterile disks were incorporated in 100 μl of plant extracts (5 mg/disk). The disk was completely saturated with the extract and allowed to dry. Mueller Hinton (MH) agar plates were swabbed with test bacteria and six extract disks with one of the standard positive control disks  was placed on the MH agar plate. DMSO was taken as the negative control. Plates were incubated overnight at 37°C.4 CFU/ml. The test was carried out in triplicates. The plates were incubated overnight at 37°C. After 18 - 24 hours, the MIC was determined and the percentage of growth inhibition was calculated by,The agar dilution susceptibility test was performed based on modified method of NCCLS and CLSI ,23 to deT: Test: SC: Solvent control; PC: positive control3, Pew's and Shinoda's tests, glycosides by Killer-Killanis test, saponin by frothing test, tannins by FeCl3 and lead acetate & terpenoids by Salkowski test. The test for hydrolysable tannins, phlobatannins, phenol, quinones and volatile oils were also carried out as in literature [To identify the phytochemical derivatives in the extracts, standard phytochemical screening was performed ,25. Alkaterature -28.Analysis of variance (ANOVA) was performed using GeneSpring GX - 7.3 microarray software to determine the significance of MIC values between the extracts against bacterial culture. The probability value (p) ≤ 0.05 was considered as significance in this study.A. nilagirica leaf extracts were examined against 11 clinical and four phytopathogens causing illness in human and damage in major crops [The antibacterial activity of or crops , respectErwinia sp. and X. campestris. On the other hand, ethanol and diethyl ether extracts showed high activity against C. michiganense and P. syringae (p ≤ 0.05). Also, the petroleum ether extracts showed 11-12 mm zone of inhibition to C. michiganense. Interestingly, hexane extract of A. nilagirica exhibits maximum inhibitory activity against all the phytopathogens in comparison to other extracts [Table Clavibacter michiganense (13 mm), Erwinia sp (13 mm), Pseudomonas syringae (12 mm) and Xanthomonas campestris (14 mm). It is understandable that hexane extract is more potent showing a higher degree of antimicrobial activity to phytopathogens in comparison to other extracts. Also, the results of hexane extract against X. campestris was appeared to be two-fold better than the previous study of Phyllanthus emblica, Acacia nilotica, Sapindus mukorossi and Terminalia chebula which shows 6.00 mm zone of inhibition at 50 gm/l concentration reported as the most effective for X. campestris [The analysis of methanol and chloroform extracts against phytopathogens showed a significant level of inhibition against mpestris . In addiA. nilagirica leaf extracts were carried out on clinical bacterial pathogens. The hexane, methanol and petroleum ether extracts exhibited significant high inhibitory zones against P. aeruginosa, P. vulgaris and S. typhi. The chloroform and diethyl ether extracts showed maximum area zone of inhibition (10 mm) for B. subtilis, compared to other extracts (8 mm). The ethanol extract exhibited 14 mm zone for E. coli, which is the maximum with respect to the positive standard streptomycin. Subsequently, antibacterial activity of methanol (12 mm), chloroform (13 mm) and diethyl ether (14 mm) extracts were found as effective for Y. enterocolitica. Moderate activities were observed against S. flexneri and E. aerogenes for chloroform and methanol extracts. Among the 11 clinical bacterial strains, Escherichia coli, Yersinia enterocolitica, Bacillus subtilis, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa and Shigella flexneri were the most susceptible bacteria to all solvent extracts. Nearly, 40% growth inhibitions were observed in concentration of 5 mg/ml. Surprisingly, no activity were observed against S. aureus, E. faecalis and K. pneumoniae suggesting their resistance to A. nilagirica extracts.Similar analysis of A. nilagirica organic solvent extracts against 15 bacterial species were carried out using the micro dilution technique. The MIC values of 6 extracts ranged from 32 to 512 μg/ml [Table Erwinia sp. and X. campestris. Diethyl ether extracts showed 32 μg/ml MIC for P. syringae and C.michiganense. In general, all extracts showed less than 128 μg/ml MIC for the tested phytopathogens. Interestingly, hexane extract had higher activity in all the phytopathogens even at a low concentration of 32 μg/ml. The MIC analysis of clinical pathogens showed that the methanol extract was highly active in comparison to other extracts, which inhibited the series of study organisms at a low concentration (32 μg/ml) except B. subtilis (64 μg/ml) and Shigella flexneri (128 μg/ml) [Table A. nilagirica extracts had very low or no activity, even in the highest concentration (512 μg/ml) MIC against S. aureus, E. faecalis and K. pneumoniae. Hence, we conclude that these organisms are resistant to A. nilagirica extracts. According to the results from zone of inhibition and MIC studies [Table The MIC tests of A. nilagirica extracts to determine the natural bioactive compound. By studying the presence of phytochemical in A. nilagirica, the medicinal value of the plant can be explained scientifically. The phytochemical screening of extracts showed the presence of major derivatives and their results were summarized [Table Phytochemical test were carried out on the ed Table . The anaEscherichia coli, Bacillus subtilis, Yersinia enterocolitica, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris and Pseudomonas aeruginosa. Also, the phytochemical screening of menthol extract showed the presence of most of the derivatives like flavonoids, terpenoids, phenol, amino acids, alkaloids and tannins. Furthermore, alkaloids [A. nilagirica against clinical pathogens.In susceptibility test, the hexane extracts showed the considerable levels of inhibition against phytopathogens. The phytopathogens test of petroleum ether extract showed low inhibition range (8 to 10 mm) in comparison to other extracts (10 to 14 mm). In conjugation with phytochemical screening of all the extracts with petroleum ether, showed the variations in abundance in alkaloids derivates. Hence, it is suggested that reduction of alkaloid abundance in petroleum ether may be the cause of decreased activity in phytopathogens. Supportive to our finding, previous studies indicate the effective role of alkaloid against phytopathogens -32. The lkaloids ,34, aminlkaloids , flavonolkaloids -38, phenlkaloids , tanninslkaloids -42, terplkaloids  of variolkaloids . Our resA. nilagirica showed the broad spectrum of antibacterial activity on the tested microorganisms. Hexane extract exhibited high inhibitory potency against phytopathogens and methanol extract showed maximum inhibition against clinical pathogens except S. aureus, E. faecalis and K. pneumoniae. The phytochemical analysis showed the presence of effective biological compounds like alkaloids, amino acids, flavonoids, phenols, tannins and terpenoids. These derivatives could be potential alternatives to the traditional chemical control of clinical pathogen and phytopathogenic bacteria. Furthermore, the development of natural antimicrobials will help to decrease the negative effects of synthetic drugs. Fractionation and characterization of these active compounds will be the future work to investigate.Extracts of The authors declare that they have no competing interests.AA has carried out the experimental part such as extraction, inoculums preparation and antimicrobial evaluation. WH supervised the work, evaluated the results and corrected the manuscript for publication. Authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/10/6/prepub


Pseudomonas putida KT2440 (P. putida KT2440) is a highly versatile saprophytic soil bacterium. It is a certified bio-safety host for transferring foreign genes. Therefore, the bacterium is used as a model organism for genetic and physiological studies and for the development of biotechnological applications. In order to provide a more systematic application of the organism, we have constructed a protein-protein interaction (PPI) network analysis system of P. putida KT2440.P. putida KT2440 which is generated from three protein-protein interaction (PPI) methods. We used PSIMAP , PEIMAP , and Domain-domain interactions using iPfam. PutidaNET contains 3,254 proteins, and 82,019 possible interactions consisting of 61,011 (PSIMAP), 4,293 (PEIMAP), and 30,043 (iPfam) interaction pairs except for self interaction. Also, we performed a case study by integrating a protein interaction network and experimental 1-DE/MS-MS analysis data P. putida. We found that 1) major functional modules are involved in various metabolic pathways and ribosomes, and 2) existing PPI sub-networks that are specific to succinate or benzoate metabolism are not in the center as predicted.PutidaNET is a comprehensive interaction database and server of P. putida KT2440 PutidaNET is freely available at http://sequenceome.kobic.kr/PutidaNET.We introduce the PutidaNET which provides predicted interaction partners and functional analyses such as physicochemical properties, KEGG pathway assignment, and Gene Ontology mapping of  P. putida KT2440 is a ubiquitous bacterium which can break down a variety of organic materials for food. Because of its versatile metabolic activities, P. putida KT2440 is thought to play a pivotal role in the recycling of organic wastes and the degrading of biogenic and xenobiotic pollutants in the environment http://ww as BIND , MINT [1 as BIND , HPRD [1 as BIND , CYGD) [ as BIND , and Bio as BIND . PSIMAP  as BIND -23. In t as BIND  with a c as BIND  domains http://string.embl.de[In order to select more reliable PPIs, we developed and used a 'combined score' between any pair of proteins which were predicted by PEIMAP, PSIMAP, and iPfam algorithms. This scoring method is also used by the STRING server g.embl.de.P. putida KT2440 proteome, we searched physicochemical properties and cross-reference databases using KEGG and GO. We used Biopython [P. putida KT2440 proteome.In order to understand the biological function of iopython  modules iopython  and Signiopython  programsPutidaNET provides cross-reference to public database information such as 1) KEGG pathways, 2) GO categories, and 3) GO-slim  through P. putida KT2440 was pre-cultured at 30°C with vigorous shaking in culture media  and then inoculated into 1 L culture media containing succinate (10 mM) or benzoate (5 mM) as a sole carbon source. The bacteria were harvested at the late exponential phase (absorbance at 600 nm. 0.7-0.8) and suspended in 20 mM Tris-HCl buffer (pH 8.0). Bacteria were disrupted by a French pressure cell  at 20,000 lb/in2, and soluble protein mixtures were prepared by centrifugation . The protein samples were fractionated by 12% SDS-PAGE. The gel lanes were divided into 42 fractions according to molecular weight, and the sliced gels were digested with trypsin . The resulting peptide extracts were pooled and lyophilized in a vacuum concentrator. Tryptic peptides were dissolved with 0.5% TFA (Trifluoroacetic acid) solution prior to further 2D-LC fractionation and used for MS/MS analysis using LTQ linear Ion Trap MS . For the database search, the P. putida protein database was downloaded from the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/. Tryptic peptides were identified using SEQUEST .In order to find significant features, we integrated PPI network and proteomic data which were produced as previously described . P. putiP. putida protein database and the reverse protein database were used to exclude any false-discovered proteins [For better accuracy of protein identification by MS/MS analysis, the proteins .We acquired the protein lists in culture media including succinate or benzoate. In order to find regulated sub-network by succinate and benzoate, we analyzed betweenness centrality (BC), the number of shortest paths going through a certain node, and degree, the number of interaction partners, using NetworkAanalyzer, a cytoscape plugin . We usedP. putida KT2440, contains 82,019 PPI partners that have been predicted. Using the PPI algorithms, we obtained 61,011 (PSIMAP), 4,293 (PEIMAP), and 30,043 (iPfam) predicted PPIs except for self interaction. These PPIs were around 74.39% (PSIMAP), 5.23% (PEIMAP), and 36.62% (iPfam) of the P. putida KT2440 proteins. Although the total number of predicted interaction targets is very large, as they are ranked by combined score, experimentalists can select high ranking (more probable) ones according to their functional interests.PutidaNET, a free accessible database with 3,254 proteins for P. putida KT2440 [P. putida KT2440. PPIs were regulated specifically by difference sets of benzoate and succinate that tend to occur at the network periphery more than the network center  and rich medium (LB) also showed that the major induced protein patterns were very similar (data not shown). Specifically induced proteins in benzoate medium were β-ketoadipate pathway enzymes for benzoate and 4-hydroxybenzoate and stress proteins. On the other hand, enzymes for TCA cycle, pyruvate metabolism, and glycolysis were increased in succinate medium, which will be increased for the utilization of succinate influx.Figure a KT2440 . And we P. putida KT2440 which has a high level of metabolic diversity for biodegradation. This high level of diversity enables the bacterium to utilize a wide range of carbon sources. The ribosome is an organelle that coordinates protein synthesis in all cells. The bacterial ribosome consists of more than 50 ribosomal subunit proteins and three rRNAs. Since bacterial cells contain vast amounts of ribosomes, most ribosomal subunit proteins can be observed as main peaks by mass spectrometry.In order to find features in protein networks, we detected functional modules as highly interconnected sub-networks. As a result, we found five functional modules with KEGG pathway information Figure , Table 1Pseudomonas putida. The PutidaNET server is the first web server that provides various kinds of functional information such as a PPI viewer, physicochemical properties, biological pathways, gene ontology, and protein-protein interaction for P. putida KT2440. It can assist researchers to access and obtain the information through an automatic annotation for queried proteins. Using proteomics data from certain medium conditions, we analyzed the characteristics of P. putida KT24400 using PutidaNET. Proteomic data gave us the quantitative information of induced proteins at benzoate or succinate culture conditions, which supplements the database. PPI combined with proteomic data can give users more specific information.PutidaNET is the integration of mutually complementary protein-protein interaction information for the systematic analysis of The authors declare that they have no competing interests.SJP, BCK, and JWR constructed the database. SWC and DP developed the web site. JSC designed the overall web site. KAL analyzed the statistics of PPI network results. SJP, JSC, DP, and JWR wrote the main draft of the paper. JB and SIK directed the study and helped with the draft manuscript.BMC Bioinformatics Volume 10 Supplement 15, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Bioinformatics, available online at http://www.biomedcentral.com/1471-2105/10?issue=S15.Other papers from the meeting have been published as part of Supplementary Figure 1-(a-e) the biological modules obtained from MCODE cytoscape plug - in. This figure shows five functional modules: a) module 1, b) module 2, c) module 3, d) module 4, and e) module 5. For example, module 1 is a functional module about ribosome pathways.Click here for file


The nematode intestine is a major organ responsible for nutrient digestion and absorption; it is also involved in many other processes, such as reproduction, innate immunity, stress responses, and aging. The importance of the intestine as a target for the control of parasitic nematodes has been demonstrated. However, the lack of detailed knowledge on the molecular and cellular functions of the intestine and the level of its conservation across nematodes has impeded breakthroughs in this application.Ascaris suum and Haemonchus contortus, we generated a large collection of intestinal sequences from parasitic nematodes by identifying 3,121 A. suum and 1,755 H. contortus genes expressed in the adult intestine through the generation of expressed sequence tags. Cross-species comparisons to the intestine of the free-living C. elegans revealed substantial diversification in the adult intestinal transcriptomes among these species, suggesting lineage- or species-specific adaptations during nematode evolution. In contrast, significant conservation of the intestinal gene repertories was also evident, despite the evolutionary distance of ∼350 million years separating them. A group of 241 intestinal protein families (IntFam-241), each containing members from all three species, was identified based on sequence similarities. These conserved proteins accounted for ∼20% of the sampled intestinal transcriptomes from the three nematodes and are proposed to represent conserved core functions in the nematode intestine. Functional characterizations of the IntFam-241 suggested important roles in molecular functions such as protein kinases and proteases, and biological pathways of carbohydrate metabolism, energy metabolism, and translation. Conservation in the core protein families was further explored by extrapolating observable RNA interference phenotypes in C. elegans to their parasitic counterparts.As part of an extensive effort to investigate various transcribed genomes from Our study has provided novel insights into the nematode intestine and lays foundations for further comparative studies on biology, parasitism, and evolution within the phylum Nematoda. Haemonchus contortus in immunity and the damage to H. contortus intestine by anthelmintic fenbendazole have highlighted the versatility of the intestine as an emerging target. However, biological information regarding fundamental intestinal cell functions and mechanisms is currently limited. Conserved intestinal genes across nematode pathogens could offer molecular targets for broad parasite control. Furthermore, qualitative and quantitative comparisons on intestinal gene expression among species and lineages can identify basic adaptations relative to a critical selective force, the nutrient acquisition. This study begins to identify intestinal cell characteristics that are conserved across representatives of two clades of nematodes (V and III) and further clarifies diversities that likely reflect species- or lineage-specific adaptations. Results consistent with functional data on digestive enzymes from H. contortus and RNAi in Caenorhabditis elegans, as examples, support the potential for the comparative genomics approach to produce practical applications. This study provides a platform on which extensive investigation of intestinal genes and a more comprehensive understanding of the Nematoda can be gained.Biological properties of the nematode intestine warrant in-depth investigation, the results of which can be utilized in the control of parasitic nematodes that infect humans, livestock, and plants. Both the importance of intestinal antigens from  Caenorhabditis elegans is capable of producing oocytes with about the same total biomass as its own body per day C. elegansThe intestine is one of the major organs in nematodes, creating a key surface at the intestinal apical membrane that interacts with the environment. While specific cellular characteristics of the intestine can be diverse among nematode species, they typically conform to polarized epithelial cells with the apical membrane composed of microvilli lining the digestive tube. In apparent contrast to other surfaces of nematodes, digestive and assimilative functions, as well as various metabolic pathways and cellular trafficking, are expected to be extremely active at the intestinal surface. For example, an adult Haemonchus contortus, a hematophagous nematode of small ruminants H. contortus intestinal cells are hypersensitive to benzimidazole anthelmintics, apparently through the target protein beta-tubulin isotype 1 Meloidogyne incognitaThree lines of evidence indicate that the intestine is an important target for the control of parasitic nematodes. First, intestinal antigens enriched for apical membrane-associated proteins have been successfully used to immunize against H. contortus females identified 51 intestinal genes including cysteine proteases H. contortus and hookworms Necator americanus and Ancylostoma caninum allowed the identification of 544 intestine-expressed genes C. elegansFive major clades (I–V) are currently recognized to comprise the phylum Nematoda Ascaris suum, which presumably feeds on the semi-digested contents in the host intestine, and the clade V blood-feeding parasite H. contortus. Nearly 10,000 and 5,000 genes were identified from the two nematodes, respectively. More importantly, given the attention to the intestine, we produced the largest collection of intestinal genes in parasitic nematodes by dissecting adult intestine from each species, a procedure that is not practical for many other nematodes because of their small sizes and the lack of laboratory culturing systems. Extensive cross-species comparisons were made among the adult intestinal genes from the parasites and those expressed in the adult intestine of the free-living bacterivore C. elegans. Both diversification and conservation of intestinal gene repertories were evident among the species investigated. The diversities of intestinal transcriptomes by clade and species may reflect the substantial life style differences among these nematodes. A group of 241 protein families were found conserved in the intestine of all three nematodes, accounting for ∼20% of the intestinal gene repertoires from the three species. These genes may include core intestinal functions that are indispensable among many nematodes. Functional annotations were generated for the intestinal genes. Molecular characteristics of the intestinal genes were further explored to highlight various physiological aspects of the nematode intestine.In this study, we sampled the transcribed genomes from several tissues and developmental stages from two parasitic nematodes: the clade III nematode www.nematode.net. ESTs were processed and clustered as described before de novo predictions Dissection of the adult intestine was carefully performed under microscopy as described previously : i) Caenorhabditis spp., all amino acid sequences in the complete genomes of C. elegans (Wormbase Release v150), C. briggsae , and C. remanei , ii) Other Nematoda, all non-Caenorhabditis nematode nucleic acid sequences in GenBank excluding those from A. suum  or H. contortus (when querying H. contortus sequences) , and iii) Non-Nematoda, all amino acid sequences in the non-redundant protein database NR excluding those from nematode species . WU-BLASTP (wordmask = seg postwe B = 1000 topcomboN = 1) was used to query the translated sequences against protein databases, and WU-TBLASTN (wordmask = seg lcmask B = 1000 topcomboN = 1) for searching against nucleotide databases E-value cutoff of 1.0e−5 was used to accept sequence similarities in all BLAST searches.Databases used for sequence comparisons wereC. elegans intestinal gene was assigned two counts for the numbers of times it was sampled by SAGE tags from either the glp-4 dissected gut or the glp-4 adult whole worm, respectively. The mutants lack the gonad when raised at 25°C, therefore contamination by other tissues is less likely P-value to represent the likelihood of intestinal enrichment for each EST cluster or C. elegans gene using these two counts. The P-value cutoff of 0.001 was chosen to define the putative intestine-enriched genes from the three nematodes.Each intestinal EST cluster was assigned two counts according to the numbers of times it was sampled from either the intestinal or non-intestinal cDNA libraries, respectively. Similarly, each C. elegans genome.A hidden Markov modeling-based algorithm, Phobius A. suum EST clusters and the complete gene set of C. elegans (the modified Wormbase Release v150 containing only the longest splicing isoform at each gene loci). InParanoid-generated main orthologous pairs, which are essentially the mutual-best matches between all the available genes from the two species, were further screened against the 447,546 A. suum Genome Survey Sequences (GSSs) that were generated recently , resulting in the final group of 1,652 putative main orthologous pairs in which the C. elegans members do not have better matches in GSSs than the A. suum EST partners assigned by InParanoid. C. elegans gonad-expressed genes were extracted from SAGE data generated from dissected gonad  For tissue-level comparisons made between intestine and gonad, InParanoid C. elegans genes). Raw BLAST results were fed to a C-language implementation of Markov Cluster (MCL) Algorithm (www.micans.org/mcl), a fast and scalable unsupervised cluster algorithm based on simulation of flow in graphs C. elegans genes .An all-against-all WU-BLASTP was performed on all the 9,918 translated intestinal genes from the three species  was measured over the complete set of 9,918 translated intestinal genes using a hypergeometric test, the p-value cutoff of 1.0e−5 was chosen for enrichment. The less informative ontologies, including those at level 4 or higher for Biological Process or Molecular Function, and those at level 2 or higher for Cellular Component, were removed from the enrichment list. Also removed were redundant ontologies by keeping only the lower level more informative ontology if the same group of genes was mapped to more than one GO term.Default parameters for InterProScan v13.1 E-value cut-off of 1.0e−10 reported by WU-BLASTP against the Genes Database Release 39.0 from Kyoto Encyclopedia of Genes and Genomes (KEGG) was first used for finding homologous matches. Then the top match and all the matches within a range of 30% of the top BLAST score, if meeting the cut-off, were accepted for valid KEGG associations An empirical mixed approach was used for mapping the novel genes to canonical pathways. The A. suum are: BI781215-BI784439, BM032617-BM034650, BM280443-BM285290, BM318846-BM319958, BM515079-BM518821, BM566483-BM567588, BM568416-BM569529, BM732977-BM734435, BM964439-BM965448, BQ094886-BQ096565, BQ380669-BQ383404, BQ835081-BQ835723, BU965907-BU966430, CA849193-CA850481, CA953713-CA955182, CB100077-CB102042, DV018957-DV019894, EB186562-EB187079. The accession numbers for ESTs from H. contortus are: CA033335-CA034379, CA868595-CA870175, CA956361-CA959150, CB018493-CB022024, CB063882-CB065260, CB099467-CB100076, CB190871-CB192419, CB331948-CB333475.Nucleotide sequences data reported in this paper are available in the GenBank, EMBL and DDBJ databases. The accession numbers for ESTs from A. suum and 6 H. contortus stage- or tissue-specific cDNA libraries, and sequenced 31,416 and 14,014 5-prime ESTs from the two species, respectively. These ESTs totaled to 13.6 and 6.3 million bases for A. suum and H. contortus, accounting for 77% and 63% of the total nucleotides from the two species currently available in public databases , all available ESTs were grouped into 17,989 A. suum and 9,842 H. contortus EST contigs, each containing ESTs derived from nearly identical transcripts according to overlapping sequences to reduce sequence redundancy A. suum and 5,058 H. contortus EST clusters based on sequence similarities identified among contigs as well as in previously identified genes  from H. contortus were constructed from dissected adult intestine with methods based on either Poly-A A. suum and 7,068 H. contortus ESTs were derived from these intestinal libraries. These ESTs occurred in 3,121 A. suum and 1,755 H. contortus EST clusters, accounting for about 30% of the total genes sampled in each nematode. Since these EST clusters contained ESTs sampled from the adult intestine, they were considered to represent adult intestinal genes, making this the largest tissue-level gene discovery in parasitic nematodes thus far  from thus far .C. elegans is a bacterivore obtaining nutrients primarily or exclusively from the consumption of bacteria. Two previous studies reported identification of genes expressed in the adult C. elegans intestine: i) sequence tags generated by serial analysis of gene expression (SAGE) from the dissected adult intestine were mapped to over 4,000 C. elegans genes C. elegans, covering over 25% of all coding loci in its entire genome   . Many ofA. suum and C. elegans and those expressed in another tissue, namely the gonad. These two species have the largest numbers of sequences available, and they also represent the most distant relationship among the three nematodes investigated. The gonad was chosen because the next largest group of genes was sampled from this tissue in A. suum after the intestine. H. contortus was excluded from this analysis because a gonad-expressed gene set was not available from this nematode. Genes expressed in the intestine and gonad were divided into four putative tissue-specific groups: i) 2,453 A. suum and ii) 2,557 C. elegans genes expressed in the intestine but not in the gonad (the two intestine groups), and iii) 2,690 A. suum and iv) 2,589 C. elegans genes that were found in the gonad but not in the intestine (the two gonad groups). The use of the similar numbers of genes in each group is expected to reduce false results caused by over-representation from any single category.To evaluate common characteristics of the nematode intestine, we first sought evidence for the molecular conservation of the tissue in the context of phylogeny. We made comparisons among genes expressed in the intestine of p-value = 2.5e−04 at the bit-score cutoff of 100 in a permutation two-tailed Z-test; p-value = 4.2e−08 at the bit-score cutoff of 50; Molecular conservation was first evaluated by comparing the numbers of putative homologous pairs identified among the intestine and gonad gene groups. The number of the putative homologs between the two intestine groups was significantly larger than that between the intestine and gonad groups  To increase the confidence of analysis, we next focused on the putative orthologous pairs predicted among the intestine and gonad gene groups, which was a smaller data set than the homologous pairs used above but with higher stringency. Among the total of 1,652 putative orthologous pairs predicted from gans see , 289 were groups , wherease groups . OverallAlthough the use of the incomplete transcriptomes and a bias towards relatively abundant transcripts in EST sampling can affect results, analyses of either homologous or orthologous pairs both provide direct support for the molecular conservation of the nematode intestine. With the obvious pattern of diversification in the nematode intestine (discussed earlier), our results indicate that a subset of the intestinal gene repertoires, which likely contribute to the intestinal characteristics conserved across diverse nematode species, remain conserved during the evolution of Nematoda.Interestingly, genes expressed in the gonad appear to be less well conserved based on both analyses. However, these results do not suggest the lack of evidence for the conservation of the gonad. Instead, the two-tissue comparisons indicate that the levels of conservation are lower in the gonad than in the intestine, suggesting that the levels of molecular conservation may differ in different nematode tissues. In fact, the conserved characteristics of the gonad may become more evident with larger sample sizes and/or by comparisons with another tissue with a lower level of conservation than the intestine, when new sequence data becomes available. Similarly, differences at the levels of molecular conservation were observed in different tissues between human and mouse, which diverged only about 25 million years ago A. suum, H. contortus, and C. elegans in a single analysis, we built protein families from the complete set of 9,918 translated intestinal genes combined from the three nematodes. A total of 5,587 intestinal protein families (IntFam) were identified conservatively based on sequence similarities by MCL clustering To compare the intestinal transcriptomes of C. elegans intestinal genes than to those from A. suum (31%) or H. contortus . These data may indicate that novel intestinal genes have independently evolved in relation to the different lineages of parasitism. Complete GO mappings for the three intestinal transcriptomes are presented in the searchable AmiGO browser at www.nematode.netThe 9,918 translated intestinal genes sampled from the three nematodes were annotated and classified using Gene Ontology tus 35%; . In addiC. elegans and B. malayi. They regulate almost every aspect of cellular activities and may phosphorylate up to 30% of entire proteomes C. elegans homologs , to the reference canonical pathways in Kyoto Encyclopedia of Genes and Genomes www.nematode.netTo identify the biological pathways that are active in the nematode intestine, we mapped the 9,918 intestinal translated sequences, and for comparison, the complete C. elegans genes . Interestingly, immune system was an enriched KEGG cellular process in the A. suum intestine; this pathway barely missed the cutoff for enrichment in H. contortus , but no enrichment was indicated for the C. elegance intestine  were extracted and extrapolated to a total of 45% of these IntFams (C. elegans members (RNA interference (RNAi) has been developed and successfully applied to genome-wide gene silencing to inhibit gene functions in  IntFams . Protein members . Among tA. suum and H. contortus from various tissues or developmental stages, accounting for 77% and 63% of total available bases for the two nematodes, respectively. The identification of 9,947 A. suum and 5,058 H. contortus genes in this study will vastly facilitate the related genome sequencing projects currently underway. The research has produced the largest samplings of the adult intestinal transcriptomes thus far in parasitic nematodes by identifying 3,121 A. suum and 1,755 H. contortus intestinal genes, making possible the extensive comparative studies with the adult intestinal transcriptome of the free-living C. elegans. We found that, even with the evolutionary distance of an estimated 350 million years separating clades III and V nematodes C. elegans. Overall, this study has contributed to a better understanding of nematode biology, providing central information for the development of novel and more effective parasite control strategies. Finally, the use of the C. elegans model to dissect basic parasite biology has been slow to evolve. Results presented here identified numerous specific areas of research where C. elegans might contribute in this way.We have performed large-scale sampling of the transcribed genomes in Figure S1A. suum and H. contortus EST Clusters. The three phylogenetically specific sequence groups used to identify sequence similarities of the intestinal genes were: i) Caenorhabditis spp., amino acid sequences from the complete genomes of C. elegans, C. briggsae, and C. remanei, ii) Other Nematoda, non-Caenorhabditis nematode nucleic acid sequences excluding those from either A. suum or H. contortus, when sequences from A. suum or H. contortus were queried, respectively, and iii) Non-Nematoda, non-nematode amino acid sequences from the non-redundant protein database NR. In total, 53%  A. suum and 75%  H. contortus EST clusters contained primary sequence similarities to known genes from other species, but similar distributions of the identified matches to various species groups were observed in the two parasites.Distribution of Sequence Similarities Identified in (0.05 MB PPT)Click here for additional data file.Figure S2A. suum and C. elegans. Significant larger number of genes in the A. suum intestine group had homologous counterparts in the C. elegans intestine group than in the C. elegans gonad group at BLAST bit-score cutoff of either 50 or 100, indicating the intestinal expression of homologous genes tend to be maintained across nematodes. However, the number of homologous pairs detected between the two gonad groups was not different from that between the gonad and intestine groups.Homologous Pairs between the Intestine and Gonad Gene Groups from (0.14 MB PPT)Click here for additional data file.Table S1EST Generation and Clustering.(0.02 MB XLS)Click here for additional data file.Table S2A. suum.Identification of 150 Intestine-enriched Genes in (0.06 MB XLS)Click here for additional data file.Table S3H. contortus.Identification of 60 Intestine-enriched Genes in (0.04 MB XLS)Click here for additional data file.Table S4C. elegans..Identification of 247 Intestine-enriched Genes in (0.07 MB XLS)Click here for additional data file.Table S5A. suum and H. contortus.Classification of Putative Protein Kinases in (0.03 MB XLS)Click here for additional data file.Table S6Gene Ontology Terms Statistically Enriched in IntFam Groups Other Than IntFam-241.(0.03 MB XLS)Click here for additional data file.Table S7A. suum.Genes Mapped to KEGG Immune System in (0.03 MB XLS)Click here for additional data file.Table S8H. contortus.Genes Mapped to KEGG Immune System in (0.03 MB XLS)Click here for additional data file.Text S1Supplementary Materials.(0.04 MB DOC)Click here for additional data file.