Wise et al. introduced a rank-based statistical technique for meta-analysis of genome scans, the Genome Scan Meta-Analysis (GSMA) method. Levinson et al. recently described two generalizations of the GSMA statistic: (i) a weighted version of the GSMA statistic, so that different studies could be ascribed different weights for analysis; and (ii) an order statistic approach, reflecting the fact that a GSMA statistic can be computed for each chromosomal region or bin width across the various genome scan studies.We provide an Edgeworth approximation to the null distribution of the weighted GSMA statistic, and, we examine the limiting distribution of the GSMA statistics under the order statistic formulation, and quantify the relevance of the pairwise correlations of the GSMA statistics across different bins on this limiting distribution. We also remark on aggregate criteria and multiple testing for determining significance of GSMA results.Theoretical considerations detailed herein can lead to clarification and simplification of testing criteria for generalizations of the GSMA statistic. Wise, Lanchbury and Lewis  introducn = 120 bins spanning the entire genome, and correspondingly 120 GSMA statistics. Wise [Levinson  describecs. Wise , the joics. Wise  aggregatXij, i = 1, ..., m, j = 1, ..., n, denote the rank of any particular linkage test statistic (e.g., LOD score) in the jth chromosomal region (bin) from the ith study, with each study being ranked separately. Levinson [n = "worst" on the basis of, say, maximum LOD score or lowest p value observed within each bin, but the reverse ranking from 1 = "worst" to n = "best" is also feasible. In practice, m can be as few as 4  null distribution of each Sj was derived in Wise [PAvgRnk, the "pointwise probability" of any Sj, is obtained from its marginal null distribution. The normal approximation to the exact distribution of the Sj is straightforward: the Sj are identically distributed, and each Sj has an approximate normal distribution with mean  and variance  under the null hypothesis that ranks are randomly assigned within each study. Koziol and Feng [m ≤ 12.We first introduce some notation. Let Levinson  rank the4 (e.g., ); and, f in Wise , PAvgRnk, with the weight wi ascribed to the ith study reflecting the relative linkage information from that study. (We are temporarily omitting the j subscript for clarity.) The normal approximation to the marginal null distribution of Sw is straightforward, and depends on the two parameters  and . The combinatorial argument utilized by Koziol and Feng [, where . We then have E[Rw] = 0, , andLevinson  proposedand Feng  to derivand Feng , we here.)(We have used Koziol and Feng , eqn. 11 up to 4th order terms, is f (z) = φ (z)[1 + c4H4 (z)], where φ(·) is the standard normal density function, , H4 (·) is the 4th degree Hermite-Chebyshev polynomial, H4 (z) = z4 - 6z2 + 3, and the constant c4 given by  (Stuart and Ord [The Edgeworth Type A series approximation to the density of c4 and φ(·) are as above, and H3(·) is the 3rd degree Hermite-Chebyshev polynomial, H3 (z) = z3 - 3z (Stuart and Ord [where Φ(·) denotes the cumulative distribution function of the standard normal distribution,  and Ord , eqn. 6.m = 20 schizophrenia genome scans, and found strong evidence for linkage on chromosome 2q, as well as suggestive evidence for linkage at several other chromosomal locations. The rank data for each scan are available online at D.F. Levinson's website (accessed July 14, 2004) [Sj, j = 1, 2, ..., 120, corresponding to the 120 bins spanning the entire genome, then their preferred weighted versions. Lewis [wi, i = 1, 2, ..., 20, are 2.32, 1.77, 1.20, 1.17, 1.17, 1.16, 1.15, 1.08, 1.03, 1.01, 0.95, 0.88, 0.80, 0.80, 0.68, 0.67, 0.59, 0.54, 0.53, 0.51, greater than a four-fold range.In practice, we would expect the Edgeworth series approximation to provide an adequate representation of the exact distribution of the weighted GSMA statistic, in a manner analogous to the unweighted case . Here, w4, 2004) , and we s. Lewis  had recos. Lewis , the ind, where  with m = 20, n = 120, by drawing each Xi as an independent random integer from 1 to 120 (that is, a uniform distribution of the integers from 1 to 120), then forming Rw with the Levinson [Rw. We then formed , and compared the empirical distribution of the 10,000 Z values with the Edgeworth approximation as described above; for comparative purposes, we also computed the normal approximation, which is based on matching the first two moments rather than the first four moments with Edgeworth. Figure Z values with both a normal approximation, panel A, and the Edgeworth approximation, panel B. Note that, even in this setting of the weighted combination of m = 20 individual GSMA statistics, the normal approximation is particularly ill-fitting in the tails. Agreement in the tails would be of particular relevance in practical applications, as these represent the areas of potentially significant findings . The noticeable disagreement in the tails between the weighted GSMA statistic and its normal approximation is largely ameliorated with the Edgeworth approximation. With attendant computational savings, the Edgeworth approximation provides a practical means of determining significance of weighted GSMA results compared to simulation; tail probabilities derived from the normal approximation should only be used with extreme caution.We simulated the null distribution of the weighted GSMA statistic Levinson  weights.Levinson , to prodj). The Levinson [Sj: their Pord refers to the probability of any observed Sk given the kth bin's place in the ordering of all of the Sj. We here derive approximations to this distribution. Let , j = 1, ..., n, and T(1) <T(2) < ... Tn) (denote the order statistics. We note first that the Tj have (approximately) a singular symmetric multivariate normal distribution, with means 0, variances 1, and correlations . That the joint distribution is singular follows from the observation that  for all i, hence,  is identically 0. If we dismiss the correlations as negligible (of absolute magnitude < 0.01 for n > 100), then the Tj are (approximately) independent, identically distributed N(0,1)  random variates, and the cumulative distribution function (cdf) Fk of the kth order statistic Tk) (is given byWe turn next to order statistic considerations (and reintroduce the subscript Levinson  order stwith Φ(·) as above (David , eqn. 2.Tj). (Note thatWe briefly examine whether correlations can be ignored when determining the distributions of the Prob (Tn) (≤ x) = Prob (T1 ≤ x,T2 ≤ x, ...,Tn ≤ x);     (2)n = 120, we depict in Figure Tn) (under independence, eqn. (1), compared to the distribution from eqn. (2) with pairwise correlations . The independence model tends to agree quite closely to the correlation model in this particular case, especially in the critical right tail, and has the virtue of numerical simplicity. We remark that one might improve slightly on the normal independence model by incorporating the Edgeworth correction into the individual cumulative distribution functions in equation (1).this latter probability may be calculated in R using the mvtnorm package , based oPAvgRnk < 0.05 and Pord < 0.05 "are the most likely to contain linked loci or to be adjacent to linked bins". Note that their criterion entails both the marginal distribution of the Tj, through PAvgRnk, and the (joint) order statistic distribution of the Tj, through Pord. We remark that there is some redundancy to the aggregate criterion {PAvgRnk < 0.05 and Pord < 0.05}, as can be seen through consideration of critical values relating to their aggregate criterion. With the normal approximation to the distribution of each normalized GSMA statistic Tj, the criterion {PAvgRnk < 0.05} is equivalent to the criterion {Tj > 1.645}. The criterion {Pord < 0.05} may be computed from eqn. (1), and depends on the ordering of the individual Tj. With n = 120, then for the ten largest order statistics T(120), T(119), ..., T(111), the criterion {PAvgRnk < 0.05 and Pord < 0.05} reduces to {Pord < 0.05}, since their 95th percentiles under their joint order statistic distribution exceed 1.645 [implying that, if {Pord < 0.05} obtains, then {PAvgRnk < 0.05} will automatically be satisfied]; and, for the remaining order statistics T(110), T(109), ..., T(1), the criterion {PAvgRnk < 0.05 and Pord < 0.05} reduces to {PAvgRnk < 0.05}, equivalently, {Tj) (> 1.645}, as their 95th percentiles under the order distribution, eqn. (1), are less than 1.645 [implying that, if {PAvgRnk < 0.05} obtains, then {Pord < 0.05} will automatically be satisfied].Levinson  had propPAvgRnk < 0.000417} (0.05 corrected for 120 tests) for evidence that a bin is likely to contain a linked locus or loci. One can improve on this procedure by using Holm's [m = 20 schizophrenia genome scans. As noted above, we used the online data to reconstruct the normalized unweighted GSMA statistics Tj,j = 1, 2, ..., 120, corresponding to the 120 bins spanning the entire genome. With m = 20 studies, we shall invoke the normal approximation to the distributions of the individual Tj.We conclude with a remark concerning multiple testing. Levinson  suggesteg Holm's  paradigmg Holm's  procedurLewis  had exteP(1) among n p-values with α/n and we find that the p-value is less than α/n. Then our multiple testing problem has been reduced by one test, and we should compare the next smallest p-value P(2) to . In general, we would compare Pi) (with . Holm's [i = 1, comparing Pi) (with , and stops as soon as Pi)  is less conservative than Bonferroni.Let us briefly review the Holm  method, . Holm's  step-dowThe Holm  method, Tj statistics, which we have recomputed from the online Levinson dataset [In Figure  dataset . On thisIn Figure P(1).] That is, in this particular instance, the unweighted GSMA statistics with either Bonferroni or Holm [In Figure  or Holm  correctiPAvgRnk formulation of Levinson [m = 20 individual GSMS statistics, the normal approximation is particularly ill-fitting in the tails. [Agreement in the tails is of particular relevance to practitioners, as these represent the areas of potentially significant findings .] As for the order statistic formulation and the aggregate criteria of Levinson [For practitioners utilizing GSMA statistics, the question arises as to whether the methods proposed here as well as in Koziol and Feng  are mereLevinson ), then tLevinson  or KozioLevinson , and shoLevinson  providesLevinson  and KoziLevinson  will typLevinson  can be bLevinson , we beliJAK conceived, designed and drafted the manuscript. ACF performed the statistical simulation.Both authors read and approved the final manuscript.


Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis.The first microbial genome sequence, The BLAST Score Ratio (BSR) approach, implemented in a Perl script, classifies all putative peptides within three genomes using a measure of similarity based on the ratio of BLAST scores. The output of the BSR analysis enables global visualization of the degree of proteome similarity between all three genomes. Additional output enables the genomic synteny (conserved gene order) between each genome pair to be assessed. Furthermore, we extend this synteny analysis by overlaying BSR data as a color dimension, enabling visualization of the degree of similarity of the peptides being compared..Combining the degree of similarity, synteny and annotation will allow rapid identification of conserved genomic regions as well as a number of common genomic rearrangements such as insertions, deletions and inversions. The script and example visualizations are available at:  Haemophilus influenzae genome sequence in 1995 [In the decade since the publication of the  in 1995 ; as of OBLAST analysis has become a ubiquitous method of interrogating new sequence data, but there are limitations to using BLAST alone as a discriminating tool. Many other methods and individuals use BLAST output E-values as a criterion for data parsing. While this measure may be efficient, the output is often skewed by both the database used for comparison and the length of the match . Small rSeveral other tools utilize the BLAST algorithms to compare nucleotide or peptide sequences from genome projects. The Wellcome Trust Sanger Institute ACT software  can dispBSR analysis is a departure from traditional genome scale analyses as it overcomes the limitations of BLAST E-values in comparative studies by normalizing the BLAST raw scores. BSR analysis is a tool for the rapid comparison of complete proteomes of any three genomes, and enables a visual evaluation of the overall degree of similarity of these proteomes and their genomic structure.1" and "Query2" respectively. Initiation of the script results in each of the putative peptides in the Reference proteome being compared to all of the other peptides in the Reference and Query proteomes using NCBI BLASTP.We have implemented the BSR algorithm using Perl. The inputs are the predicted proteomes of each of the three genomes under analysis, formatted as multi-FASTA files. An additional file for each of the proteomes is required. This file must contain a unique identifier, matching the FASTA header of the corresponding peptide in the multi-FASTA file, the relative genomic location of the start and stop of the coding region as well as the annotation for each peptide. The user selects one proteome as the "Reference"; the other two proteomes are termed "Query1 and Query2 proteomes with each best BLAST raw score recorded as Query1 and Query2, respectively  output contains an ordered list including the Reference peptide unique identifier, annotation, and Reference BLAST raw score, in addition to the unique identifier of the best hits in the Query proteomes, corresponding BLAST raw scores and the calculated BSR. Additionally, four unique files are generated corresponding to the peptides within the four quadrants delineated in Figure Following calculation of the BSRs, a number of output files are generated, including both text and graphical formats. The text files are tab-delimited for ease of parsing; filenames are derived from the named proteome files used as input into the script. The R_Q1_Q2.txt  between the two genomes , howevere Figure .The Gnuplot, PostScript and xfig outputs allow publication-quality, global visualization of the similarity and synteny of the selected genomes. However these formats do not allow the annotation associated with individual peptides to be viewed interactively. To overcome this limitation, additional XML files for the similarity and synteny plots described above are generated. These files are the input for the freely available GGobi software. GGobi is a data visualization system for viewing high-dimensional data . The tooThe GGobi package also allows the expansion of the BSR approach to include more than three genomes or other additional parameters associated with proteomic or genomic data, enabling interactive, user-driven exploration of these complex datasets. The current BSR implementation uses three genomes as input; however, additional genomes can readily be added as new dimensions simply by repeating the analysis with the same Reference genome and varying the Query genomes. Additional non-BSR dimensions are readily included, such as pI or %GC, or factors such as surface localization or some other feature of the peptides of interest.C. pneumoniae AR39 (GenBank Accession Number AE002161) is more similar to C. caviae GPIC (GenBank Accession Number AE015925) than C. muridarum strain Nigg (GenBank Accession Number AE002160) as 7.3 % of the proteome is shared between only C. caviae GPIC and C. pneumoniae AR39 compared to only 1.6% between C. caviae GPIC and C. muridarum strain Nigg. Finally, Figure C. caviae proteome has no significant hit to any of the peptides in the Query proteomes, although many of these peptides (78.2%) are currently annotated as hypothetical.Genome structure is often altered during the evolution of species . VisualiC. caviae GPIC and C. pneumoniae AR39 reveals that the genomes contain two points of inversion  and greater than 50% amino acid identity. This group of peptides is more readily identified by BSR analysis than BLAST E-value, which is artificially low due to the small peptide size. Additionally, peptides that have a BSR score < 0.4 but a BLAST E-value < 1 × 10-15 correspond 7.8% of the proteome. These represent divergent peptides with an artificially high BLAST E-value score resulting from limited regions of identity. The BSR analysis more accurately classifies these peptides based on the amino acid identity over the entire peptide. As the BSR comparison utilizes a single genome as a reference, the BSR score is calculated using a unidirectional best BLAST hit. However, when the Chlamydial proteomes were compared only one case in over 1000 could be found with a BSR score > 0.4 that was not also a bidirectional best BLAST hit.In the analysis of the Chlamydial proteomes using BSR score and BLAST E-values approximately 1% of peptides examined have a BSR score > 0.4 and BLAST E-value > 1 × 10The BSR approach allows rapid evaluation of the level of conservation of any three proteomes and the degree to which the genome structure between the three genomes is similar. While in this report we discuss the applications of this approach to whole genomes, the analysis has been performed on portions of genomes such as genomic or pathogenicity islands, plasmids and phage to identify peptide similarity and regional structure.More genome sequences are being generated from closely related organisms – a trend which shows no sign of abating. The BSR approach has become a crucial tool in our comparative genomics armamentarium and has been utilized in a number of genomic comparisons, revealing regions of similarity and difference between both closely and distantly related organisms ,15,16.Project name: BSR.plProject homepage: Operating System: Unix and MacOS XProgramming language: PerlOther requirements: Perl Statistics::Descriptive module License: NoneAny restrictions to use by non-academics: NoneBSR – BLAST score ratio; BLAST – basic local alignment search tool.DAR, GSAM and JR conceived and implemented the first versions of BSR and prepared the manuscript. All authors have read and approved the final manuscript.


Ilex (Aquifoliaceae) and discrepancy between plastid and nuclear phylogenies is the rule in this genus. These observations suggest that inter-specific plastid or/and nuclear introgression played an important role in the process of evolution of Ilex. With the aim of a precise understanding of the evolution of this genus, two distantly related sympatric species collected in Tenerife (Canary Islands), I. perado and I. canariensis, were studied in detail. Introgression between these two species was previously never reported. One plastid marker (the atpB-rbcL spacer) and two nuclear markers, the ribosomal internal transcribed spacer (ITS) and the nuclear encoded plastid glutamine synthetase (nepGS) were analyzed for 13 and 27 individuals of I. perado and I. canariensis, respectively.Intra-specific and intra-individual polymorphism is frequently observed in nuclear markers of I. perado, they are highly polymorphic in I. canariensis. The presence of pseudogenes and recombination in ITS sequences of I. canariensis explain this polymorphism. Ancestral sequence polymorphism with incomplete lineage sorting, or past or recent hybridization with an unknown species could explain this polymorphism, not resolved by concerted evolution. However, as already reported for many other plants, past or recent introgression of an alien genotype seem the most probable explanation for such a tremendous polymorphism.The plastid marker is intra-specifically constant and correlated with species identity. On the other hand, whereas the nuclear markers are conserved in I. canariensis, but I. perado is suspected. The introgression would be unilateral, with I. perado as the male donor, and the paternal sequences would be rapidly converted in highly divergent and consequently unidentifiable pseudogenes. At least, this study allows the establishment of precautionary measures when nuclear markers are used in phylogenetic studies of genera having experienced introgression such as the genus Ilex.Data do not allow the determination with certitude of the putative species introgressing  Ilex [Aquifoliaceae comprise one genus, Ilex  and apprIlex [atpB-rbcL spacer, rbcL and trnL-F) is highly correlated with the geographic distribution of extant species. Four chloroplast clades are found: one exclusively Eurasian clade, one exclusively American clade and two different North-American/Asian clades (one of them comprising most of the deciduous species among other evergreen species). On the other hand, the nuclear phylogeny  is incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. The nuclear phylogeny is not correlated with the geographic distribution of extant species.The molecular phylogeny of the genus Ilex ,3 shows trnL-trnF sequencing and nuclear ITS sequencing, a study of Asian Ilex of the Bonin Island and of the Ryukyu Island [Ilex leucoclada M. is highly polymorphic [Ilex probably experienced frequent incomplete lineage sorting and nuclear and/or cytoplasmic introgression, making the study of its history very complex.Any of the plastid or the nuclear phylogeny corroborates previous morphological or biosystematic studies . Using cu Island  confirmeymorphic . During Ilex [Ilex, three species are tetraploid  and one species is hexaploid (I. pedunculosa), indicating probable hybridizations between species having divergent genomic background .Few data are reported on the chromosome number of Ilex . The basIlex contain polymorphic nuclear sequences (ITS and 5S rDNA spacer). Except for I. purpurea and I. guianensis, only one individual was studied per species. The sampling being too low for a correct evaluation of this intraspecific polymorphism, an exhaustive study of one plastid marker (the atpB-rbcL spacer) and two nuclear markers  was undertaken on several individuals of I. perado and I. canariensis collected in Tenerife (Canary Islands). These species were chosen because, based on DNA data, they are not closely related [I. canariensis is endemic of Canary Islands, whereas I. perado has a wider distribution in Spain, Portugal, North-Africa and Canary Islands. Natural or artificial hybridization between both species was never reported. The data show that, contrarily to I. perado, I. canariensis has highly polymorphic ITS and nepGS sequences. The aim of this study was to (1) explain the polymorphism observed in ITS of I. canariensis by an investigation of its pattern of substitution and its functionality, (2) determine the evolutionary mechanisms responsible of this polymorphism and (3) focus on ITS evolution and consequences for phylogenetic reconstruction of the genus Ilex.A previous study  showed t related ,3 and aratpB-rbcL spacer phylogram obtained from the alignment of the sequences of I. perado and I. canariensis collected in Tenerife. All individuals of I. perado have the same atpB-rbcL spacer sequence. For I. canariensis, 26 individuals have the same atpB-rbcL spacer sequence and three substitutions are observed (in a T-rich variable region) for specimen 39. This plastid marker perfectly agrees with species determination.Figure I. canariensis are extremely polymorphic, it was interesting to observe their relationships with available ITS sequences previously investigated species by Manen et al. [I. perado and I. canariensis sequences found in Tenerife altogether with 43 ITS sequences of other Ilex species. Thick bars indicate internal branches conserved in the consensus tree. The closest possible outgroups for the genus Ilex, are Helwingia and Phyllonoma. However these genera are so isolated systematically that their use to root Ilex should be taken with care. On 13 individuals of I. perado, only one substitution (a transition) is observed in ITS. On the other hand, ITS sequences of most individuals of I. canariensis are polymorphic and few sequences are identical. The divergence between all ITS sequences observed in I. canariensis is much higher than between available ITS sequences of all other species investigated. The GC content is 57% for I. perado and from 45 to 62% for I. canariensis. Regarding their GC content, three groups of ITS are found in I. canariensis: a clade with 45% GC, a clade with 61% GC in average and several clades with 53–54% GC in average. The GC content of other investigated species range from 55 to 61%  which are involved in common pairing relationships on the structure . In orde to C6 whIlex was calculated by maximum likelihood from the ITS data of Manen et al. [I. perado sequences and for the I. canariensis sequences found in the GC 62% clade. This increased rate is statistically significant according to the Kruskal-Wallis rank test [The GC content, indicated for all ITS sequences of Figure n et al.  and usedank test .As expected for pseudogenes ,11, the I. perado, many ITS sequences found in I. canariensis have substitutions at some of these conserved sites. Only the GC 61% clade of I. canariensis comprises non-substituted conserved 5.8S sites  shows tI. canariensis experienced frequent recombinations: in the entire ITS matrix of I perado and I. canariensis, the DnaSP program [I. perado and in clade 2 of I. canariensis. Clades 5 and 6 are highly recombined. An example of recombined ITS sequences of I. canariensis from clade 5 is shown in Figure Most ITS sequences of  program  detects q (0.554) was greater than for all 1000 clonal replicates , indicating a history of recombination (significance 0.001).In order to exclude the possibility that the observed pattern of substitution is the result of homoplasy and to confirm that these sequences are actually recombined, maximum likelihood tests were carried out. Using PIST , the maxThe LARD maximum-likelihood method  was applnepGS of I. perado. On the other hand, I. canariensis shows polymorphism for this gene. Thirty sites differentiate I. perado from I. canariensis, of which eight are polymorphic in I. canariensis, either heterozygous or homozygous  could be explained by a release of evolutionary constraint and a subsequent high rate of substitution. Indeed, ITS sequences have functional constraints in relation with the processing of the rRNA precursor producing the functional 18S, 26S and 5.8S subunits.In conclusion, the high divergence found in ITS sequences of I. canariensis. Moreover, recombinants could result from "jumping" PCR reaction [nepGS for Oxalis [Gossypium [Evidence for recombination in divergent sequences is not obvious. It is difficult to recognize homoplasy generated by recombination from actual homoplasy . Statistical methods  are still too rudimentary to precisely describe the recombination events in the set of ITS sequences found in reaction -23, wherr Oxalis  and on fossypium .I. canariensis ITS sequences experienced intra-molecular recombinations  calculated with DnaSP  underests Figure . The facThe distribution of informative characters shown in Figure Begonia [Microseris [Quercus [Amelanchier [Paeonia [Buddia, Gossypium, Nicotiana, Tripsacum [Armeria [Recombination in highly polymorphic ITS sequences seems a rule in plants. This is not surprising because the mechanisms of concerted evolution in rDNA arrays are based on crossing-over and gene conversion. It has been reported in Begonia , Microsecroseris , Quercus[Quercus , Amelanclanchier , Paeonia[Paeonia , Buddia,ripsacum , Armeria[Armeria  and otheI. canariensis is much higher than between ITS sequences of all other species investigated, knowing that, according to their GC content . Only the GC 45% clade does not group with the bulk of I. canariensis ITS sequences. Its position is not defined and varies in the vicinity of a Eurasian lineage (I. latifolia, I. leucoclada, I. maximocziana, I. rugosa and I. perado). Thus data do not support a particular relationship of most I. canariensis ITS pseudogenes with another Ilex species, except for the pseudogenes with a GC content of 45%, that are frequently observed in I. canariensis.Assuming that multiple ITS sequences found in he genus ,3 were ie Figure . Most fuI. canariensis, the most probable candidate would be the sympatric species I. perado. It can not be ruled out however that the distribution of I. canariensis was much wider in the past [I. latifolia, I. leucoclada, I. maximocziana, I. rugosa and I. perado. Pseudogene sequences (particularly the ITS sequences of clade GC 45%) being too divergent and of different nucleotide composition, the observed relationship of clade GC 45% with the group of species comprising I. perado is questionable because of possible spurious long branch attraction. However, the data of the nuclear encoded plastid glutamine synthetase (a nuclear single copy locus) are not conflicting with an introgression of I. perado in I. canariensis. All the eight polymorphic sites observed in I. canariensis always comprise one allele shared with I. perado. Another possibility is that these ITS pseudogenes represent a relictual ancestral polymorphism in the course of elimination by lineage sorting or concerted evolution. In fact ancestral polymorphism could also be the result of ancient introgressions. The data accumulated here do not allow a definitive conclusion.In the case of hybridization involving the island species the past ,33 and tI. perado and I. canariensis is confirmed, the introgression would be unidirectional because ITS sequences of I. perado do not show any polymorphism. This situation is reminiscent of the unilateral hybridization observed between Begonia formosana and B. aptera, where on 60 ITS sequences analysed in natural or artificial hybrids, 58 sequences are clustering with the ovule donor B. formosana, and only 2 are found clustering with the pollen donor B. aptera [I. perado precedes the one of I. canariensis, thus the loading of still living I. perado pollen grains on young effective I. canariensis stigmates is more favored than the contrary. Moreover, there are much more male than female I. perado plants in Tenerife [If a putative cryptic hybridization between . aptera . UnidireTenerife ,35. ThesIlex [Ilex. Thus, precautionary measures should be taken when studying nuclear ITS sequences in the genus, particularly the search for recombinant and pseudogenes. Particular PCR conditions should be used [Ilex is recommended. This study will probably make phylogenetic interpretations easier and will certainly help to the understanding of the complex evolutionary history of Ilex [This study was undertaken with the aim to study and overcome the problem of ITS polymorphism found in many species of Ilex . IntrogrIlex ,4 and hiIlex  have alr be used ,19,23. R be used  were howI. perado ssp. platyphylla Webb. & Berth. and 27 individuals of I. canariensis Poir. were collected in Tenerife (Las Mercedez, Aqua Garcia and Aqua Mansa) and genomic DNA was extracted from dry leaves as previously reported [Thirteen individuals of atpB-rbcL spacer was sequenced for the 40 specimens of Tenerife according to Cuénoud et al. [I. perado and five individuals of I. canariensis produced a perfectly readable ITS sequence with no polymorphism. On the other hand, 22 individuals of I. canariensis produced an unreadable highly polymorphic ITS sequence. Cloning in E. coli was necessary and four clones per individual were sequenced. For all specimens, the nuclear encoded plastid glutamine synthetase (nepGS) was amplified and sequenced according to Emshwiller and Doyle [I. canariensis, but as indels are not involved in the polymorphism, sequences were readable and polymorphic sites were coded according to the international code for nucleotide polymorphism .The plastid d et al. . In a fid et al. . All indnd Doyle . PolymorI. canariensis and I. perado were aligned with ITS sequences of 43 species of Ilex previously studied in Manen et al. [Phyllonoma and Helwingia, the closest relatives of the genus Ilex  allowing for different rate of transitions and transversions as well as unequal base frequencies] using the unique maximum parsimony tree obtained from the ITS matrix of Manen et al. [Ilex and Helwingia and Phyllonoma as outgroup. For comparisons, the number of substitutions (Kimura 2-parameter) was calculated from this ancestral sequence for I. perado and I. canariensis ITS sequences using PAUP.In order to examine the pattern of substitution of ITS sequences found in n et al.  based onIlex ancestral sequence for I. perado and I. canariensis ITS sequences, according to Buckler et al. [The high frequency of deamination-like substitutions (C -> T and G -> A at CpG and CpNpG sites) is typical for pseudogenes and was also calculated from the reconstructed r et al.  and Muirr et al. . This frI. perado and I. canariensis 5.8S sequences was calculated.Based on the alignment of fifty 5.8S sequences including vertebrates, invertebrates, fungi and plants , 59 totI. perado and I. canariensis and in different ITS clusters observed in I. canariensis. This program is based on linkage desequilibrium, neutrality tests and substitution distribution along the locus. ITS sequences showing strong evidence of recombination, as detected by DnaSP, were submitted to PIST [I. canariensis ITS sequences (see results) was constructed with base frequencies, ti/tv ratio, proportion of invariable sites and gamma shape parameter estimated under the HKY model [39] and these parameters were used to calculate the tree scores of simulated clonal sequences.The minimum number of recombination events RM  was calc to PIST  to calcuThe recombination points of three selected sequences (see results) showing evidence of recombination were calculated by maximum likelihood using LARD  with theITS matrix in NEXUSClick here for file


Schistosoma mansoni and Plasmodium chabaudi develop higher P. chabaudi parasitaemia and had a higher mortality rate. This important observation deserved to be further investigated among human populations.Helminthic infections concomitant with malaria are common in inter-tropical areas. A recent study showed that mice co-infected with S. mansoni, and 175 were not. The incidence rate of malaria attacks was significantly higher among S. mansoni-infected individuals, particularly those carrying the highest worm loads, as compared to uninfected subjects (26.6% versus 16,4 %). In contrast, the rate of malaria attacks was lower, without reaching significance, in medium grade S. mansoni infections. Thus, infection by S. mansoni affects susceptibility to malaria, but this can vary according to the intensity of parasite load. The immunological mechanisms underlying this dual effect need to be further explored.Malaria attacks were recorded in 512 children aged 6–15 years living in Richard Toll  among whom 336 were infected by  Schistosoma mansoni and Plasmodium chabaudi co-infected mice develop higher P. chabaudi parasitemia and higher mortality rate [Malaria is prevalent in many parts of tropical Africa, where other concomitant parasitic infections are common. However, little is known about how concurrent infections affect the expression to and/or pathogenesis of each other. A recent study showed that ity rate . This obity rate  and by fity rate .S. mansoni infection [In the Senegal river basin, where malaria is hypoendemic, extensive irrigation programmes have been developed, promoting a spread of nfection . The expThe study took place in Gallo Malick, a district of Richard Toll, which is located near a canal ensuring the irrigation of rice and sugar cane fields. The population was estimated in 1998 to reach 3,685 inhabitants. Annual rainfall amounts to around 250 mm water. The district is approximately 1 km long and 500 m wide. A dispensary is set up in the centre of the district and is thus easily accessible.In September 1998, after obtaining agreement from the parents, two stools examinations were carried out in all children aged between 5 and 15 years. From 25 mg of stools, two slides were prepared, according to the modified Kato-Katz method [Schistosoma haematobium eggs, were performed. Ten ml of urine were filtered through a 12 μ millipore membrane and the eggs on the whole filter surface were counted. A questionnaire was issued to check habits with regard to protection against mosquitoes and usage of anti-malarial drug-prophylaxis. It was also ascertained that none of the included children had been administered anti-S. mansoni treatment (praziquantel) over the past year (September 97 – September 98). In March 99, all children who presented with at least one S. mansoni positive stool examination were treated with praziquantel (40 mg/kg).The follow-up was implemented between September 1998 and April 1999. In January 1999, a further stool examination and a urine filtration for the detection of Any child presenting with a body temperature > 38°C or having a history of fever-in the 24 hours preceding the consultation and with a parasite density ≥ 5000 parasites/μl of blood was defined as suffering from a malaria attack.S. mansoni at least once was defined as S. mansoni positive. If both examinations proved positive, the highest parasitic burden was kept for further analysis. Parasite loads were classified into four categories: A) 1 to 100, B) 101 to 400, C) 401 to 1000, and D) > 1000 eggs per one gram of stools.Any child whose tool tested positive for S. mansoni but presenting other intestinal parasitic infection were excluded from the analysis.In order to exclude possible interactions with other helminthic infections, children from the group not infected by S. mansoni. This is in agreement with the distinct sources of infection, Anopheles larvae breeding in small water collections in gardens, and Schistosoma vectors in rice fields and irrigation canals.When houses where children had a malaria attack and those where the children were carrying schistosomiasis were located on a map, the two pathologies were found to be distributed in a uniform way in the whole district, without evidence of clustering of double infection malaria-S. mansoni categories eggs load and age.Comparison between children presenting at least one malaria attack and children not having presented any malaria attack was performed using a forward logistic regression model using STATA statistical software. The variables included in the model were sex, S. mansoni infections using a lifetime estimate table from the EGRET programme (Statistics and Epidemiology Research Corporation, Seattle, Washington). Any p value lower than 0.05 was considered significant.The period between the beginning of the study and the date of a malaria attack was compared between groups of S. mansoni eggs and 189 were negative for both stool examinations.In September 1998, 525 children underwent two stool examinations. Three-hundred and thirty-six-were found to carry Schistosoma haematobium (2.7%). Among them, 11 were also positive for S. mansoni. The stool examination showed the presence of 31 other intestinal parasitic infections (Ascaris and Trichuris). Patients negative for S. mansoni and carriers of another intestinal (12 subjects) or urinary parasite (2 subjects) were excluded of the analysis.Urine filtration detected only 13 children excreting eggs of S. mansoni (65.7%) and 175 (34.3%) not infected). The prevalence increased as a function of age, reaching 80% in children older than 11 years with > 30% of children presenting with high schistosome loads, i.e. > 1,000 eggs/ 1 g of stools.In total, 511 children were included in the analysis  were attributed to a malaria attack, according to the criteria described. Only 10 children underwent two malaria attacks. In total, 18.9 % (97/511) of the children included in the analysis presented with a malaria attack at least once.S. mansoniior not. However, the malaria attack prevalence was also the same whether or not the children were sleeping under a mosquito bednet.Chemoprophylaxis for children is not recommended by the National Control Programme, but 7.9 % of the parents reported that they were giving a chemoprophylactic treatment to their children. 58.9 % stated they used a mosquito bednet. These proportions were the same in the two groups of children, infected by S. mansoni and 16.6% (29/175) in those non-infected (p = 0.40). The malarial incidence, however, varied depending on the load of eggs. It was high in all groups, but the highest (26.6 %) in those carrying the highest worm loads (> 1,000), except in subjects presenting medium loads (>100 and < 400 eggs/g of stools) where malarial incidence was lower than in S. mansoni negative individuals (9.4 %). Sex and age had no significant influence, which is not surprising since this is a mesoendemic area. Using a logistic regression model and taking the negative group as reference, the difference was significant with the group carrying a high load of eggs (RR = 2.24 (1.2 – 4.2) , the malaria incidence was significantly increased for low S. mansoni egg carriage (1–100) (RR = 2.51 (1.05–6) as well as high S. mansoni egg carriage (>1,000) RR = 3.12(1.33–7.29).The incidence rate of malaria attacks was 20.2 % (68/336) in the group of children concomitantly infected by Out of 10 children who presented two malaria attacks, nine were infected by S.m.; four presented a schistosome load higher than 1,000 eggs/g of stools, and three > 400 and <1,000.S. mansoni loads and other groups increased over time during the follow-up  or a low load of eggs (1–100), whereas a lower attack rate was observed in children presenting a medium egg load (>100 and <400 eggs/g), although this opposite trend did not reach significance.The analysis of the incidence of the malaria attacks by amount of parasite load in Protection against falciparum malaria has been found to be associated with the preferential production of the cytophilic classes, IgG1 and IgG3, of antibodies , this beS mansoni, but only for those having the highest loads. The opposite effect was found in individuals carrying medium schistosome infections. As immunological studies could not be performed, one can only speculate about this biphasic effect. The production of cytophilic classes against malaria requires that the T-cells helper effect is provided by Th1 type of T-cells, however cytokines produced by T-cells specific for schistosome eggs or worms can influence responses to malaria. It has been reported that, during the first phase of S. mansoni infection, T-cells are stimulated which secrete cytokines belonging, in majority, to the Th1 type. The switch towards Th2 type cytokines occurs later on and is dependent on egg production [S. mansoni eggs.In the present study, this observation has been confirmed with yet another worm infection, oduction . In the Obviously, further studies, particularly of T-cell responses to both malarial and schistosome antigens are required to sort out this issue.S. mansoni infection can increase the susceptibility to malaria in subjects excreting high schistosome egg loads.Helminthic infections are a fact of life in malaria endemic areas and their influence on the course of infection and the epidemiology of malaria is a fascinating though neglected area of research. This study shows that CS, PM, JA, PC, MD were responsible for field and laboratory studies. JYL was responsible for the statistical analysis. PD inspired and designed the study


The discussion about the interplay between theory and intervention has a long history and much has been written and published. The companion perspective papers by Rothman and Jeffery provide alternative points of view on this important and complex question. Their papers are not intended to be critical reviews of the literature. Instead, the aims of the two papers are to delineate the problem, present the different perspectives of the authors on the issue, and suggest constructive steps researchers can take to move the field forward.We hope that these articles generate a lively response from our readers and spur innovative ideas for strategies to cultivate a healthy developmental interplay between theory and intervention in behavioral nutrition and physical activity research.We invite you to consider the intellectual and practical challenges posed by the two authors and perhaps respond with your perspectives, which may be published in the IJBNPA.We look forward to hearing from you soon.Simone A. French, Ph.D.Tony Worsley, Ph.D.Co-Editors, IJBNPA


Multiple studies have linked fungal exposure to asthma, but the link to severe asthma is controversial. We studied the relationship between asthma severity and immediate type hypersensitivity to mold  and non-mold allergens in 181 asthmatic subjects.Aspergillus, Alternaria, Cladosporium, Penicillium and Candida) and 4 other common inhalant allergens (D. pteronyssinus, Grass Pollen, Cat and Dog).We recruited asthma patients aged 16 to 60 years at a University hospital and a nearby General Practice. Patients were categorized according to the lifetime number of hospital admissions for asthma (82 never admitted, 53 one admission, 46 multiple admissions). All subjects had allergy skin prick tests performed for 5 mold allergens . Multiple mold reactions were also much commoner in the group with multiple admissions . The number of asthma admissions was related to the number and size of positive mold skin allergy tests (Spearman Correlation Coefficient r = 0.60, p < 0.0001) and less strongly correlated to the number and size of non-mold allergy tests (r = 0.34, p = 0.0005). Hospital admissions for asthma patients aged 16–40 were commonest during the mold spore season (July to October) whereas admissions of patients aged above 40 peaked in November-February (Chi Squared, p < 0.02).Skin reactivity to all allergens was commonest in the group with multiple admissions. This trend was strongest for mold allergens and dog allergen and weakest for These findings support previous suggestions that mold sensitization may be associated with severe asthma attacks requiring hospital admission. Alternaria species has been reported to be especially common in patients with life-threatening asthma [Most asthma patients have mild symptoms which are well controlled with anti-inflammatory and bronchodilator therapy but a minority of asthma patients have severe airway inflammation and airflow obstruction requiring multiple hospital admissions. The reasons for these differences in asthma severity are complex and not fully understood . At leasg asthma . Asthma g asthma -11. Moldg asthma -17.Indoor mold exposure might also contribute to asthma severity. Many patients believe that their asthma is aggravated by damp housing, especially if there is visible mold growth . It has These studies prompted us to undertake a study of asthmatic patients in Salford in Greater Manchester (North-West England) to assess whether atopy in general and mold sensitization in particular was associated with increased asthma severity. There are few agreed definitions of severe asthma so we used hospital admissions and treatment category (according to British Thoracic Society Asthma Guidelines) as surrogate markers for asthma severity .This study was undertaken in Hope Hospital, a 900 bedded University Hospital in Salford, Greater Manchester, UK. The study was designed to record the atopic status for mold and non-mold allergens of asthma patients at every level of severity from very mild to very severe . We studied patients with severe asthma during hospital admissions or at subsequent visits to the hospital chest clinic. Patients were recruited opportunistically during hospital admissions or during routine consultations with the Respiratory Nurse Specialist (RNS) in the hospital Chest Clinic. Recruitment was evenly spread over 30 months from January 1996 to June 1998. During this period, the Respiratory Nurse service was under development with only one part-time RNS available to serve a large population of hospital in-patients and ambulatory care patients at chest clinics. Ward-based doctors and nurses referred patients with acute asthma to the RNS on an opportunistic basis based on availability of the part-time RNS and whether the ward teams were aware of the developing RNS service. About 20% of all adult asthma patients admitted during the study period were referred to the RNS and most of these were recruited in the study (provided the RNS had time available to do so). Although recruitment was not systematic (based mainly on availability of RNS service), we were not aware of any potential bias which might have affected the recruitment process and we believe that the recruited patients were typical of adult patients with acute asthma admitted to our hospital. We recruited ambulatory patients  at the same hospital chest clinic and in a single Primary Care practice where 25 patients were recruited during routine consultations with the practice nurse. This recruitment was also opportunistic, based mainly on the availability of time to complete the study protocol during busy clinics. We believe that these patients were typical of patients seen at hospital chest clinics and General Practice asthma clinics in this area. Patients were grouped according to their lifetime history of hospital admissions for asthma (multiple admissions, single admission, or no admissions). Patients with no hospital admissions were further categorized according to the treatment steps described in the British Thoracic Society guidelines for asthma management; Step 1 requiring only occasional bronchodilator treatment, Step 2 requiring low-doses (<800 mcg per day) of inhaled steroid, Step 3 requiring high doses of inhaled steroids or long acting beta agonists, Step 4 requiring additional therapy such as domiciliary nebulized therapy . We atteAll patients were Caucasians who were lifelong residents of the United Kingdom. Inclusion criteria were a diagnosis of asthma by the patient's doctor, age 16 to 60 and ability to give informed consent. All subjects gave written informed consent prior to partaking in the study which was approved by the Salford Research Ethics Committee. Exclusion criteria included a diagnosis of COPD, non-European ethnic group  and consumption of any antihistamine in the previous 48 hours. All subjects completed a questionnaire concerning respiratory symptoms, smoking status and allergies.Dermatophagoides pteronyssinus, cat, dog, mixed grass pollen, Aspergillus fumigatus, Alternaria alternata, Cladosporium herbarum, Penicillium notatum and Candida albicans.All tests were conducted by one author (LCH) or by one other respiratory nurse specialist using a standardized technique in an open manner. A small drop of allergen was placed on the volar surface of the forearm. Allergens were purchased from Allergopharma (Reinbek, West Germany), a single batch of each allergen was used throughout the study. We used skin test lancets with a 1 mm tip . The lancet was introduced vertically into the skin through the allergen solution. Allergens studied were: negative control, histamine 0.1%, Aspergillus and a 5 mm reaction to Cladosporium would have a "mold sensitization score" of (6-1)+(5-1) = 9 mm.Weal diameter (if any) was recorded at 15 minutes. If a weal was asymmetrical, the mean of two perpendicular measurements was calculated. Weals less than 3 mm greater than the negative control reaction were regarded as negative in accordance with guidance issued by the European Academy of Allergology . We deviAs a subsidiary study , we also studied the seasonality of asthma admissions in Salford by reviewing the electronic records of 520 asthma admissions under the care of two pulmonary physicians who kept a computerized database at this hospital between 1995 and June 2000 . This covered a wide time-span before, during and after the period of the mold sensitization study; some of the allergy study patients were admitted and recruited during this time but they represented only a small (and random) proportion of the admissions studied for seasonality. Asthma admissions were analyzed by month of admission and divided into 3 four-month "seasons"; March to June ; November to February ; . PatientStatistical analysis was performed using Prism II software . Chi squared test was used to compare the number of positive allergy skin tests between groups of asthma patients. Spearman Correlation Coefficient was used to compare each patients lifetime number of hospital admissions with their "Mold sensitization score" (as described above). Mann Whitney tests were used to compare mean sensitization scores between groups of patients.One hundred eighty-one asthmatic patients were recruited. Their characteristics are given in table Positive skin tests to all allergens were commoner in the group with severe asthma  than patients with milder asthma. Table . Atopic Alternaria) to 41% (Cladosporium) in the severe asthma group compared with 0–10% in the milder asthma groups.Mold sensitization was uncommon in mild asthma but very common in asthma patients with multiple admissions Figure . There wAspergillus and Candida precipitins and specific IgE were not measured in this study. None of these patients had any clinical features suggestive of allergic bronchopulmonary aspergillosis (ABPA) such as pulmonary infiltrates, bronchiectasis or marked eosinophilia. The number of admissions correlated with the number and size of positive skin tests using the scoring system described previously. For mold sensitization, the Spearman Correlation Coefficient was 0.60 (two-tailed p < 0.0001) and for non-mold allergens was 0.34 (two-tailed p = 0.0005). The cumulative "mold sensitization score" and the "non-mold sensitization score" for each group of patients is shown in table Aspergillus, one Penicillium).Of the patients not admitted to hospital, 32 had very mild asthma (BTS Step 1), 25 had mild-moderate asthma (BTS Step 2) and 25 had moderate to severe asthma (BTS Steps 3–4). There was no significant difference in mold or non-mold sensitization between these groups of non-admitted patients with different grades of asthma severity.Our review of asthma admissions to this hospital between 1995 and 2000 identified 520 patients admitted under the care of the two chest physicians who kept a computerized database . There were 173 asthma admissions in the 16–40 age group, these admissions peaked in late summer and fall (figure Alternaria or Cladosporium is a powerful risk factor for severe asthma[D. pteronyssinus) sensitization in our patients with more severe asthma [Although the present study was larger than most previous studies of mold sensitization in severe asthma, it must be regarded as a "pilot study" due to the non-systematic recruitment of asthma patients and the cross-sectional nature of the study. Our data indicate that mold (and dog) sensitization is common in patients with severe asthma requiring multiple hospital admissions in Manchester. The results are consistent with previous evidence that atopy  is related to asthma severity or bronchial hyper-reactivity -6,12-17.re asthma in severre asthma,14. We he asthma ,25. HoweThere has been some debate about the best cut-off point for weal size to define a positive skin-prick test. We accepted the European Academy of Allergology figure of 3 mm greater than the negative control . HoweverA key question is whether severe asthma is actually caused by sensitivity to molds or is simply associated with it. In any case, mold sensitivity will certainly not be the only cause of severe attacks of asthma; upper respiratory tract virus infections and some drugs being two of other well documented causes. The greater degree of mold sensitization in the severe asthma group could simply reflect an extreme example of the generalized increase in atopy amongst this group. However, we believe that mold allergy may be responsible for severe asthma attacks for several reasons.Alternaria-sensitive had their near-fatal asthma episodes in summer and early fall when mold spore levels would be expected to be high [First, the temporal relationship between high environmental spore counts and asthmatic attacks is strong. Airborne spore levels may be up to 1000 times higher than pollen levels . The dat be high .Cladosporium in July, Alternaria and hyaline basidiospores in August, uredospores in September and coloured basidiospores in October. The data from Derby (52 miles south-east of Manchester) are similar[Cladosporium, Alternaria, Penicillium and Aspergillus with maximal levels between June and October [A. alternata and C. herbarium was common and associated with asthma severity – OR of 2.03 for the former, of 3.2 for the latter fungus and 2.34 for both. No such association was found for pollens or cats, although sensitization to house dust mite was slightly more frequent in those with severe asthma (OR 1.61) [Second, the seasonal  peak of asthma admissions occurs when ambient air counts of molds are high. We have documented a late summer-fall peak of asthma admissions involving young adults in Manchester which coincides with the summer-fall peak of asthma deaths in UK patients aged under 35 years ,21. These similar. In addi October . A pan-E October . SensitiOR 1.61) . The preThird, there is evidence that indoor mold exposure may contribute to asthma severity. Many patients report respiratory symptoms in damp and moldy houses and a review of nine population-based studies found that seven reported one or more positive associations between fungal levels and health outcomes . The stuAspergillus in particular is a major respiratory allergen causing the vast majority of cases of allergic bronchopulmonary mycosis [Aspergillus and Candida allergens may share IgE-binding epitopes [Aspergillus and a few other common airborne fungi have and it is likely that sensitization to other fungi will be discovered in the future.Fourth, we know that  mycosis -33. It iepitopes . Howeverepitopes . Few funCandida is present in the gut of most, if not all, humans. The difference may relate to the nature or intensity of exposure to mold allergens or to their ability to become airborne and to gain entry to human airways due to their small size. Also many potent allergenic proteins have been described in Aspergillus and some in other fungi [Aspergillus is inhaled, which is almost hourly. Some fungal antigens are proteases (Asp f5, Asp f10, Asp f13, Asp f15, Asp f18) and as DP1 is also a protease – a similar pathogenic role can be postulated [It is not known why mold allergens should produce more severe airway disease than other common allergens such as house dust mites, cat dander, or grass pollen. Fungi are very common in the environment and er fungi . There istulated ,36.It therefore seems likely that there is a causal relationship between mold allergy and asthma severity for some younger asthma patients. Our seasonal admissions data suggest that up to 6% of adult asthma admissions in Manchester and 19% of asthma admissions in the 16–40 age group may be attributable to mold allergy. These unlucky individuals seem to have more severe asthma than patients with sensitization to other common allergens and an increased risk of fatal or near-fatal asthma or hospital admissions during the mold spore season, especially on days when the local mold spore count is high. The risk may be further increased if the patient lives in a damp, moldy house or attends a damp moldy school ,30,37. ICandida albicans, is a yeast, does not become airborne but does produce hyphae in tissue. Thus it is possible that human asthma due to fungal allergens may have three sources of allergen exposure – outdoor mold spores, indoor mold spores and endogenous fungal growth on body surfaces including the skin and gut [One of the fungi which we investigated and for which antigenic extracts are available,  and gut .Alternaria sensitization in Italian patients with respiratory symptoms ranges from 2% in Northern Italy to 29% in Southern Italy [Fungal sensitization is relatively uncommon in our British asthma patients and in Finnish schoolchildren  comparedrn Italy . Some ofrn Italy . Also furn Italy ,16,17 suThe findings of this study support previous suggestions that mold sensitization may be associated with severe asthma attacks requiring hospital admission.BTS Guidelines = British Thoracic Society Guidelines for Asthma Management, RNS = Respiratory Nurse SpecialistThe author(s) declare that they have no competing interests.ROD originated and co-ordinated the study and contributed to the analysis of the data and preparation of the paper.LCH contributed to the design of the study and was the main clinical investigator. She also contributed to the analysis of the data and preparation of the paper.DWD contributed to the design of the study and contributed to the analysis of the data and preparation of the paper.The pre-publication history for this paper can be accessed here:


Public repositories of microarray data contain an incredible amount of information that is potentially relevant to explore functional relationships among genes by meta-analysis of expression profiles. However, the widespread use of this resource by the scientific community is at the moment limited by the limited availability of effective tools of analysis. We here describe CLOE, a simple cDNA microarray data mining strategy based on meta-analysis of datasets from pairs of species. The method consists in ranking EST probes in the datasets of the two species according to the similarity of their expression profiles with that of two EST probes from orthologous genes, and extracting orthologous EST pairs from a given top interval of the ranked lists. The Gene Ontology annotation of the obtained candidate partners is then analyzed for keywords overrepresentation.We demonstrate the capabilities of the approach by testing its predictive power on three proteomically-defined mammalian protein complexes, in comparison with single and multiple species meta-analysis approaches. Our results show that CLOE can find candidate partners for a greater number of genes, if compared to multiple species co-expression analysis, but retains a comparable specificity even when applied to species as close as mouse and human. On the other hand, it is much more specific than single organisms co-expression analysis, strongly reducing the number of potential candidate partners for a given gene of interest.CLOE represents a simple and effective data mining approach that can be easily used for meta-analysis of cDNA microarray experiments characterized by very heterogeneous coverage. Importantly, it produces for genes of interest an average number of high confidence putative partners that is in the range of standard experimental validation techniques. The availability of genome sequences from several model organisms, including humans, and of high-throughput technologies to study gene function is dramatically changing the approach to biological problems. In particular, the consolidated reductionist gene-by-gene strategy is being replaced by a 'modular approach', in which several genes are studied simultaneously to gather a more comprehensive picture of the many different cellular processes : in liviIt is well recognized that one of the most important and widespread mechanisms used by cells to regulate functional modules is the coordinate transcriptional and/or post-transcriptional modulation of mRNA levels of the interacting genes. Therefore, DNA microarrays represent a fundamental tool to unravel biological complexity on a genome-wide scale. Information concerning the expression of thousands of genes, and also of different transcripts from the same gene, can be obtained in a single experiment, and the relationships among gene expression patterns can be studied systematically . The extBesides being useful to other researchers to confirm the published results, the deposited datasets can be used as a substrate for new analysis, aimed at discovering functional modules by searching for related expression profiles. Recent studies have shown that, if the expression of two or more genes is consistently related throughout many independent microarray datasets, the genes display a significant degree of functional similarity ,8. HowevHomo sapiens, Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans was very effective in identifying new genes involved in core biological functions [Phylogenetic conservation has been recently proposed as a very strong criterion to identify functionally relevant co-expression links among genes . Signifiunctions . AlthougSince regulatory regions diverge much more rapidly than coding sequences ,13, a siAdditionally, when using microarray data to establish significant correlations among gene expression profiles, almost invariably the information obtained with probes covering different gene portions is averaged . Though Coexpression-based Linking of Orthologous ESTs) a new data mining method for the identification of transcripts showing evolutionary conserved co-expression in cDNA microarray datasets. This approach is based on the pairwise comparison of data from two species. The predictive capability of the method was proved by comparing human with mouse data. Our results show that CLOE is a valuable tool for biologists that can be used to identify putative partners for genes of interest and/or to predict some of their functional properties.In this study we describe CLOE   . We then generated the respective ranked lists, subdivided them in 1% rank intervals and analyzed the number of orthologous pairs in corresponding rank intervals. As a control, we performed the same analysis on an equal number of randomly chosen (and hence non-orthologous) human-mouse EST pairs. The analysis was repeated three times with essentially identical results.-94, chi square test). The difference was still very significant in the 2% rank interval, even though with a much higher p value (p = 1.3·10-10), but was not detectable below that threshold. Interestingly, a slight enrichment was also observed in the last rank interval . The latter observation is consistent with the previously noted fact that negative correlations tend to be less common and significant than positive correlations [As shown in Figure Saccharomyces cerevisiae [Drosophila melanogaster [Caenorhabditis elegans [We next investigated the effectiveness of our approach, by comparing it to single and multiple organisms co-expression analysis. To address this point, we analyzed the ability of the three methods to predict known physical and functional interactions among mammalian proteins. Protein-protein complexes have begun to be determined on a genome-wide scale only for revisiae , Drosophnogaster  and Caen elegans , but no  elegans  . On the other hand, the average percentage of CLOE links that correspond to a documented protein-protein interaction was 6.6 %, i.e. approximately 5 times higher than that obtained with the single organism method  ratios.Within these datasets, the number of ESTs representing a given transcription unit, as well as the number of valid experiments for each EST, are highly variable. While this feature would pose serious problems, if one should attempt to average the data of all the probes belonging to the same gene, it may offer extremely valuable information when every EST is considered independently, since each clone explores the expression properties of a particular group of exons, in a particular combination of experimental situations.Non-correlated or anti-correlated expression between two well-represented EST probes belonging to the same gene would strongly suggest that they correspond to alternatively expressed transcripts. For this reason, we decided not to merge the data of probes belonging to the same gene, but to treat separately every EST probe in the datasets.The choice of the most representative EST for the genes of interest represents a particularly critical aspect of our procedure. If interested to a single gene, the more exhaustive solution to this problem would be to generate and evaluate a list of candidate partners for every possible pairwise combination of ESTs probes. However, this would greatly complicate the analysis should one be interested in analyzing the potential partners of many genes. An alternative possibility is to focus, for every Unigene cluster, on the most representative ESTs, i.e. the probes with the highest number of experiments. For these reasons, the decision about what ESTs to analyze is left to the end user.Our implementation of the method can accept as input both a UniGene cluster ID or the results of a BLAST search performed with the sequence of interest against the EST database. In both cases, it retrieves a list of all probes found in the two datasets for the orthologous UniGene clusters.To help the user decide which ESTs to analyze, the program provides basic information about all the EST probes representing the gene of interest in the two datasets. Moreover, to help the user identify the most representative EST probe in each dataset, i.e. the probe with the highest number of experiments, it also provides the number of valid data points for every probe. Finally, to assess the redundancy of the information provided by the different probes, i.e. whether they represent different experiments and display similar/different expression profiles, the program calculates, for every pair of probes belonging to the same UniGene cluster, the number of common experiments and the Pearson correlation coefficient between their expression profiles.r) with every other EST in the respective dataset. The raw r is the normalized for the number of common data points (n) obtained for the analyzed ESTs. This is done by multiplying r for , since the statistical significance of r is a function of the product ·r. Such normalization is particularly important when, as in our case, n has a very wide range of variation. The ESTs are then ranked by decreasing normalized score. Finally, a user-defined top percentage of the two ranked lists is compared to identify those ESTs that are associated to the same InParanoid cluster ID. A non-redundant list of the positive InParanoid clusters, sorted by the average highest rank obtained in the two organisms, represents the main output of the program ;readme.txt (detailed instructions for using our program).MP: development of most of the software, execution of the described analysis.PP: development of the routines used to calculate the normalized Pearson, supervision of the statistical analysis.LS: assessment of the biological significance of results.FD: supervision of the project, manuscript writing.


This article reports a cDNA collection representing more than 2000 new, full-length transcripts from a high-quality cDNA library. A large number of cDNA inserts were sequenced from a high-quality library of chicken bursal lymphocyte cDNAs. Comparisons to public gene databases indicate that the cDNA collection represents more than 2,000 new, full-length transcripts. This resource defines the structure and the coding potential of a large fraction of B-cell specific and housekeeping genes whose function can be analyzed by disruption in the chicken DT40 B-cell line. Large-scale genomic and cDNA sequencing projects have revealed thousands of new genes whose open reading frames (ORFs) are highly conserved during vertebrate evolution, but whose precise cellular functions remain unclear. Although functional analysis by gene disruption is possible after transfection of murine embryonic stem cells and the breeding of knockout mice , these win silico gene structure prediction methods have a high error rate and often do not correctly annotate the intron-exon structure of genes. Only full-length cDNAs unambiguously define the boundaries of the transcription units within whole-genome assemblies and cloned full-length cDNAs are also of immense practical value to complement mutant phenotypes and artificially express the encoded protein [The recent release of the chicken genome sequence  greatly  protein . For the protein ,12. Alth protein  and othe protein , relativHere we describe a project to sequence and characterize a large number of full-length cDNAs from bursal lymphocytes. The corresponding genes are likely to be expressed in DT40 and this should facilitate their analysis by targeted gene modifications. In combination with the recently released cDNAs from other tissues , the burThe overall strategy for producing the greatest possible number of new full-length cDNAs expressed in bursal lymphocytes is outlined in Figure The plasmids corresponding to the remaining 2,796 ESTs were chosen for full insert sequencing by bidirectional primer walks. Once the end of the walks had been reached, the sequences of the full-length cDNA inserts were assembled. From the BLAST search results the most likely methionine start codon was assigned to each sequence. About 15% of the cDNA sequences showed evidence for premature frameshifts in the form of short ORFs and stretches of conserved sequence in a different frame further 3'. If overlapping ESTs were found in the public databases, the cDNA sequences were edited to correct the likely reverse transcription error, otherwise these sequences were discarded.A total of 2,272 high-quality chicken full-length cDNA clones were sequenced and assembled, manually annotated with respect to their likely translation start codon and deposited both at The Bursal Transcript Database website  and in tThe most remarkable feature noted in the analysis of 5' UTRs of the bursal cDNAs is a very high GC content (67%). This supports the observation that the GC content of 5' UTRs is particularly high in warm-blooded species . On the The accurate prediction of the translation start codon remains difficult and in some cases our annotations remain tentative. Sequences surrounding the translation start codons are not random and in mammals match the consensus GCCRCCaugG (where aug is the start codon and R is either A or G) . The mosA detailed analysis shows that the riken1 collection of cDNA sequences contains 4,406 AUG codons upstream of the annotated translation start codons in 2,218 of the bursal cDNAs. Nine hundred one of these alternative start codons were in the same reading frame as the annotated ORF. An in-frame stop codon within the 5' UTR region was present downstream of 501 of these 901 alternative start codons. The total number of ORFs present in 5' UTR regions of riken1 cDNAs was 1,289.We have checked whether the context of the annotated AUG start codons differs from the context of the alternative upstream AUG sequences of the bursal cDNAs. We therefore extracted 10-bp long sequences surrounding the annotated start codons and the alternative upstream AUGs and visualized sequence variability using the sequence logo software  Figure . The annAll full-length cDNAs were compared to the collection of transcripts predicted from the chicken genome sequence by the Ensembl system . The traFigure When compared to available chicken ESTs or cDNAs in the public databases, some of the bursal cDNAs showed significant structural differences most likely due to differential transcript processing. In addition, the bursal cDNA collection has been used to define a large number of intragenic single-nucleotide polymorphisms (SNPs) .All full-length cDNAs were compared to the Pfam database , which sTables There are 22 full-length cDNAs in our collection containing Pfam domains annotated by the GO term 'molecular function unknown'. Experimental information concerning the molecular mechanisms of action is very sparse or nonexistent for proteins sharing these evolutionarily conserved domains. Highly similar human proteins exist for the chicken proteins, an example being the human protein BM02. Taking into account the ease of targeted genome modification and availability of numerous functional assays, the DT40 cell line is an attractive model system to provide first insights into the functions of the evolutionarily conserved domains described above.All the full-length cDNA sequences are stored within the Bursal Transcript database . This dain vitro or for complementation of mutant DT40 phenotypes with the added convenience that the cDNA sequences are not only known, but also available as cloned pieces of fully sequenced DNA.The cDNAs from bursal lymphocytes represent one of the largest full-length cDNA collections in the chicken, comprising about one third of all currently available, experimentally verified transcripts and will be of general interest to researchers using the chicken as an experimental model as well as to the poultry industry. The resource has already been integrated with the chicken genome sequences to build a unigene catalog , to defiLMO1 locus. This sequence was present as a stuffer of the lambda vector used for the library construction and the clones containing it were removed from further analysis. In total, the 5' single-pass sequencing of 14,976 clones yielded 11,116 high-quality ESTs of the riken1 library.The riken1 library was synthesized from mRNA of 2-week-old CB strain bursal lymphocytes using the biotinylated cap trapper method ,31. The BLAST searches against the 'All non-redundant GenBank CDS' database showed that approximately 80% of the 5' EST sequences matched GenBank entries with a score of at least 50. This score threshold was chosen because it allowed us in most cases to align the putative start codon of the query sequence to the EST. These sequences were chosen and clustered  to removSufficient plasmid template for numerous sequencing reactions was prepared from the clones corresponding to the selected ESTs. All plasmids were then sequenced with a primer complementary to a plasmid sequence 3' of the cDNA insertion site. Subsequently, custom-made 20-mer primers based on available sequences were used for sequencing until the 3' and 5' ends of the cDNA inserts were reached. All sequences were processed as described previously , except Gallus gallus ESTs from the public databases. If the short ORF could be corrected by adopting the sequence of an overlapping EST, the cDNA sequence was edited. The type of editing was recorded and the corresponding riken1 clone was annotated as likely to be defective. In total, 293 cDNAs were removed because either a likely artificial frameshift could not be corrected by using sequences of overlapping ESTs or they contained multiple stop codons in all three reading frames or they showed evidence for unspliced introns. All cDNA clones are freely available upon request to the corresponding author and their sequences have been submitted to the EMBL public database (accession numbers AJ719267-AJ721138 and AJ851370-AJ851825).The integrity of the conserved ORF within each assembled cDNA sequence was manually examined by inspecting BLAST search results against the public protein and EST databases. To facilitate this task, a new EstSet module was added to FOUNTAIN . The useThe sequences surrounding the annotated start codons (10 bp upstream and downstream) were exported and submitted for information content visualization by the WebLogo software . Subsequ-6) run on the binary database files downloaded from the National Center for Biotechnology Information (NCBI). Functional classes were assigned according to Pfam to GO mapping provided by the InterPro database. The XML information exchange standard was used to interface the BLAST program outputs with the FOUNTAIN package.The riken1 cDNAs were compared with the collection of predicted chicken transcripts downloaded from the Ensembl ftp site  using th


PLoS Biology, Thomas Bartman et al. use the powerful tools afforded by zebrafish genetics to dissect the early steps of heart valve formation. In the process, they provide evidence for a causal relationship between the early function of the heart and its final structure.The heart starts beating and pumps blood through the body long before it has achieved its mature architecture. In theory, this provides a chance for cardiac function to sculpt cardiac structure, an intriguing possibility for developmental biologists, and one of potentially great clinical import for cardiologists seeking to identify the causes of  cardiac anomalies. In this issue of At the time of its first beat, the vertebrate heart is little more than a tube, lined on its outside by a myocardial cell layer whose contractions (the heartbeats) power blood flow, and on its inside by an endocardial cell layer, an extension of the inner wall of the connecting blood vessels. What it lacks still are valves and septae, the fibrous gates that subdivide the mature heart into atrial and ventricular chambers, and control the directionality of blood flow. These structures derive from the endocardium in a process that begins—shortly after the establishment of blood flow—with the local accumulation of endocardial cells into what are known as endocardial cushions (ECs).cardiofunk (cfk), which was devoid of ECs. Genetic mapping of the cfk mutation revealed a single sequence change in a gene encoding a novel actin molecule that is most closely related to the sarcomeric actins found in sarcomeres, the contracting organelles of muscle cells. The result was surprising because contractions are not a property of endocardial cells. Using RNA detection assays, the authors show that the cfk gene is in fact expressed in the myocardium, rather than in the endocardium. It therefore appears that the inability to form ECs in cfk mutants does not reside in the endocardium per se, but is an indirect consequence of a myocardial anomaly.The zebrafish lends itself well to large-scale genetic screens, and powerful genomic tools are now available to efficiently identify the gene affected by any mutation. The authors have used genetic screens to identify several mutations that affect early cardiac function or morphology. Heart anomalies are easy to detect in zebrafish, and can be examined in real time and in live specimens because the embryos develop outside the mother and are fully transparent. Using a fluorescent molecular marker highly expressed in the ECs, the authors narrowed in on mutations that result in valve defects, and identified a mutant they named cfk mutation introduces a single amino acid change in the actin protein, and through detailed biochemical analyses, the authors show that the mutant actin is impaired in its ability to assemble into fibers in vitro. What might be the consequence in vivo? The authors note that cfk mutants display abnormal heart contractions prior to the development of their EC defect. Support for the notion that myocardial contractions are required for EC formation comes from the examination of silent-heart (sih) mutants. sih mutants, which lack a heatbeat, have been shown to harbor a mutation in troponin T, one of the motors of actin contractions; the authors find that sih mutants also fail to develop ECs. The mechanisms linking myocardial contractions and cushion formation remain unclear. Blood flow may be a trigger, though the authors find that ECs can develop even in the presence of pharmacological compounds that abolish it. The characterization of additional mutants should help answer this question.The Valve or septal defects represent 40% of cardiac anomalies in humans. Bartman and colleagues suggest that, by analogy with zebrafish, some may result from congenital defects affecting very early myocardial function. Their work thus opens new avenues for the early detection of human cardiac malfunctions and malformations.