C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps).Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server  and their sequences as additional files with this paper.We have performed an analysis of CTLD family composition in Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates.The vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the  Its m and ice -9. In se and ice -12.C. elegans [D. melanogaster [Three studies using the whole-genome approach have been published analyzing the distribution of the superfamily in  elegans , D. melanogaster  and humanogaster . An earlnogaster  attemptenogaster  providedA number of fish CTLDcp sequences have been reported separately in the literature and public sequence databases. The best-studied and most distinct set are serum antifreeze proteins (AFPs) from several cold-water-living species ,16,17. TSalmo salar serum lectin (SSL) [Gillichthys mirabilis (long-jawed mudsucker), annotated as "mannose receptor C" [Apart from AFPs, several other soluble bony-fish CTLDcps have been described: 5 isoforms of in (SSL) , three cin (SSL) , skin muin (SSL)  and two in (SSL) , two lecin (SSL) , a carp in (SSL) , goldfiseptor C" .Fugu [Known membrane-bound CTLDcps from bony fishes include a polycystic kidney disease protein 1 (PKD1) orthologue from Fugu , a rainbFugu , and a sFugu . AlthougFugu  do not cThe only known CTLDcp sequence from cartilaginous fishes is a tetranectin homologue from reef shark cartilage .Fugu rubripes genome, available since 2002 [The nce 2002 , is the nce 2002 .Fugu rubripes genome assembly are publicly available. The second version of the assembly (v.2), constructed from 4.1 million sequencing reads (5.4 X sequence coverage), was reported in the original publication announcing the completion of the Fugu rubripes genome sequencing [Fugu rubripes genome annotation. Although EnsEMBL and JGI annotations and genome browsers are different, they share the same gene and transcript structure predictions created by the EnsEMBL pipeline.Currently three versions of the quencing . The thiquencing . The JGIquencing  are the Fugu genome sequence targeting different protein families have been published recently [Fugu rubripes genome.Several analyses of the draft recently -39, whicAt the time this study was started, annotation of the v.3 assembly was not yet published; hence, most of our analysis was done with v.2 of the assembly and later mapped to the v.3 assembly. From our experience, there is no substantial difference between v.2 and v.3 assemblies in the amount of sequence information and its quality, although the v.3 assembly contains longer scaffolds due to more extensive linkage. Despite very high similarity at the sequence level, the v.3 assembly annotation contains no history information that would provide links between contigs, genes, and transcripts in the second and the third versions of the assembly. None of the stable identifiers for genes, transcripts or peptides from v.2 are present in v.3. This information cannot be generated by usual procedures used in EnsEMBL (e.g. ID Mapping Application, which is a part of EnsEMBL Java APIs) and has to be obtained by sequence comparisons. This lack of correspondence creates difficulties for the sequence analyzer and end point reader. To facilitate analysis and allow comparison, references to feature identifiers for both of the assemblies are given in Table Fugu rubripes, most of the EnsEMBL gene structure predictions are based on homology with known protein sequences from other organisms, mostly mammals. We expected a significant fraction of CTLDcps to be conserved between fish and human, and, therefore, to be predicted correctly by EnsEMBL in the Fugu genome. So our first approach to detecting Fugu CTLDcps was to search a sequence database of predicted Fugu proteins with a hidden Markov model (HMM) for the CTLD. This search returned 69 significant matches. Some of the identified genes had a description assigned to them, apparently derived from the description of the sequence they were found to be homologous to. These descriptions, however, could not be used as a reliable basis for assigning orthology and paralogy relationships. For example, a sequence, which we later identified as an Endo180 orthologue (SINFRUG00000058766 in v.2 assembly annotation) is described as "80 KDA SECRETORY PHOSPHOLIPASE A2 RECEPTOR PRECURSOR PLA2", while another gene, which we designated as an aggrecan orthologue (SINFRUG00000069597 in v.2 annotation) was annotated as "ADRENOLEUKODYSTROPHY PROTEIN (ALDP)". Therefore, we reviewed domain architecture and sequence similarity matches for each of the sequences found to verify phylogenetic relationships.Due to almost complete lack of cDNA or EST sequences for Fugu CTLDcps were used to initially cluster the set of Fugu proteins and detect approximate orthology/paralogy links. Inparanoid has an important advantage over phylogenetic tree reconstruction software, as it does not require a multiple alignment of sequences but creates a distance matrix of the local pairwise alignments. This method assigned putative human orthologues to 25 Fugu proteins. Orthology relationships for the other 44 sequences from the set were established by individual analyses.The results of Inparanoid  comparisFugu CTLDcps were soluble proteins, whereas very few human CTLDcps are. Simple comparison with the GenScan [Fugu CTLDcps. GenScan predictions, in turn, could not be used as a basis for our analysis because they sometimes contain regions that are absent from human or mouse orthologues, and often merge neighboring genes. Another general problem was observed with proteins that had a previously unknown domain architecture (see below). In such cases individual domains were split into separate gene models.While analyzing phylogenetic relationships predicted by Inparanoid, we discovered several systematic and sporadic mistakes in the EnsEMBL gene predictions. The most widespread mistake was a failure to include exons encoding TM domains into gene structure prediction. Consequently, almost all EnsEMBL-predicted  GenScan  featuresFugu genome shows that, similarly to the human and mouse genomes, the selectin cluster is well conserved and contains all three selectin genes in tandem (SELE, SELL, SELP), located on scaffolds 1045 (32046–41921) and 166 (83937–93826) in the v.2 and v.3 Fugu genome annotations, respectively. However, the EnsEMBL annotation contains a prediction of two overlapping genes , one of which is located in the intron of the other .After all these searches, we had identified a set of 94 ps Table , which iFugu CTLDcps using the classification scheme for human CTLDcps based on domain composition; this comprises 14 groups [Fugu CTLDcps that did not have mammalian homologues we detected a distinct group of soluble dual-CTLD sequences, which we have called F1  group. Gene structure prediction for members of the U group is the lowest in quality, due to lack of supporting evidence apart from similarity to CTLD sequence profiles and GenScan predictions.We have clustered 4 groups . Link/PT1 Figure . The remFugu genome , while in human this family has 3 members encoded by a gene cluster on Ch 17 (ASGR1, ASGR2, HML2). The Fugu sequence was identified as an HML2 orthologue by phylogenetic analysis based on the alignment of CTLD sequences. Another clearly identifiable member of group II is the orthologue of scavenger cell receptor C-type lectin (SRCL), which is duplicated in Fugu and is 50% identical to the human SRCL. The rest of the group II Fugu CTLDcps (DC-SIGN-F1 – DC-SIGN-F8, XLCMCL) do not have clearly identifiable orthologues among known human CTLDcps, although phylogenetic analyses based on CTLD sequence alignment indicate that they are homologous to members of the group II subgroup containing DC-SIGN, Mincle and Dectin-2, which also appear as top hits in BLAST searches. However, this subset of group II Fugu sequences co-clusters in phylogenetic trees and is not similar enough to any tetrapod sequence to establish orthology. Four of the sequences (DC-SIGN-F2, DC-SIGN-F3, DC-SIGN-F4, DC-SIGN-F5) are located in a cluster on scaffold 75 in the v.3 assembly. Two members of the subgroup (DC-SIGN-F1 and DC-SIGN-F6) have unstable placements in phylogenetic trees, and may appear on a branch containing human/mouse group V sequences, if the latter are included in the alignment. This association is, however, unstable and may be due to mistakes in CDS prediction or phylogeny reconstruction. Alternatively, it is possible that these sequences are homologous to the common predecessor of group V and group II CTLDcps.We found only one representative of the asialoglycoprotein receptor (ASGR) family in Fugu has two collectins, there are no orthologues for mannose binding proteins (MBPs) or pulmonary surfactant proteins (PSP), which are the best studied members of the group in human. Both of the Fugu collectins (COLEC10, MGC3279) are well conserved compared with their human orthologues and co-cluster with them in phylogenetic trees. No functional information is available for the novel collectin MGC3279, which was discovered in a large-scale cDNA sequencing project and maps to chromosome 2p25.3 in the v.31 NCBI assembly of the human genome, but the exceptionally high level of conservation between human and fish (~76% identity) strongly suggests that it is functional and important in both organisms. COLEC10 (collectin liver 1, CL-L1) was originally reported as limited to birds and mammals [Although  mammals  based onFugu and have the same genome arrangement.As already mentioned, all three selectin genes found in other vertebrates are present in Fugu orthologues for all four human group VI members: macrophage mannose receptor (MManR), DEC-205 (CD205), phospholipase A2 receptor (PLA2R) and Endo180. In addition, there are 5 sequences (MManR-F1 – MManR-F5) showing high similarity to members of the group, four of which do not contain the minimal number of CTLDs (8) present in the known group VI sequences  with multiple CTLDs clustered in two groups: 5 at the N terminus and 10 (7 in v.3 assembly) at the C terminus, with an LCCL domain [named after its presence in Limulus factor C, cochlear protein Coch-5b2, and late gestation lung protein Lgl1; [There is one more potential group VI member in in Lgl1; ] and a cin Lgl1; , while Fin Lgl1; . AlthougFugu orthologues for all known human members of groups VIII and IX. One member in each of these groups is duplicated in Fugu (Layilin and Tetranectin).We have identified Fugu PKD1L2 sequence is very similar to the sequences of human and mouse PKD1L2 cDNAs, even though the latter were deposited in GenBank at the beginning of June 2003 – after GenScan prediction. This example indicates that ab initio GenScan predictions on the Fugu genome can be very accurate.In addition to the PDK1 orthologue, which was identified previously , there iFugu (EMBPL). Although the similarity between the mammalian and the fish sequences is very low (~30% identity), several observations suggest that the Fugu EMBP-like sequence is an orthologue of one of the two mammalian genes. First, the overall domain architecture of the fish protein is similar to that of the EMBPs. Although the fish CTLD has a neutral pI (7.1), it is preceded by a 30-residue peptide with a predicted pI of 3.62, analogous to the longer acidic neck of the mammalian EMBPs. In the existing classification [We found a single sequence resembling mammalian eosinophil major basic proteins (EMBPs) in fication , the preFugu, with one gene duplicated (C1qRP). In addition, a novel member of the family conserved between Fugu and mammals was identified, which we named CETM (for CTLD, EGF, TransMembrane domain) , almost identical to each other and positioned in tandem on scaffold 1930 in the v.2 assembly. In v.3 of the assembly, the AFPL-encoding region was rearranged and one of the AFPL loci disappeared. The intron-exon structure of the CTLD-encoding region is identical to the structure of the sea raven antifreeze protein gene  with thrFugu and often are highly conserved between fish and human ; we will consider them as a single group despite their different domain architectures. Predicted members of the CD44 family ), however, are much more divergent from their human homologues, and it is not clear whether the two loci found in Fugu are orthologues of the two human genes or paralogues which arose by duplication of an ancestral gene.All link domain-containing proteins identified in mammals are represented in Fugu, we were expecting to also find duplicate copies of all HAPLN members. However, orthologues of only three HAPLNs were found (CRTL1, BRAL1, HAPLN3), two of which are linked to hyalectans in the same way as in mammalian genomes (CRTL1 with Versican, BRAL1 with Brevican). The state of the assemblies does not allow to determine conclusively whether HAPLN3 is linked to Aggrecan or not. Only two of the Fugu lectican gene duplications are accompanied by corresponding HAPLN genes: Aggrecan-F1 is linked to HAPLN3-F1 and the CRTL1 paralogue is present downstream to Versican-F1 in two tandem copies (CRTL1-F1 and CRTL1-F2). In neither version of the assembly could the HAPLN4 homologue be identified downstream to Neurocan or Neurocan-F1. Sequence conservation levels within the HAPLN proteins compared with their human orthologues is quite high (e.g. 76% identity for CRTL1).In a recently published comprehensive study of another family of the Link group, the hyaluronan and proteoglycan binding link proteins (HAPLN), four homologues were identified in vertebrates  each linked to one of the four lecticans . As all Fugu sequences is only moderate (~30%). Another homologue with the same domain structure and similarity to the F1 group members, which was returned as the top-scoring hit by BLAST searches in the nrdb, is the SCARF2 protein from a planarian Girardia tigrina [Drosophila (NP_609962), which presumably corresponds to the single member of group B in the Drosophila CTLDcp classification of Dodd and Drickamer [The members of this group are soluble proteins with two or three CTLDs, which we initially characterized as fragments of putative macrophage mannose receptor paralogues. However, phylogenetic analysis showed that these proteins constitute a separate group, with no mammalian orthologues detectable in sequenced genome and protein databases. The domain structure prediction is confirmed by three zebrafish cDNAs (CAE17649, CAE17650, CAE17651), which have the same domain organization, although conservation between zebrafish and rickamer , was alsFugu orthologues for several uncharacterized mammalian CTLDcps (Bimlec, MGC3279, KIAA0534, CETM, SEEC, CBCP, NLSLH) that are well conserved between Fugu and mammals. Most of the predictions were supported by mammalian cDNA sequences from public databases, but for two of them (NLSLH and CBCP) no full-length cDNA from any organism was found in DBs. The high level of genomic sequence conservation over evolutionary time from fish to human, as in the case of NLSLH, and the presence of partial cDNA and EST sequences from rodents and human, as in the case of CBCP, were strongly suggestive that the predictions are correct. The novel CTLDcps that could be attributed to one of the 14 known groups have been discussed in the preceding sections for the corresponding groups; those that do not fit into the existing classification are described below.Discovering novel superfamily members in existing database sequences is one of the most important and exciting outcomes of a systematic computer-based study such as this. We predicted putative Fugu (~50% identity), and the compact structure of the predicted Fugu gene. CBCP has been placed in a new CTLD group, XVII; its domain structure is shown in Figure A large ~2100 aa) proteoglycan (CBCP), containing a set of chondroitin sulphate proteoglycan (CSPG) repeats , which a100 aa prSCP, EGF, EGF, CTLD-containing protein)  was sequenced in the NEDO high-throughput sequencing project. The predicted Fugu SEEC is 63% identical to the human sequence. The sperm-coating glycoprotein (SCP) domain, which is present in a broad set of organisms from yeast and plants to mammals, but whose function is unknown [Another protein with a novel domain organization, whose prediction is strongly supported by available cDNAs, is SEEC ( unknown , is rare unknown , and SEEFugu (NLSLH and NLSLH-F1) but only moderately conserved (32% identity) between Fugu and human. The putative human orthologue is located on Ch1q25.1 about 18 Mb further from the centromere than the selectin cluster and is supported only by EST sequences (AA912157, AA889574), but not cDNAs. No conserved domains except for the CTLD could be detected in the human and Fugu NLSLH loci so, if the predictions are correct, NLSLH is a soluble single-CTLD-containing protein. Carbohydrate-binding motifs are not present in the NLSLH and NLSLH-F1 CTLDs.A predicted protein named "novel L-selectin homologue" (NLSLH) because its CTLD is most similar to selectin CTLDs is duplicated in Finally, a type I transmembrane protein Bimlec, whose prediction is supported by a full-length human cDNA, was placed in a new group XV.Fugu-specific CTLDcp paralogues  were between 1.5–2.5, which indicates a complete saturation of the synonymous sites Figure . Ks valuFugu CTLD sequences with their vertebrate orthologues present in GenBank, and built linearized phylogenetic trees based on the alignments. As human and mouse sequences were invariably available, the divergence time between these two species [96 Myr; 60] was used to calibrate the clock, together with the divergence time between Actinopterygii and Sarcopterygii [430 Myr; 32]. Symmetrical tree topology ((H, M) (F, F1)), expected for a Actinopterygian-specific duplication, was revealed by at least one phylogeny reconstruction method we used for the following six homologue groups (data not shown): brevican, neurocan, MManR, SRCL, tetranectin and HAPLN3, with duplications dated 369, 284, 397, 377, 360 and 312 Myr, respectively. A typical tree with symmetrical topology is shown for MManR in Figure In order to date the duplications based on molecular clock measurements, we aligned duplicated Fugu rubripes has some limitations, as the genomic sequence is incomplete, fragmented and sometimes misassembled, and the expressed sequence information is scarce. On the other hand, many of the genomes that are currently being sequenced will be released and remain for sometime in the same state as the Fugu genome data are now. Indeed, more than a year after the initial release [Fugu genomic data [v.3 assembly and EST sequencing project; [A systematic study based on draft-quality whole-genome data for an organism like  release  very fewproject; ] have beab initio predictions combined with manual curation and interspecies comparison have proven to be very accurate (e.g. see PKD1L example), thanks to the compactness of the Fugu genome, smaller ratio between intron and intergenic region sizes compared with mammalian genes, wealth of data for comparative analysis etc. We do not expect that sequencing the remaining 5% of the Fugu genome, which is mostly heterochromatic regions, will lead to discovery of many new CTLDcps. From the comparison of the Fugu CTLDcp repertoire discovered by us and found in other fish species independently, the only surprising omission in our results is a MBP orthologue. MBP sequences have been found in several other fish species. Their absence in Fugu may represent a bona fide gene loss. As to the fragmentation, only a few of the CTLDcps are split between scaffolds, namely versican and some MManR paralogues  and VII (lithostathine) in the Fugu genome is at odds with the conclusions of two studies describing group V CTLDcp evolution in chordates. A recent paper describes possible CD94 homologues (cichlid killer cell lectin receptor, cKLR) in bony fishes Paralabidochromis chilotes and Oreochromis niloticus, which are encoded by a large multi-gene family with at least 10 members [Our conclusion on the absence of group V CTLDcps in the  members . Another members .2+/carbohydrate-binding residues, and phylogenetic analysis based on the CTLD alignment. The latter consideration is mentioned as the most important one. However, as the authors themselves note, bootstrap values for placing cKLR on the group V branch, are "low to moderate". Indeed, we found that in phylogenetic trees built using different methods (maximum parsimony, distance estimation method with PAM matrix followed by neighbor-joining tree reconstruction, maximum likelihood) from the ClustalW alignments of cKLR sequences with group V and group II CTLD sequences from Fugu, mouse and human, cKLR placement is unstable. As shown in Figure Fugu-specific subset of group II CTLDcps (DC-SIGN-F1 – DC-SIGN-F8), most of which do contain residues required for Ca2+/carbohydrate binding. On a tree built by the maximum parsimony method, we found cKLR on a separate branch equally related to group II and group V sequences (not shown). Also, a BLAST search with the complete cKLR sequence (GI 31789959) in the non-redundant NCBI protein database returns members of the ASGR subgroup of group II as top matches. Therefore, we judge that sufficient support for assignment of cKLR to group V is lacking and the question of the presence of the NK-cell receptor family in fishes is still open.The decision by Sato et al.  to assigAs to the putative CD94 homologue from tunicates, it is indeed more similar to CD94 than to any other CTLDcp. However, the low level of sequence homology and the lack of evidence for existence of group V CTLDcps in more advanced taxa does not allow a confident statement that the sequence from tunicates is a CD94 orthologue, rather than a result of convergent evolution.Mycobacterium tuberculosis (reviewed in [Fugu may reflect a larger role for innate immunity in host defense in lower vertebrates. Interestingly, multi-copy clusters comprising at least 10 genes encoding close cKLR homologues were identified in another cichlid fish species Oreochromis niloticus [Unlike pairwise unlinked duplications (see below), tandem duplications and other gene family expansions are limited to two groups, namely the DC-SIGN subgroup of group II and MManR. In mammals, members of these subgroups play an important role in innate immune responses. In particular, DC-SIGN is actively studied due to its ability to bind and internalize a broad range of bacterial and viral pathogens, including HIV-1 and iewed in ), while iewed in . Expansiiloticus , which sFugu collectin group – another CTLD group directly involved in innate immunity in mammals. Moreover, the mannose binding protein (MBP), which is the best-studied mammalian collectin involved in lectin complement activation pathway, was not detected by us. The absence of MBP orthologues in Fugu is rather puzzling, as MBP sequences have been found in several other fish species , and arsequence , althougequenced ; they hana picta  or lack  roretzi .Fugu (F1) may be the first vertebrate group that has detectable homologues in invertebrates. Alternatively, it is possible that none of the Fugu F1 group members are in fact orthologous to the invertebrate sequences, as sequence similarities are only moderate (~30% ID) and the domain architecture is simple and could have evolved independently in different lineages. However, several observations suggest that at least F1 members from Fugu and zebrafish and SCARF proteins from Girardia tigrina evolved from the same predecessor. First, similarity levels between fish sequences and between fish and planarian sequences are about the same. It is unlikely that the fish sequences are unrelated, which implies that F1 members are evolving quickly, and only major structural features of these molecules are under selective pressure [Fugu (V and VII) have lost sugar-binding properties. This is also the case for the antifreeze proteins from fish and snake venom CTLDcps, which have only been found in the corresponding clades. Third, similar domain organization (two CTLDs, no transmembrane domain) is also observed in two other known groups of invertebrate CTLDcps: immulectins from various insect species [C. elegans, classified as group D1 by Drickamer and Dodd [Drosophila. Altogether, this indicates that domain structure alone cannot establish an evolutionary link between the fish and invertebrate sequences. Hence, the suggestive link between the F1 group fish members and the planarian and Drosophila proteins is even more interesting.Previous whole-genome studies of the CTLD superfamily in two invertebrates ,14 failepressure . Second, species ,72 and nand Dodd . DespiteThe existing classification of CTLDcps is generally accepted and popularly used in studies of the superfamily and recently has been updated . The claOur newly discovered CTLDcps, with a few exceptions, do not fit into the existing classification because of their unique domain architecture. We propose several new groups to accommodate the novel CTLDcps which have been found in both higher and lower vertebrates and are supported by cDNA sequences:• XV – Bimlec (type I transmembrane protein), which in phylogenetic trees is not placed on the same branch as group VIII sequences, has a distinct exon-intron structure of the CTLD region and a neck not similar to the neck region of the group VIII sequences;• XVI – SEEC, based on unique domain architecture;• XVII – CBCP, based on unique domain architecture;Additional groups may be required for the sequences not supported by sufficient expression data (NLSLH) and other sequences from the "unclassified" group whose presence in higher vertebrates is not clear. Also, clade-specific groups, such as fish antifreeze proteins (AFP), dual-CTLD sequences (group F1) predicted by us and so far identified only in fish, or snake venom CTLDcps which lack orthologues in other vertebrates, are required.It has been suggested previously ,48 that The hypothesis that whole-genome duplications were one of the main driving forces in vertebrate evolution, providing genetic material for increased diversity and progressive development , and thaFugu.Although many fish genes are indeed duplicated ,77,80-82Fugu genome 300–400 Myr ago. While we cannot determine definitively the nature of the duplications , a pronounced bias in the number of two-member paralogue groups strongly suggests that there was a single large-scale or whole-genome duplication event in fish.Our results clearly show that tandem gene copying is a mechanism of CTLD family evolution and led to generation of three gene clusters: DC-SIGN-F2 – DC-SIGN-F5 (4 genes), CRTL1-F1 and CRTL1-F2, and AFPL-F1 and AFPL-F2. Members of the two latter clusters are nearly identical and may be an assembly artifact. Twelve other duplicated genes are not linked in the current assembly and have sequences much more diverged than tandem duplicates. Of the 12 genes only MManR, which has 3 paralogues, is present in more than two copies. We consider this is important evidence in favor of a whole-genome duplication, as sporadic duplications cannot explain such a strong bias towards two-member paralogue groups. Unfortunately, the results of duplication time estimations are less conclusive as they give only a broad timeframe for the possible duplication events of about 300–400 Myr. As in the case of some other fish gene families reported previously ,62,87,88Fugu.Another interesting observation is that CTLDcp genes were either duplicated, or retained after a large-scale duplication, in a pronounced selective manner. One group (I) is duplicated completely, while in other groups only partial duplications are found. Interestingly, group I (lecticans), which in tetrapods contain four large (>2000 amino acids) proteins, very similar to each other in sequence and domain structure, is a good candidate for demonstrating the 2 R hypothesis. If the four lecticans arose as a result of the alleged two rounds of the whole-genome duplication early in vertebrate history, the fact that the family was also completely duplicated in fish and retained after the duplication appears very non-random and implies some functional explanation. In the human genome, all four genes encoding lecticans are located on different chromosomes (1, 5, 15 and 19), but it is not clear whether they are linked in Fugu genome we identified 7 group VI sequences, some of which are fragmented . Though in the d Figure . A wholeFugu rubripes. Although the sequence assembly is in the draft state and lacks physical mapping information and native cDNA sequences that could be used to make and verify gene predictions, the quality of the data is good enough despite these limitations to answer many important questions. Our study demonstrates that all but two groups of CTLDcps present in mammals are also found in fish, that most of the groups have the same composition as in mammals, and that the missing groups are the evolutionarily most dynamic ones involved in physiological processes that may be specific to higher vertebrates. We also identified at least one distinct fish-specific CTLD group, which could be the first known vertebrate CTLD group also found in invertebrates.We have performed an analysis of the CTLD superfamily composition in Fugu genome makes it an extremely convenient reference sequence for identification of new genes based on supporting similarity features, and we were able to identify and predict the structure of several new CTLD-containing genes highly conserved between Fugu and human. The new sequences are supported by cDNA and EST sequences from databases and have previously unknown domain architectures. We are now characterizing some of these sequences experimentally. We also show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event in fish.The compactness of the Corrected gene predictions are made available as a distributed annotation system (DAS)  resourceFugu rubripes genome assembly [Fugu CTLDcp genes discussed. Version 3 of the assembly can be found on the main EnsEMBL web site [Searches and gene annotations were done on version 2 of the assembly  downloadassembly ,92. Whenassembly  matches assembly  and on oassembly , which iweb site . The EnsFugu sequences found was compared with the 95% non-redundant set of sequences of human CTLDcps that could be found in the Entrez proteins database, using the Inparanoid program [We used a multi-step approach to find genes encoding CTLDs. First, a hidden Markov model (HMM) profile of the CTLD was used to scan a FASTA database of EnsEMBL-predicted genes with the hmmsearch program from the HMMER package . To dete program . All of Because of systematic and sporadic errors in EnsEMBL gene predictions, we had to manually revise the structure of each of the 69 genes encoding proteins detected by the HMM-based search. This was done using the Apollo genome annotation software  connecteFugu scaffold sequences with a CTLD HMM built in a global alignment mode. This was done to detect well conserved CTLDs while avoiding many false positives.a) Similarity features detected by GeneWise  search ob) Same as a), but with an HMM built in the local alignment mode; this was done to detect highly conserved fragmented CTLDs;Fugu scaffold sequences using all known human CTLD sequences; this was done to detect CTLDs that are less conserved;c) Similarity features detected by a TBLASTN search of d) ORFs encoding putative transmembrane (TM) domains. To create this track a database of all possible ORFs longer than 45 bp was produced and translated into protein sequence using the EMBOSS programs. This was then scanned with the TMHMM program  to detecTo verify whether there are CTLDs that were not covered by EnsEMBL gene predictions, we searched for all significant CTLD similarity features detected by GeneWise which do not overlap with any of the genes analyzed in the first stage. This step led to detection of 25 new CTLD-coding genes, including most of the ones that have previously uncharacterized domain organization. At the next stage we analyzed the loci with different CTLD similarity features detected by genewisedb search with a local alignment HMM. Finally, the features identified by BLAST and not overlapping with already detected genes were analyzed. This set of features mostly contained only partial CTLD matches.We translated both the new and already predicted gene CDSs into protein sequences and performed another Inparanoid comparison. Phylogenetic relationships were analyzed with the programs from the Phylip package . ClustalBioPerl  and EnsETo estimate the proportion of substitutions in synonymous sites, we aligned translated sequences of the duplicated CTLDcp-encoding genes with ClustalW, using either whole sequence or sequence for the CTLD-encoding region only, and built nucleotide sequence alignments based on the protein alignments. Ks estimations were performed with four methods: Lynch and Connery  and Li , both imFugu paralogues and their mammalian orthologues were made with ClustalW. The MEGA2 program [Duplication dating using the calibrated molecular clock approach was performed as in . Alignme program  was used program ,60,83.AFP, antifreeze protein;AFPL, AFP-like;Calx-β and CTLD-containing Protein;CBCP, CDS, coding sequence;CTLD, EGF, TransMembrane domainCETM, CTLD; C-type-lectin-like domain;CTLDcp; CTLD-containing protein;DAS, distributed annotation system;EMBP, eosinophil major basic protein;EST, expressed sequence tag;HMM, hidden Markov model;MBP, mannose-binding protein;Myr, million years;Novel L-SeLectin Homologue;NLSLH, PKD1, polycystic kidney disease protein 1;SCP, EGF, EGF, CTLD;SEEC, TM, transmembrane.ANZ carried out the bioinformatics studies and participated in the interpretation of its results. JEG conceived the study and participated in the interpretation of its results. Both authors participated in writing the manuscript and approved its final form.Fugu CTLD genes. Transcript sequences for re-annotated The file contains cDNA sequences (in FastA format) for all CTLDcp-encoding genes that were re-annotated by us (sequence identifiers starting with ANU).Click here for fileFugu CTLD genes. Protein sequences for re-annotated The file contains protein product sequences (in FastA format) for all CTLDcp-encoding genes that were re-annotated by us (sequence identifiers starting with ANU).Click here for file


Collecting and managing information is a challenging task in a genome-wide profiling research project. Most databases and online computational tools require a direct human involvement. Information and computational results are presented in various multimedia formats (e.g., text, image, PDF, word files, etc.), many of which cannot be automatically processed by computers in biologically meaningful ways. In addition, the quality of computational results is far from perfect and requires nontrivial manual examination. The timely selection, integration and interpretation of heterogeneous biological information still heavily rely on the sensibility of biologists. Biologists often feel overwhelmed by the huge amount of and the great diversity of distributed heterogeneous biological information..We developed an information management application called GeneNotes. GeneNotes is the first application that allows users to collect and manage multimedia biological information about genes/ESTs. GeneNotes provides an integrated environment for users to surf the Internet, collect notes for genes/ESTs, and retrieve notes. GeneNotes is supported by a server that integrates gene annotations from many major databases (e.g., HGNC, MGI, etc.). GeneNotes uses the integrated gene annotations to (a) identify genes given various types of gene IDs (e.g., RefSeq ID, GenBank ID, etc.), and (b) provide quick views of genes. GeneNotes is free for academic usage. The program and the tutorials are available at: GeneNotes provides a novel human-computer interface to assist researchers to collect and manage biological information. It also provides a platform for studying how users behave when they manipulate biological information. The results of such study can lead to innovation of more intelligent human-computer interfaces that greatly shorten the cycle of biology research. In a genome-wide profiling project, researchers usually select many sets of genes (or ESTs) for further investigation based on the computational analyses of the experimental data. For example, a set of genes is selected because they are clustered together based on their mRNA levels. To generate or support biologically meaningful hypotheses, researchers need to selectively and systematically collect information about the genes/ESTs from various sources. For example, huge amount of gene annotations have been accumulated over decades in distributed databases. In addition, there are plenty of online computational tools (e.g., BLAST , TMHMM , MEME 33, etc.) ® annotation files, which can be downloaded from the web site of Affymetrix.We maintain a server Figure  that intGeneNotes manages information by projects Figure . Each prGeneNotes offers many useful functions. The most attractive feature to biologists is the function for creating, collecting, and managing notes for genes/ESTs. Basically, there are three ways for creating/collecting notes. First, GeneNotes has an embedded Web browser, which allows the user to surf the Internet. The user can selectively record useful information segments shown in the Web browser or save a web page as a note. Saving a web page as a note is especially useful when the web page contains the non-text results (e.g., images, logos, etc.) generated by a remote computational server. GeneNotes automatically stores the sources of notes and allows users to trace back to the sources of notes later. Second, users can create text notes to summarize their thoughts/comments about genes/ESTs. Finally, users can create notes linked to local files (e.g., images, PDF files, word files, etc.). The user can directly modify a text note and add annotations to non-text notes. Each note has an editable title. A meaningful title helps the user recall what information the note contains. When the number of notes is large, it may become difficult for users to browse and locate notes. GeneNotes allows users to organize notes into directories, which can be defined by categories.Currently, GeneNotes runs on Microsoft Windows 2000/XP and uses Microsoft Data Access Components (MDAC) 2.7 to manipulate the note database. MDAC 2.7 provides the same Data Access core components as Windows XP SP1. Windows 2000 users need to download and install MDAC 2.7 or the latest version of MDAC, which are free at the web site of Microsoft. We are considering developing a platform independent version of GeneNotes and using other free database software such as MySQL .® annotation files, etc. It allows users to edit the project, e.g., add/delete gene lists, create/change the note database, add/delete Affymetrix GeneChip® annotation files, etc. The user can select a gene list in the project explorer. The selected gene list will be displayed in the gene list window. When a gene is selected in the gene list window, its properties (e.g., name/symbol/synonyms, RefSeq IDs, GenBank IDs, UniGene ID, etc.) will be displayed in the gene property window and its Gene Ontology annotation will be visualized in the Gene Ontology visualization window. The gene property window also displays a set of hyperlinks that are linked to the records of the selected gene in remote biological databases . All windows are dockable. Users can configure of the interface by dragging and dropping the window to virtually wherever they want. If a window is set as auto-hide, it will hide itself when it is inactive so that the computer screen can be used to display other windows.GeneNotes provides a novel human-computer interface for researchers to effectively and efficiently manage multimedia biological knowledge. There are many online databases (e.g., Entrez Gene , GeneCarProject name: GeneNotes.Project home page: Operating system(s): Windows 2000/XPProgramming language: C#License: Free for non-commercial usage.PH was responsible for the conception, design, implementation, and testing of the GeneNotes program. WHW was responsible for its conception, design, and testing and provided overall project coordination. Both authors have read and approved the final manuscript.


Escherichia coli K12 revealed an asymmetry in the distribution of transcriptional patterns along the bacterial chromosome and showed that spatial patterns of transcription could be modulated pharmacologically and genetically.Analysis of the transcriptional activity in  Escherichia coli K12 as a function of the position of genes on the chromosome.Although genes on the chromosome are organized in a fixed order, the spatial correlations in transcription have not been systematically evaluated. We used a combination of genomic and signal processing techniques to investigate the properties of transcription in the genome of Spectral analysis of transcriptional series revealed the existence of statistically significant patterns in the spatial series of transcriptional activity. These patterns could be classified into three categories: short-range, of up to 16 kilobases (kb); medium-range, over 100-125 kb; and long-range, over 600-800 kb. We show that the significant similarities in gene activities extend beyond the length of an operon and that local patterns of coexpression are dependent on DNA supercoiling. Unlike short-range patterns, the formation of medium and long-range transcriptional patterns does not strictly depend on the level of DNA supercoiling. The long-range patterns appear to correlate with the patterns of distribution of DNA gyrase on the bacterial chromosome.Localization of structural components in the transcriptional signal revealed an asymmetry in the distribution of transcriptional patterns along the bacterial chromosome. The demonstration that spatial patterns of transcription could be modulated pharmacologically and genetically, along with the identification of molecular correlates of transcriptional patterns, offer for the first time strong evidence of physiologically determined higher-order organization of transcription in the bacterial chromosome. Significant progress has been made recently in our understanding of how DNA is packaged into chromosomes, particularly at low compaction levels determined by supercoiling and/or protein-dependent condensation . Availabin vivo  and orgain vivo ; diffusiin vivo ; the chr[in vivo ,6; chrom[in vivo ,8; and a[in vivo ,9.Although the molecular bases of these structural features are not known, the bacterial chromosome can be viewed as a cellular organelle, whose dynamics may be coupled to the state of the cell. In turn, the state of the cell is reflected in the whole-genome transcriptional activity . TherefoCovariation in transcriptional activity along the chromosome determine spatial transcriptional patterns ,15. SuchEscherichia coli K12. Moreover, we demonstrate that such patterns are affected by genetic and environmental factors, thereby offering the first biologically relevant insights into the nature of the spatial organization of transcription in bacteria.Using signal processing and statistical techniques, we systematically examined transcriptional activity of genes as a function of their position in the bacterial chromosome. Here we report the discovery of stable, short- and long-range patterns in genome-wide transcription in 600 = 0.5 in LB or M9 medium supplemented with 0.2% glucose (cultures reached stationary phase at OD600 = 3.5 in LB medium and at 2.5 in M9 medium). Samples of total RNA extracted using hot-phenol method [We modeled transcriptional activity of the chromosome as a one-dimensional spatial series of transcript abundances. Transcript abundances were measured in cells grown in batch cultures to ODl method  were labl method , accessiIndependent of the growth conditions, the ACF could be characterized as a decaying function whose largest statistically significant portion assumes positive values and corresponds to relatively short gene-to-gene distances  and by the range of stable correlations . Following the two-parameter exponential fitting of at least eight independently measured ACFs, we determined that the transcriptional correlations decay by half in the rich medium over 7.5 kilobases (kb)  apart. To investigate such long-range transcriptional patterns in more detail we decomposed the original signal of transcript abundances into a series of harmonics. The frequency spectrum of transcript abundances is shown in Figure s Figure : 690-1 k-1 kb-1, 300-1 kb-1, 365-1 kb-1 and 555-1 kb-1. The wavelet spectrum also shows that patterns of transcriptional activity are not symmetrical with respect to the chromosome. The dominant patterns are localized largely in the left replichore  and appear to be bounded by the origin of replication. The most dominant pattern wave in transcriptional series, represented by a period of about 600 kb, spreads for 2.3 megabases (Mb), from the origin of replication to the terC site. The second most pronounced pattern (around 125 kb) consistent with the Fourier spectrum results from the transcriptional activity between the origin of replication and terG, about 1.3 Mb away. While significant components of the wavelet spectrum consistent with the Fourier spectrum were largely distributed on the left replichore, the scales unique to the wavelet spectrum were narrowly distributed along the scattered parts of the right replichore.The Fourier transform provides signal average characteristics and does not determine the frequencies at a particular spatial locality. To localize significant frequencies determined by Fourier transform and to find potentially significant local spikes of transcriptional activity, we subjected the spatial series of transcript abundances to a wavelet transform. The wavelet analysis revealed significant spectral components at the scales very similar to significant periods in the Fourier spectrum: approximately 125 kb and 600-700 kb Figure . In addip < 0.05) diminished by (in some case) 30 minutes with the drug . We rationalized that if transcription is not only inhibited, but modulated, globally, we might be able to track changes in the transcriptional spectra. We used a topoisomerase inhibitor, norfloxacin, to try to modulate transcription in the cell by inhibiting topoisomerase activities. As supercoiling and transcription in the cell are expected to be tightly coupled , we antiIn addition to modulation of the transcriptional spectra by non-equilibrium perturbations, we were interested in steady-state alterations in the process of transcription that would not be associated with irreversible changes in bacterial physiology. Such alterations may result from a compensation to a partial reduction in gyrase activity. A transient increase in transcription of the gyrase genes has been observed following inhibition of gyrase activity . We ratigyrA and/or gyrB transcripts. We characterized one spontaneous mutant of gyrase, resistant to 0.8 μg/ml of norfloxacin, which carries the D82G mutation in the gyrA allele and causes elevated levels of gyrA and gyrB mRNA in vivo and lowers supercoiling activity of DNA gyrase in vitro (K.S.J, Hiroshi Hiasa and A.B.K, unpublished work). This mutant had a normal rate of growth and cell density in stationary phase (less than 5% different from the isogenic wild-type strain in LB). As predicted, the compensatory transcriptional mechanism was not limited to transcriptional regulation of gyrase expression but had a global effect. Using microarrays we estimated that steady-state transcriptional activity of as many as 847 genes had changed in the mutant relative to the isogenic wild-type strain. The microarray results were in part confirmed by reverse transcription-PCR (RT-PCR) for 10 out of 10 randomly chosen differentially expressed genes with an observed change in transcript abundance of 50% or greater. The distribution of differentially expressed genes along the chromosome is shown in a histogram in Figure terE site through the terG site appeared to be most affected in the mutant.Using resistance to low concentrations of norfloxacin as a screening method, we selected mutants with increased levels of If transcriptional activity of the chromosome is differentially perturbed, that is, some regions are being more affected than others, then patterns that existed in unperturbed chromosome may no longer spread across regions with such an 'out of sync' activity, resulting in a partial spatial confinement of originally wider patterns. Moreover, if transcriptional activity is inhibited in a spatial locality, it may cause reduction, or even complete elimination, of local patterns. The wavelet power spectrum in Figure E. coli [While global and local patterns of transcription are likely to be due to multiple causes, our data suggest DNA gyrase as an important factor in pattern formation. It has been suggested that DNA gyrase is not randomly distributed along the chromosome of E. coli . We examE. coli genome sequence [et al. have shown that the size of a supercoiled domain in the E. coli chromosome is of the order of 10 kb [The development of new high-throughput technologies for parallel analysis of gene expression  and the sequence  have enaof 10 kb . The simThe observed changes in the long-range correlations could not be explained by changes in supercoiling because they are observed in the mutant as well as after drug treatment. It is more likely that changes in the medium- and long-range transcriptional patterns are associated with a change in the distribution of gyrase binding to the chromosome. The similarity between patterns of gyrase binding and transcription provides the basis for such a conjecture. It also seems plausible that coherent transcription, or changes in transcription, among clusters of functionally related genes  could beet al. [Analysis of the structure in any signal can be complicated by instrument biases. Such biases in microarray measurements were originally pointed out by Speed and co-workers . Althouget al.  revealedE. coli chromosome. These patterns can be classified on the basis of overall similarity in transcriptional activity of individual genes as well as on the basis of regional similarities. Three major spatial patterns have been identified: short-range correlations that are stable, on average, over 7 kb and could extend up to 15-16 kb; medium-range correlations over 100-125 kb; and long-range correlations over 600-800 kb. These patterns are experimentally stable and can be reproducibly detected in mid-exponential cells grown in batch culture. The growth rate and medium composition appear to have very minimal effects on pattern formation. However, these patterns could be modulated by perturbing DNA gyrase. The significant patterns of gyrase distribution on the chromosome match those of transcriptional activity. Among several proteins (see Results) whose distribution we mapped on the chromosome in mid-exponential culture, the pattern of gyrase binding was the only one coinciding with the patterns of transcription. Although it remains to be seen whether the observed patterns resulted from coherent transcription of functionally related genes regularly distributed along the chromosome and/or through chromosomal organization, the findings presented here are the first evidence of physiologically determined higher-order organization of transcription in any chromosome studied to date.This study demonstrates the existence of spatial patterns of transcription in the E. coli K12 strain MG1655 obtained from the ATCC. Mutant gyrAR alleles used in this study carried D82G (this study) and S83L [gyrAR alleles were linked to a Tn10 marker and P1 transduced into E. coli K12 MG1655 as described previously [dnaC2 [All experiments in this study were carried out in and S83L  mutationeviously . MG1655 y [dnaC2  was usedE. coli were designed, printed and probed as described previously [600 of 0.5-0.6 using the hot-phenol method [Whole-genome DNA microarrays of eviously ,18. To eeviously . The releviously . The RNAl method  at 1% faFollowing the removal of the array-, pin- and dye-specific effects ,41, the j = 0,1,2,...,J, where yx is the series value at the index corresponding to a given ORF location,  is the mean over all N observations and J is the number of genes in the genome minus 1. The standard error of autocorrelation estimates is determined as (N)-1/2, where N is the number of samples in the series.with Y that corresponds to the relative abundance of mRNA. This variable could be measured as a function of the position, x, on the E. coli chromosome. Values of Y are discretely recorded following two-color hybridization on the whole-genome DNA microarrays. Thus there is a finite series of values of x, {ix}, i = 1, 2, 3,...., N  and corresponding values of Y, {iY}, i = 1, 2, 3,...., N. We call {iY} a spatial series or sequential series. For the sake of convenience we approximated positions of the centers of mass of individual genes, {ix}, as evenly spaced. A process is a rule or procedure that generates a sequential series, that is, a prescription for determining the values Y for a given set of values of x. We defined a spatial series recorded in one whole-genome hybridization as one realization of the process.Information about the process of transcription could be extracted by identifying a pattern in the observed variations in gene activities followed by a search for the cause or explanation of the pattern. For instance, the pattern may consist of a defined dependence of transcriptional activity as a function of a chromosomal position, or it may consist of the dependence of transcriptional activity as a function of time following a treatment or during the cell cycle. This functional dependence may express itself as a linear variation or harmonic oscillation partially hidden behind the noise. We considered a physical variable Yi} and not specific values that are the same at every realization. However, defining such a distribution did not seem feasible. Instead, we determined significant autocorrelation components in the spatial series by assessing the effect of experimental error on the significance of correlations. Following the recording of spatial series in at least three independent biological replicates, we simulated realizations of the process of transcription by resampling relative abundances in a gene-specific manner (see [The nature of the data that we register using microarrays as well as the nature of the process itself is likely to be such that the rule(s) generating a sequential series specify the probability distribution of {ner  final concentration. Following incubation with monoclonal antibodies against the GyrA subunit of DNA gyrase (TopoGEN), the protein-DNA complexes were precipitated using Protein A-agarose beads (Sigma). Ligation-mediated PCR  was used to amplify DNA following the reversal of cross-links. Sonicated genomic DNA served as a reference in two-color hybridizations, in which both the sample and the reference were labeled in the Klenow reaction following random amplification by LM-PCR.Chromatin immunoprecipitation of DNA sequences bound by DNA gyrase and their detection using whole-genome DNA microarrays were adapted from . BrieflyAdditional data file A table of the intensities used in the analysis of spatial patternsClick here for additional data file


In vivo evaluation requires invasive imaging procedures that cannot be repeated serially.Endothelial function in hypercholesterolemic rabbits is usually evaluated ex vivo measurements.We evaluated a non-invasive ultrasound technique to assess early endothelial function in rabbits and compare data with in vivo by transcutaneous vascular ultrasound of the abdominal aorta. Ex vivo endothelial function was evaluated on isolated aortic rings and compared to in vivo data.Twenty-four rabbits (fed with a cholesterol diet (0.5%) for 2 to 8 weeks) were given progressive infusions of acetylcholine (0.05–0.5 μg/kg/min) and their endothelial function was assessed ex vivo results.Significant endothelial dysfunction was demonstrated in hypercholesterolemic animals as early as 2 weeks after beginning the cholesterol diet . Unexpectedly, response to acetylcholine at 8 weeks was more variable. Endothelial function improved in 5 rabbits while 2 rabbits regained a normal endothelial function. These data corroborated well with in vivo by transcutaneous vascular ultrasound of the abdominal aorta in the rabbit and results correlate well with ex vivo data.Endothelial function can be evaluated non-invasively  Historically, evaluation of endothelial function in small animals has been performed on isolated vessel segments, or vessels exposed by surgical procedures. Very few attempts were made to develop a method of analysis of endothelium-dependent relaxation in vivo-5. In thNon-invasive methods to study endothelial function in humans  have been used for many years and have yielded an important amount of data -12. Unfoin vivo in rabbits and to compare this non-invasive method with results obtained ex vivo on isolated aortic rings.The objective of the current study was to assess the reliability of transcutaneous vascular ultrasound in order to evaluate endothelial function Acetylcholine, nitroglycerin and sodium nitroprusside were from Sigma (Markham, ON, Canada). Angiotensin II and endothelin-1 peptides were acquired from Peninsula Laboratories Inc. (San Carlos, CA)Guide to the care and use of experimental animals published by the Canadian Council on Animal Care and the protocol was approved by the Animal Protection Committee of the Université Laval. Sixteen rabbits were divided in two groups (n = 8) and all animals were fed with standard rabbit chow supplemented with 0.5% cholesterol (w/w) (Harlan, Indianapolis, IN) for 2 or 8 weeks respectively. The other 8 animals received normal rabbit chow for eight weeks . After 2 weeks, 8 randomly chosen cholesterol-fed rabbits were killed; the others were kept alive for an additional 6 weeks  as for the normal control group.Twenty-four male New Zealand White rabbits (3–4 kg body weight) were used in this study. Animals were treated in accordance to the ex vivo experiments. Plasma samples were drawn from the marginal ear vein every week and plasma cholesterol levels were determined using a commercially available spectrophotometric assay kit .When animals were sacrificed abdominal and thoracic aortas were excised and immediately rinsed in freshly prepared Krebs buffer in preparation for the Ultrasound evaluation of endothelial function of the abdominal aorta was performed at baseline, 2 weeks and 8 weeks. Rabbits were sedated using midazolam (0.5 mg/kg), butorphanol (0.5 mg/kg) and ketamine (30 mg/kg) IM. Marginal ear vein and artery were cannulated for drug infusions and arterial blood pressure monitoring, respectively Figure . Heart rOnce the imaging of the aorta was considered optimal, the animals received the following drug perfusions I.V sequentially for 2 minutes each: 1) saline at 1 ml/min; 2) acetylcholine (Ach) at 0.05 μg/ml/min and Ach at 0.5 μg/ml/min. Nitroglycerin (5 μg/ml/min) was used as positive control. Typical arterial blood pressure recordings are illustrated in Figure 2 where D is the diameter of the aorta. Area was expressed in percent of change from baseline. Inter and intra-observer variability was assessed on 10 randomly selected studies.Video sequences from the first 15 seconds (void volume) of drug infusion at baseline and between 40 to 60 seconds of drug infusion were digitized (Dazzle Video Creator, Dazzle Multimedia, Fremont CA) and stored on a computer for analysis Figure . Still fAt the end of the protocol rabbits were given a sub-lethal dose pentobarbital (25 mg/kg) and were sacrificed by exsanguination. The middle part of the descending thoracic aorta as well as the abdominal aorta were removed and dissected free of adhering fat and connective tissues. The aorta was placed in warm Krebs solution. Rings of 5 mm thickness were suspended in individual organ chambers filled with 5 ml of oxygenated Krebs (37C pH 7.4). The segments were connected to force transducers and any variations in force were recorded continuously .-9 to 10-5 M), angiotensin II (10-10 to 10-7 M) and endothelin-1 (10-9 to 10-6 M). Results were compared to the initial response obtained with KCL 80 mM.Baseline contractile response was evaluated by a 30 to 60 minutes exposition to KCl (80 mM) where the rings were gradually stretched to a resting tension of 2 g until steady state was reached. Following this initial experiment, contractile capacity was further evaluated by exposing the rings to other vasoconstrictors. Briefly, when the rings had recovered their resting tension after the initial KCL exposure, they were exposed sequentially to cumulative concentrations of L-phenylephrine (PE, 10-6 M). Cumulative concentrations of acetylcholine (10-9 to 3 × 10-6 M) or sodium nitroprusside (10-10 to 3 × 10-5 M) were used. Sodium nitroprusside was used as a non-endothelial dependant vasodilator while acetylcholine evaluated the endothelial-dependant vasodilatation. Relaxation was expressed as a percent of change from the pre-contracted tension with PE.Relaxation studies were performed after a precontraction with PE . Differences between the various conditions in the Total cholesterol levels were measured weekly in the serum of cholesterol-fed rabbits. As illustrated in figure As expected, saline alone had no effect. Low doses of acetylcholine (ACh 0.05 and ACh 0.5 μg/kg/min) had only a minor and transient lowering effect on blood pressure. As illustrated in Figure in vivo results, we performed sections isometric contraction-relaxation experiments on isolated aortic rings. In Figure In order to confirm the validity of the We then studied the endothelium-dependent relaxation using acetylcholine on our aortic rings after a pre-contraction with phenylephrine (1 μM). As illustrated in Figure in vivo, the endothelial function of the animals fed 8 weeks with the cholesterol diet was heterogeneous. In those animals hypercholesterolemia had no effect on the acetylcholine-induced relaxation of thoracic aortic rings while for the abdominal aortic sections; the response to acetylcholine was highly variable. As illustrated in Figure As seen Our results clearly show that endothelial function can be assessed non-invasively by transcutaneous ultrasound of the abdominal aorta in hypercholesterolemic rabbits. The method was easily feasible in all animals and yielded very reproducible results. We also show that this in vivo method correlates very well with the ex vivo evaluation of endothelial function on isolated aortic rings. To our knowledge, this is the first demonstration of such a comparison.ex vivo evaluation of endothelial function on aortic rings [ex vivo data [Ultrasound imaging of the brachial artery in response to reactive hyperaemia has been used in many studies in humans -12. Normic rings . Our metivo data ,14.in vivo and ex vivo. This transient improvement of endothelial function in the early phases of the atherosclerotic process has never been described before to our knowledge and the underlying mechanisms responsible for this paradoxical response need to be explored. This dysfunction may relate to an initial stress response of the aortic endothelium to hyperlipidemia then evolving with the development of atherosclerosis lesions.The extent of endothelial dysfunction observed after 2 weeks of hypercholesterolemic diet was surprising although this parameter has not been studied very much after such a short exposure to hypercholesterolemia in rabbits [in vitro data. A transient improvement in endothelial function can occur after 8 weeks of hypercholesterolemia in some animals for reasons that remain unclear.Endothelial function can be evaluated non-invasively in rabbits using a standard vascular ultrasound probe by a trans-abdominal approach. Results correlate well with


PPARγ agonists ameliorate insulin resistance and dyslipidemia in type 2 diabetic patients. Adiponectin possesses insulin sensitizing properties, and predicts insulin sensitivity of both glucose and lipid metabolism. In diet-induced insulin resistant rats and ZDF rats, the current studies determined the correlation between PPARγ agonist-upregulated fatty acid binding protein(FABP3) mRNA in adipose tissue and PPARγ agonist-elevated serum adiponectin, and the correlation between PPARγ agonist-elevated serum adiponectin and PPARγ agonist-mediated efficacy in insulin sensitization and lipid lowering.Parallel groups of SD rats were fed a high fat/sucrose (HF) diet for 4 weeks. These rats were orally treated for the later 2 weeks with vehicle, either PPARγ agonist GI262570 (0.2–100 mg/kg, Q.D.), or GW347845 (3 mg/kg, B.I.D). Rats on HF diet showed significant increases in postprandial serum triglycerides, free fatty acids (FFA), insulin, and area under curve (AUC) of serum insulin during an oral glucose tolerance test, but showed no change in serum glucose, adiponectin, and glucose AUC. Treatment with GI262570 dose-dependently upregulated adipose FABP3 mRNA, and increased serum adiponectin. There was a positive correlation between adipose FABP3 mRNA and serum adiponectin (r = 0.7350, p < 0.01). GI262570 dose-dependently decreased the diet-induced elevations in triglycerides, FFA, insulin, and insulin AUC. Treatment with GW347845 had similar effects on serum adiponectin and the diet-induced elevations. There were negative correlations for adiponectin versus triglycerides, FFA, insulin, and insulin AUC . In ZDF rats treated with PPARγ agonists pioglitazone (3–30 mg/kg, B.I.D.) or GW347845 (3 mg/kg, B.I.D.), there were also negative correlations for serum adiponectin versus glucose, triglycerides, FFA .in vivo PPARγ activation and PPARγ agonist-induced efficacy on insulin resistance and dyslipidemia in rats.This study demonstrated that (a) PPARγ agonists improved insulin sensitivity and ameliorated dyslipidemia in HF fed rats and ZDF rats, which were correlated with serum adiponectin; (b) Serum adiponectin was positively correlated with adipose FABP3 mRNA in GI262570-treated rats. These data suggest that serum adiponectin can serve as a biomarker for both  Type 2 diabetes mellitus (T2D) and the metabolic syndrome are characterized by resistance to the action of insulin in peripheral tissues, including skeletal muscle, liver, and adipose. Activation of the peroxisome proliferator-activated receptor gamma (PPARγ) improves insulin sensitivity and lowers circulating levels of glucose, triglycerides and free fatty acids without stimulating insulin secretion in rodent models of T2D ,2. PPARγdb/db mice [Adiponectin, an adipose-specific plasma protein, possesses insulin sensitizing and anti-atherogenic properties . It has /db mice . Yang et/db mice . Tschrit/db mice .While PPARγ agonists increase plasma adiponectin and adiponectin levels predict insulin sensitivity, there is not a clear demonstration of the relationships among PPARγ agonist-increased adiponectin and PPARγ agonist-mediated efficacy on insulin sensitivity/in vivo PPARγ activation. Therefore, the current studies were designed to define these relationships and assess serum adiponectin as a biomarker for in vivo PPARγ activation and PPARγ agonist-induced efficacy on insulin sensitization and lipid lowering.Rats on the HF diet for 4 weeks showed marked insulin resistance and dyslipidemia, indicated by significant increases in postprandial serum levels of triglycerides, free fatty acids, insulin, and area under curve (AUC) for serum insulin during OGTT. But the HF diet did not cause changes in postprandial serum glucose or OGTT glucose AUC compared with rats on normal diet, consistent with an insulin resistant, pre-diabetic phenotype. Serum adiponectin level in rats on HF diet was slightly higher than that in normal diet rats at week 2, but back to the same level at week 4 Table .As showed in Fig. Treatment of rats on HF diet with GI262570 for 2 weeks significantly decreased the diet-induced elevations in postprandial serum triglycerides, free fatty acids, insulin, and insulin AUC in a dose-dependent manner , and that PPARγ agonist GI262570 dose-dependently increased FABP3 mRNA. AP2 was abundant in epididymal fat tissues, and was only slightly increased by GI262570 in a non-dose-dependent manner (data not shown). LPL was decreased in high fat diet fed rats, which was reversed by GI262570 but not dose-dependently (data not shown). With in vivo chronic exposure, the effect of PPARγ agonists on gene expression is difficult to separate from the effects on differentiation. In general we find aP2 a better marker of adipocyte differentiation than PPARγ activation. Since PPARγ agonist-mediated action in vivo may vary with organs/tissues  ,21 and d1C, and ameliorated insulin resistance in type 2 diabetic patients [Adiponectin has been demonstrated to have an insulin sensitizing effect . Circulapatients . HoweverOther adipokines, such as leptin, are important in obesity and insulin resistance. Unlike adiponectin, leptin is positively correlated with fat amount, mass and percentage . It has These studies demonstrated that in both diet-induced and genetic rat models of insulin resistant (metabolic) syndrome the full PPARγ agonists GI262570, GW347845, and pioglitazone significantly elevated serum adiponectin levels, increased adipose transcription of the PPARγ response gene FABP3, and were efficacious as expected. This is the first demonstration of correlation among PPARγ agonist-increased serum adiponectin, PPARγ agonist response gene mRNA, and PPARγ agonist-mediated efficacy in insulin sensitivity and lipid lowering. These data indicate that serum adiponectin can serve as a biomarker for both in vivo PPARγ activation and PPARγ agonist-induced efficacy in rats.2. White adipose tissue  were saved for determining mRNA levels of PPARγ response gene FABP3. In Zucker rats, serum was collected after 2 weeks of dosing for determining postprandial levels of glucose, insulin, triglycerides, free fatty acids, and adiponectin. Zucker rats were then euthanized with CO2.All procedures performed were in compliance with the Animal Welfare Act and U.S. Department of Agriculture regulations, and were approved by the GlaxoSmithKline Animal Care and Use Committee. Male caesarian derived Sprague Dawley rats (SD, 225–250 g) (Charles River, Indianapolis, IN) were fed rodent chow Purina 5001 (Harlan Teklad, Indianapolis, IN). Male Zucker diabetic fatty (ZDF) and male Zucker lean rats (8 weeks old) (Genetic Models, Indianapolis, IN) were fed Formulab Diet 5008 (PMI Feeds, Richmond, IN). After an adaptation period of 1 week, SD rats were fed a HF diet  for 4 weeks. SD Rats fed chow Purina 5001 served as normal diet control. SD rats on HF diet were treated with vehicle (0.5% hydroxypropyl methylcellulose and 0.1% Tween 80), PPARγ agonist GI262570  .Serum adiponectin of SD rats was determined by using adiponectin RIA kit (Linco Research, MO), according to the manufacture's instruction. Serum adiponectin of ZDF rats was determined by using adiponectin ELISA kit , according to the manufacture's instruction.® Chlorhexidine Gluconate, Zeneca Pharmaceuticals, Delaware). A longitudinal incision was made over the right external jugular vein. 5–10 mm of the vein was exposed by blunt dissection. Jugular cannula (Access™ Technologies, IL) was inserted into the vein for about 1 inch. The cannula was secured using sterile sutures. The cannula was routed subcutaneously, exteriorized between the scapulae. The cannula was then filled with dextrose-heparin solution (50:50), and heat sealed.Under anesthesia with isoflurane, surgical site was prepared using standard aseptic technique (with Hiboclens® XL Glucometer (Bayer, Tarrytown, NY). Serum was collected for insulin measurement. Area under curves (AUCs) for glucose and insulin during OGTT were calculated by using WinNonlin™ Noncompartmental Model 200.Rats implanted with jugular cannula were fasted overnight. The following morning, dextrose (0.5 g/ml in water, 2 g/kg body weight) was administered by oral gavage. Blood samples (0.3 ml/time) were obtained from the jugular cannula before gavage, 10, 20, 30, 45, 60, 90 and 120 min after gavage. Blood glucose was immediately measured by using EliteSerum insulin of SD rats level was determined using Rat Insulin ELISA kit , according to the manufacture's instruction. Serum insulin level of ZDF rats was determined using Igen's M-SERIES M-8 Analyzer .® method [free™ kit (Ambion – according to protocol). The samples were then quantitated by RiboGreen™ (Molecular Probes – according to protocol). GAPDH gene expression was analyzed in the absence of reverse transcriptase to ensure the samples were free of genomic DNA. The samples were then converted to cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems – according to protocol). Samples were diluted to a final concentration of 5 ng/ul of cDNA. PCR results were generated using the 5' nuclease assay (TaqMan) [Total RNA in epididymal fat pad was isolated by the TRIZOL® method . All RNA(TaqMan)  and the There was a minimum of 5 rats for each data point. Data are presented as mean ± SEM. Correlation between two parameters and the significant level of correlation were analyzed by Pearson correlation analysis. Differences between vehicle and treated groups were analyzed by two-way ANOVA. P less than 0.05 was taken to be significant.QD: Once a dayBID: Twice a dayPCR: Polymerase Chain ReactionGAPDH: Glyceraldehyde-3-Phosphate DehydrogenaseANOVA: Analysis of VarianceBY is the principal investigator. LC, LGC, JM, and DW participated in the in vivo experiments. KC and JS performed the real time PCR. KB, SS and GP participated in study design and manuscript preparation.


Emphysematous cystitis is a rare disease entity caused by gas fermenting bacterial and fungal pathogens. Clinical symptoms are nonspecific and diagnostic clues often arise from the unanticipated imaging findings. We report a case of 52-year-old male who presented with fever, dysuria and gross hematuria who was found to have emphysematous cystitis. Escherichia coli emphysematous cystitis that resolved with antibiotic treatment.Emphysematous cystitis is an uncommon, but severe manifestation of infection of the urinary bladder produced by gas forming organisms. The presentation may be atypical and contrary to the degree of inflammation, patients may present with subtle clinical findings. A high index of suspicion, especially in susceptible populations, is needed. We report a case of middle-aged, poorly controlled, diabetic male who presented with dysuria, fever and hematuria and was found to have A 52-year-old male presented with fever, chills and blood in the urine. Four days prior to admission, he noted increased urinary frequency, urgency, occasional incontinence and burning sensation during micturition. His past medical history is significant for coronary artery disease, hyperlipidemia, hypertension and insulin dependent diabetes mellitus (most recent HgbA1c of 11.7 two weeks prior). He had 66-pack year history of smoking. Medications on admission included, aspirin, atenolol, cyclobenzaprine, gabapentin, gemfibrozil, glipizide, NPH insulin, hydrochlorthiazide/triamterene, nifedipine, nitroglycerin patch, omeprazole, and sertraline.On physical examination, his temperature was 100.9°F, pulse 88, BP 123/71 mm/hg. Cardiac and pulmonary examinations were unremarkable. His abdomen was soft, nontender with positive bowel sounds and no organomegaly detected. The rectal examination was normal with a non-tender, smooth prostate. Neurological exam was exam was unremarkable.3, hemoglobin 15.1 gm/dl, sodium 128 mmol/l, potassium 4.1 mmol/l, chloride 91 mmol/l, bicarbonate 24 mmol/L, glucose 273 mg/dl and an anion gap of 13. Urine analysis revealed red, cloudy urine with pH 5.0, nitrite positive and numerous white and red blood cells on microscopy. A pelvic X-ray (figure Escherichia coli and based on the sensitivities he was discharged home on ciprofloxacin.Laboratory evaluation revealed white cell count 17,400 cells/mmE. coli [Enterobacter aerogenes, Klebsiella pneumonia, Proteus mirabilis, Staphylococcus aureus, streptococci, Clostridium perfringens [and Candida albicans [Emphysematous cystitis is a rare entity characterized by pockets of gas in and around the bladder wall produced by bacterial or fungal fermentation ,2. Patiefringens , and Canalbicans . The mecDiagnostic entities associated with gas in the genitourinary tract include emphysematous pyelonephritis, emphysematous pyelitis, and gas-forming renal abcess. Patients with emphysematous cystitis are not as acutely ill as those with pyelonephritis or pyelitis. Abdomino-pelvic CT scan can further delineate the extent of disease. It is important to differentiate emphysematous cystitis from emphysematous pyelonephritis, in which gas involves the renal parenchyma, since the latter has an increased mortality and generally requires nephrectomy. In contrast surgical intervention is rarely needed in emphysematous cystitis except when an anatomical abnormality like an obstruction or stone is present .The source of this gas within the urinary tract is from infection, trauma, vesico-enteric fistulas from radiation therapy, rectal carcinoma, diverticular disease or Crohn's disease and iatrogenic causes, such as diagnostic or surgical instrumentation. History, physical exam and imaging are the best modalities to differentiate the above etiologic causes. Fistulous tracts, abscess, can be excluded on CT scan.Emphysematous cystitis requires aggressive treatment with parenteral antibiotics and bladder drainage . DelayedEmphysematous cystitis most often is not diagnosed by routine or systematic approach. It is a rare entity, detected on imaging, and the physician should be cautious, tailor the diagnostic approach to individual patients based on the suspicion, available clinical and radiological data, and consider emphysematous cystitis in the differential diagnosis of hematuria in a patient with known risk factors.


Pregnant women are a major risk group for malaria in endemic areas. Only little information exists on the compliance of pregnant women with malaria and anaemia preventive drug regimens in the rural areas of sub-Saharan Africa (SSA). In this study, we collected information on malaria and anaemia prevention behaviour in pregnant women of rural Burkina Faso.Cross-sectional qualitative and quantitative survey among 225 women of eight villages in rural northwestern Burkina Faso. Four of the villages had a health centre offering antenatal care (ANC) services while the other four were more than five kilometers away from a health centre.Overall ANC coverage (at least one visit) was 71% . Malaria and anaemia were considered as the biggest problems during pregnancy in this community. ANC using women were quite satisfied with the quality of services, and compliance with malaria and anaemia prevention regimens (chloroquine and iron/folic acid) was high in this population. Knowledge on the benefit of bed nets and good nutrition was less prominent. Distance, lack of money and ignorance were the main reasons for women to not attend ANC services.There is an urgent need to improve access of rural SSA women to ANC services, either through increasing the number of rural health centres or establishing functioning outreach services. Moreover, alternative malaria and anaemia prevention programmes such as intermittent preventive treatment with effective antimalarials and the distribution of insecticide-treated bed nets need to become implemented on a large scale. Each year between 75.000 and 200.000 infant deaths are attributed to malaria infection in pregnancy globally, and between 200.000 and 500.000 pregnant women develop severe anaemia as a result of malaria in Sub-Saharan Africa (SSA) . PregnanP. falciparum parasitaemia, with high rates of maternal morbidity including fever and severe anaemia, with abortion and stillbirth, and with high rates of placental malaria and consequently low birth weight in newborns caused by both prematurity and intrauterine growth retardation [Pregnancies in women living in malaria endemic regions are associated with a high frequency and density of ardation ,3,5.In order to reduce malaria-related ill health, regular chemoprophylaxis has been recommended to all pregnant women living in malaria-endemic areas . Most AfWhile insecticide-treated bed nets and curtains (ITN) have been shown to substantially reduce malaria morbidity and mortality in children, results from initial trials on the efficacy of ITNs for malaria prevention in pregnancy produced conflicting evidence and were classified as inconclusive by the Cochrane Collaboration ,9. HowevIn Burkina Faso, the official policy for malaria and anaemia prevention in pregnant women comprises of chloroquine and iron/folic acid supplementation respectively. However, in the rural areas of Burkina Faso little information exists on coverage with ANC and on compliance with preventive regimens. We assessed the coverage of antenatal care and investigated the knowledge and adoption of preventive practices with respect to malaria and anaemia in users and non-users of antenatal care.Centre de Recherche en Santé de Nouna (CRSN) in Nouna Health District, northwestern Burkina Faso. The CRSN research zone consists of Nouna town and 41 of the surrounding villages with a total population of around 60.000 inhabitants. The Nouna area is a dry orchard savanna, populated mainly by subsistance farmers of different ethnic groups.The study took place in the rural part of the research zone of the Malaria is holoendemic but highly seasonal in this part of Westafrica . Most maThe official policy for malaria and anaemia prevention during pregnancy in Burkina Faso consists of a curative dose of 1 500 mg chloroquine during three days followed by a weekly dose of 300 mg chloroquine and a combined dose of daily 200 mg iron and 0.25 mg folic acid. This regimen should be followed from the first ANC visit until six weeks after delivery. Since the year 2002 it is the official policy in Burkina Faso to offer ANC services free of charge. This includes ANC card, physical examination and counselling, and malaria/anaemia prevention drugs. However, urine examination, gloves, and drugs for other concomitant diseases still have to be paid for. Urine examination and gloves usually cost 150–200 F CFA (1 Euro = 650 F CFA).The study was cross-sectional and descriptive in nature, using both qualitative and quantitative methods for data collection. The study was implemented in May and June 2003. The research team comprised of the investigators and six trained local interviewers familiar with the common spoken local languages and French. The questionnaires were pre-tested before administration.The study took place in eight of the 41 villages of the CRSN study area. Villages were selected as follows: At a first stage, the four villages of the CRSN study area where a health centre exists were purposely selected. To account for the socio-demographic variability and geographical accessibility, in each of these health centre defined sub-areas, another village distant of at least 5 kilometres to the health centre was randomly selected.We conducted six Focus Group Discussions (FGD). Two with pregnant women users of ANC, two with husbands of pregnant women users of ANC, and two with pregnant women non-users of ANC. Respective FGDs were held with groups of six to 12 participants from study villages with and without a health centre.Key informant interviews were conducted with four maternity health workers, seven traditional birth attendants and 29 women group leaders. The interviews assessed their knowledge, attitudes and practices about malaria and anaemia prevention in pregnancy.The design of the quantitative survey instrument was informed by the results of the qualitative interviews. A structured questionnaire was administered to all women from the eight study villages who had delivered a life child during the last six months (n = 225). Information on births was available through the existing Demographic Surveillance System (DSS) in the study area . The queOn the ANC cards, the estimated age of pregnancy at first visit was reported through the fundal height in centimetres, as women were not able to recall their last menstruation period during ANC visits. These data were afterwards transformed into a specific scoring system.We used two definitions for malaria prophylaxis. A complete curative dose of 1 500 mg chloroquine followed by regularly weekly 300 mg doses afterwards (complete prophylaxis), and an incomplete regimen consisting of only 300 mg weekly doses (incomplete prophylaxis). A combined dose of daily 200 mg iron and 0.25 mg folic acid was defined as a complete prophylaxis.The data were entered in Microsoft Access 2000, cleaned, and then analysed with Epi Info 2000. Univariate analysis was done with chi-square test or Fisher's exact test to compare proportions for categorical variables. Results were considered to be significant when the 2-sided P value was <0.05.We received ethical approval from the institutional Ethical Committee at the Department of Tropical Hygiene in Heidelberg, Germany, and the local Ethical Committee in Nouna, Burkina Faso. Oral informed consent was obtained from all participants.The characteristics of participants on the quantitative survey are shown in table In this study population, the minimal ANC coverage (defined as at least one ANC visit during pregnancy) was 159/225 (71%) and 63/225 (28%) if we consider the optimal frequency of at least three ANC visits . Minimal ANC coverage was 97/102 (95%) in villages with a health centre vs 62/123 (50%) in remote villages (p < 0.001), while the optimal ANC coverage was 55/102 (54%) in villages with a health centre vs 8/123 (7%) in remote villages (p < 0.001).Among ANC users, 27%, 40% and 33% of women visited ANC services one time, two times or more than two times respectively during their pregnancy. The first ANC visit of ANC users was in 14% during the first trimester, in 57% during the second trimester, and in 27% during the third trimester.Malaria and anaemia were considered as the most common diseases during pregnancy by the majority of the participants in the FGD, key informant interviews and survey women. In the Dioula language (lingua franca in the region) malaria is equivalent to "Soumaya", light to moderate anaemia to "Djolidessé" and severe anaemia to "Djoliban". Most women in the FGD were knowledgeable about the malaria prevention effect of chloroquine and the anaemia prevention effect of iron/folic acid.The knowledge of malaria and anaemia prevention measures by ANC users is given in table Table Of ANC users, the great majority reported to be satisfied with the quality of ANC services. Only 16% of ANC users were aware of the fact that ANC services have recently become free of charge, and 42% reported that they still had paid for services. Most (73%) ANC users had paid between zero and 200 F CFA, while 12% and 6% had paid between 200 and 1.500 and between 1.500 and 7.500 F CFA respectively.Apart from distance to the next health centre, lack of resources and ignorance were the most frequent stated reasons during qualitative and quantitative interviews why women did not attend ANC services. The majority of ANC non-users reported no specific prophylaxis during their pregnancy, but 6% took irregularly self medication for malaria/anaemia prevention. Moreover, 30% of ANC non-users had sought advice from traditional birth attendants (TBA).Typical statements in the FGDs with non-users of ANC services werecollected as follow:"ANC is not free of charge, you must pay for ANC card and the medicine and it could be up to 500 CFA and for me I find it is expensive. Our husbands find it expensive too, that is why the majority of us can not attend ANC services".• "I don't know the advantages of ANC services, some of the pregnant women if they don't fell sick they wouldn't accept to attend ANC ".• "There is lack of advices from health workers, if you go to the ANC for the 1sttime, they give drugs, they don't tell you when you should come back and how many visits you should attend. That is how they do here".• "If you are not sick, you don't pay for drugs, what you pay for is the ANC card at 150 F CFA...."• " But even the ANC card is free of charge, only the gloves cost 100 F CFA...."• "I had not yet heard about the free ANC services ... Since I know now that ANC is free of charge I will attend the services."• "We do not have money to go to the health centre it will be nice to have one in our village".• Itching, vomiting and fatigue were the most frequently stated side effects of malaria/anaemia prevention drug regimens during interviews, sometimes leading to non-compliance. Most women also stated that bed nets are considered too expensive for their household.The main findings of this study are (1) that coverage of antenatal care is far from complete, particularly in villages without a health centre, (2) that malaria- and anaemia-related knowledge and compliance with preventive measures is comparatively high with a wide gap between users and non-users of antenatal care and (3) that health services need to improve their response to the women's need for preventive care in pregnancy.In this community-based study from a rural area of Burkina Faso, we found an overall ANC coverage of 71%, which is however not representative given the used methodology. Most women had two ANC visits during their pregnancy, mainly during the second and third trimester. As we found ANC coverage to be much higher in villages with a health centre compared to villages quite distant from a health centre, and as we included similar numbers of health centre and non-health centre villages, our ANC coverage figure is likely to be an overestimate. The national demographic and health survey in Burkina Faso claims an ANC attendance (at least one ANC during one pregnancy) of 59% . AlthougOther reasons given for non-use of ANC services included ignorance and lack of money. This confirms similar observations from other rural African areas . InteresMalaria and anaemia were seen as important disease entities during pregnancy in our interviews. Moreover, most women interviewed were quite knowledgeable about effective malaria/anaemia prevention measures. However, compared to ANC users ANC non-users were significantly less knowledgeable about malaria/anaemia prevention measures. The interpretation is not straight forward because more knowledgeable women may be more likely to attend antenatal care or increased knowledge may be the result of health education in anatenatal care; most likely both effects contribute to the observed gap between ANC users and ANC non-users.There is now a broad agreement on the need of new strategies for community participation on the implementation of effective malaria control activities, as well as for a better education of service providers of both the public and the private sector .It was reassuring to find nearly all of the women with reported ANC use to have an ANC card in their house. Specific prescriptions were found on most cards, but in particular malaria prophylaxis prescriptions were often not complete. This explains why self-reported compliance with recommended prevention regimens was sub-optimal with regard to malaria. Such discrepancies have been observed already in a recent study in pregnant women of Nouna town . HoweverPlasmodium falciparum has put into question the efficacy of this well-known drug [The best model for effective and cost-effective malaria prevention during pregnancy in SSA still needs to be developed. Chloroquine has been the mainstay for malaria control in sub-Saharan Africa (SSA), but the emergence of chloroquine-resistant own drug . The firown drug . Althougown drug . Currentown drug . Compareown drug ,25. The own drug ,13,25.None declared.CM, AJ and OM designed the study. FS and BK were responsible for the conduct of the study in Burkina Faso. CM analysed the data. All authors contributed to the interpretation of the data, helped write the paper, and read and approved the final manuscript.The pre-publication history for this paper can be accessed here: